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Analytical investigation of the proteasome inhibitor Bortezomib and the total synthesis of specialized pro-resolving lipid mediators

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Analytical investigation of the proteasome inhibitor Bortezomib and the total synthesis of specialized pro-resolving lipid mediators

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Content ANALYTICAL INVESTIGATION OF THE
PROTEASOME INHIBITOR BORTEZOMIB AND
THE TOTAL SYNTHESIS OF SPECIALIZED
PRO-RESOLVING LIPID MEDIATORS
by

STEPHEN J. GLYNN

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)

December 2015

Copyright 2015 Stephen J. Glynn

I
Acknowledgments
To my parents (Brian & Veronica), for your encouragement, support and
unconditional love, you have inspired me to eclipse my wildest dreams. Without
you I would never have had this moment. To my older brother, Michael Glynn for
your immense support, love and care you have always been my best friend and
greatest role model. To Kelly Chuh your love, drive, ability, and beauty brighten
each day and challenge me to strive for the impossible. To my committee
members, Dr. Prakash, Dr. Louie, Dr Pratt and Dr. Zhang for their invaluable
advice and guidance. To all the my senior members of the Petasis group past and
present for being that exemplary scientist and creating an environment of growth
and learning. Thank you very much for your patience and willingness to teach. To
the current Petasis lab members for being such wonderful lab mates and friends.
To all my phenomenal friends, who stood by me through thick and thin. And
most importantly, to my mentor Nicos for taking me on board and guiding me
through numerous research projects, but always giving me enough room to grow.

II

Abstract
This body of work seeks to use synthetic and analytical chemistry
techniques to improve the understanding of biologically interesting compounds.
This has been completed in multiple projects investigating the anti-cancer
therapeutic Bortezomib and natural metabolites of essentially fatty acids with
high potency promoting the resolution of inflammation and tissue regeneration.
Chapter 1 provides a detailed analytical investigation towards the
characterization and evaluation of the interactions of FDA approved anti-cancer
therapeutic Bortezomib with dietary polyphenols. The green tea polyphenol
epigallocatechin-3-gallate was reported to effectively antagonize the ability of
Bortezomib to induce apoptosis in cancer cells. This interaction was attributed to
the formation of a covalent adduct between a phenolic moiety of
epigallocatechin-3-gallate with the boronic acid group of Bortezomib. However,
the structural details of this boron adduct and the molecular factors that
contribute to its formation and its ability to inhibit Bortezomib’s activity
remained unclear. This work describes the use of NMR spectroscopy to
characterize the structures and properties of the boron adducts of
epigallocatechin-3-gallate and related polyphenols.
The remaining chapters are dedicated to the design, synthesis and activity
of compounds enzymatically produced from essential omega-3 and omega-6 fatty
acids during a host’s inflammatory immune response, and their synthetic
analogs. This includes a brief review of the class of specialized pro-resolving lipid

III

mediators (SPMs) including their isolation, identification, and biological activity.
It has long been postulated that fatty acids such as docosahexaenoic acid present
a variety of health benefits including the production of SPMs containing anti-
inflammatory, pro-resolving, and tissue regenerative properties. This work helps
support these claims by producing the design and synthesis of several SPMs
derived from docosahexaenoic acid providing a molecular basis for some of
theses health effects. Including the design and synthesis of resolvin D4, aspirin-
triggered resolvin D4, maresin 1, maresin 2, maresin conjugate in tissue
regeneration 1 and 2, 16S-17S-epoxy-neuroprotectin D1, and a library of benzo
SPM based analogs.

IV

Table of Contents

Acknowledgments .......................................................................................... I
Abstract…..................... ................................................................................ II
Table of Contents ........................................................................................ IV
Table of Figures ........................................................................................... IX
Table of Schemes .......................................................................................... X
Chapter 1. Analytical Investigation of the Proteasome Inhibitor
Bortezomib Introduction ......................................................... 1
1.1 Introduction ...................................................................................... 1
1.2 Results and Discussion ...................................................................... 8
1.2.1 Structure elucidation of the BZM/EGCG adduct by
1
H NMR ... 8
1.2.2 Quantification and structural analysis of the BZM/EGCG
adduct by
11
B NMR ................................................................... 10
1.2.3 Comparison by
11
B NMR of selected polyphenols for their
ability to form boron adducts with BZM .................................. 12
1.2.4 Determination of the equilibrium constant of the BZM boron
adduct by
11
B NMR and
19
F NMR ............................................. 15
1.2.5 NMR evaluation of polyphenol adducts of boron drugs .......... 16
1.3 Conclusion ........................................................................................ 17
1.4 Experimental ................................................................................... 18
1.5 References ....................................................................................... 19

V

Chapter 2. DHA Derived Lipid Mediators in Inflammation Resolution 26
2.1 Introduction .................................................................................... 26
2.2 Inflammatory Response and the Resolution of Inflammation ...... 27
2.3 Identification of DHA-Derived SPMs ............................................. 29
2.4 Biological Activity of DHA Derived SPMs ...................................... 30
2.5 The Chemistry of Lipid Mediators .................................................. 33
2.6 Conclusion ....................................................................................... 35
2.7 References ....................................................................................... 36
Chapter 3. Total Synthesis of Resolvin D4 and Aspirin-triggered
Resolvin D4 ............................................................................ 39
3.1 Introduction .................................................................................... 39
3.2 Biosynthesis of RvD4 and AT-RvD4 ............................................... 39
3.3 Results and Discussion .................................................................... 41
3.3.1 Synthesis of RvD4 and AT-RvD4 ............................................. 41
3.3.2 Synthesis of RvD4 and AT-RvD4 Building Blocks ................... 42
3.3.3 Construction of RvD4 and AT-RvD4 ....................................... 45
3.4 Conclusion ....................................................................................... 46
3.5 Experimental ................................................................................... 47
3.6 References ....................................................................................... 68
Chapter 4. Total Synthesis of Maresin 1 ................................................... 71
4.1 Introduction ..................................................................................... 71
4.2 Biosynthesis of MaR1 ....................................................................... 71
4.3 Results and Discussion .................................................................... 72

VI

4.3.1 Retrosynthesis of MaR1 ............................................................ 72
4.3.2 Synthesis of MaR1 Building Block ........................................... 73
4.3.3 Construction of MaR1 ............................................................... 74
4.4 Conclusion ....................................................................................... 75
4.5 Experimental ................................................................................... 76
4.6 References ....................................................................................... 85
Chapter 5. Total Synthesis of Maresin 2, 13S, 14S-epoxy-maresin, and
Maresin sulfido-conjugates: MCTR1-methyl ester, and
MCTR2-methyl ester .............................................................. 87
5.1 Introduction .................................................................................... 87
5.2 Biosynthesis of the Macrophage-derived Pro-resolving Family .... 88
5.3 Results and Discussion .................................................................... 89
5.3.1 Retrosynthesis of MaR2 ........................................................... 89
5.3.2 Synthesis of MaR2 building blocks .......................................... 90
5.3.3 Construction of MaR2 .............................................................. 92
5.3.4 Retrosynthesis of MCTR Series ................................................ 92
5.3.5 Construction of MCTR1 and MCTR2 Methyl Esters ............... 93
5.4 Conclusion ....................................................................................... 94
5.5 Experimental ................................................................................... 95
5.6 References ..................................................................................... 108
Chapter 6. Total Synthesis of 16S-17S-epoxy-neuroprotectin
D1/protectin D1 ..................................................................... 110
6.1 Introduction ................................................................................... 110

VII

6.2 Biosynthesis of the Protectin Family of Lipid Mediators .............. 110
6.3 Results and Discussion ................................................................... 111
6.3.1 Retrosynthesis of 16S, 17S-epoxy Protectin ............................ 111
6.3.2 Synthesis of 16S, 17S-epoxy Protectin Building Block ............ 112
6.3.3 Construction of 16S, 17S-epoxy Protectin ............................... 113
6.4 Conclusion ...................................................................................... 114
6.5 Experimental .................................................................................. 114
6.6 References ...................................................................................... 121
Chapter 7. Total Synthesis of Benzo-SPM Analogues ........................... 123
7.1 Introduction .................................................................................. 123
7.2 Results and Discussion .................................................................. 126
7.2.1 Retrosynthesis of Benzo-lipoxin A 4 Analogs .......................... 126
7.2.2 Synthesis of Building Blocks for Benzo-lipoxin A 4 Analogs .. 127
7.2.3 Construction of Benzo-lipoxin A 4 Analogs ............................. 128
7.2.4 Retrosynthesis of Benzo-RvD1 Analogs ................................. 129
7.2.5 Synthesis of Building Blocks for Benzo-RvD1 Analogs ......... 130
7.2.6 Construction of Benzo-RvD1 Analogs ..................................... 131
7.2.7 Retrosynthesis of Alkyne-benzo-lipoxin A 4 Analogs ............. 132
7.2.8 Synthesis of Building Blocks for Benzo-alkyne-lipoxin A 4
Analogs ................................................................................... 133
7.2.9 Construction of Benzo-alkyne-lipoxin A 4 Analogs ................ 134
7.2.10 Retrosynthesis of Click-SPM Analogs .................................. 135
7.2.11 Synthesis of Building Blocks for Click-SPM Analogs .......... 136

VIII

7.2.12 Construction of Click-SPM Analogs ..................................... 136
7.3 Conclusion ...................................................................................... 137
7.4 Experimental ................................................................................. 138
7.5 References ..................................................................................... 178
Bibliography ............................................................................................... 181
Appendix.
1
H and
13
C Spectra of Substrates ............................................ 195

IX

TABLE OF FIGURES
Figure 1-1. Structure of Boron-based Proteasome Inhibitors .................. 1
Figure 1-2. Structure of EGCG and Other Dietary Polyphenols ............... 4
Figure 1-3. 400 MHz
1
H NMR Analysis of BZM/EGCG Adduct .............. 9
Figure 1-4. 400 MHz
11
B NMR Analysis of BZM/EGCG Adduct ............. 11
Figure 1-5. 400 MHz
11
B NMR Analysis of BZM with Select
Polyphenols ..................................................................................... 13
Figure 1-6. Use of
11
B NMR and
19
F NMR to Calculate the Equilbrium
Constant of the Boron Adduct of 4-fluorocatechol (FCT) with
BZM ................................................................................................. 16
Figure 2-1. Dual Anti-inflammatory and Pro-resolving Actions of
Specialized Pro-resolving Mediators .............................................. 28
Figure 2-2. Biosynthetic Derivation of Lipid Mediators From
Omega-3 and Omega-6 Fatty Acids ................................................ 30
Figure 2-3. History of the Synthesis of Lipid Mediators ........................ 35
Figure 7-1. LXA 4, RvD1 and the Library of Benzo-SPM Analogs ......... 125

X

TABLE OF SCHEMES
Scheme 3-1. Biosynthesis of Resolvin D4 and Aspirin-triggered
Resolvin D4 ..................................................................................... 40
Scheme 3-2. Retrosynthesis of Resolvin D4 and Aspirin-triggered
Resolvin D4 ..................................................................................... 42
Scheme 3-3. Synthesis of Building Block 3.5 .......................................... 43
Scheme 3-4. Synthesis of Building Block 3.6 and 3.7 ............................. 44
Scheme 3-5. Construction of RvD4 and AT-RvD4 From Building
Blocks 3.5 and 3.6 or 3.7 ................................................................. 46
Scheme 4-1. Biosynthesis of Maresin 1 ................................................... 72
Scheme 4-2. Retrosynthesis of Maresin 1 ............................................... 73
Scheme 4-3. Synthesis of Building Block 4.5 .......................................... 74
Scheme 4-4. Construction of MaR1 From Building Blocks 4.4
and 4.5 ............................................................................................. 75
Scheme 5-1. Biosynthesis of the Macrophage-derived Pro-resolving
Family .............................................................................................. 88
Scheme 5-2. Retrosynthesis of Maresin 2 ............................................... 89
Scheme 5-3. Synthesis of Building Block 5.3 .......................................... 91
Scheme 5-4. Synthesis of Building Block 5.4 .......................................... 91
Scheme 5-5. Construction of MaR2 From Building Blocks 5.3
and 5.4 ............................................................................................. 92
Scheme 5-6. Retrosynthesis of MCTR1 and MCTR2 Methyl Esters ....... 93
Scheme 5-7. Construction of MCTR1 and MCTR2 Methyl Esters

XI

From Building Blocks 5.19 and 5.14 ............................................... 94
Scheme 6-1. Biosynthesis of the Protectin Family of Lipid Mediators . 111
Scheme 6-2. Retrosynthesis of 16S, 17S-epoxy Protectin ...................... 112
Scheme 6-3. Synthesis of Building Block 6.3 ......................................... 114
Scheme 6-4. Construction of 16S, 17S-epoxy Protectin ......................... 113
Scheme 7-1. Retrosynthesis of Benzo-lipoxin A 4 Analogs .................... 126
Scheme 7-2. Synthesis of Building Block 7.7 ........................................ 129
Scheme 7-3. Synthesis of Building Block 7.2 ........................................ 128
Scheme 7-4. Construction of Benzo-lipoxin A 4 Analogs From
Building Blocks 7.2 and 7.7 ........................................................... 129
Scheme 7-5. Retrosynthesis of Benzo-RvD1 Analogs ........................... 130
Scheme 7-6. Synthesis of Building Block 7.15 ........................................ 131
Scheme 7-7. Construction of Benzo-RvD1 Analogs From Building
Blocks 7.2 and 7.15 ........................................................................ 132
Scheme 7-8. Retrosynthesis of Benzo-alkyne-lipoxin A 4 Analogs ....... 133
Scheme 7-9. Synthesis of Building Block 7.23 ...................................... 133
Scheme 7-10. Synthesis of Building Block 7.24 .................................... 134
Scheme 7-11. Construction of Benzo- alkyne-lipoxin A 4 Analogs
From Building Blocks 7.23 and 7.24 ............................................. 134
Scheme 7-12. Retrosynthesis of Click-SPM Analogs ............................ 135
Scheme 7-13. Synthesis of Building Block 7.35 and 7.36 ...................... 136
Scheme 7-14. Construction of Click-SPM Analogs From Building
Blocks 7.2 and 7.26, 7.35 or 7.36 .................................................... 137

1

Chapter 1. Analytical Investigation of the
Proteasome Inhibitor Bortezomib Introduction
1.1 Introduction
Some passages have been quoted verbatim from the following source
Glynn et al.
1
The proteasome inhibitor Bortezomib (BZM), also known as PS-341
and now marketed as Velcade
TM
(Figure 1-1, 1), is FDA approved for the
treatment of multiple myeloma and mantle cell lymphoma.
2–7
A novel structural
feature of BZM is its boronic acid group, which forms reversibly a borate adduct
with threonine found on the chymotrypsin-like active site of the 26S proteasome,
serving as a transition state mimic. Despite extensive efforts in drug discovery
aimed at the development of boron compounds,
8–14
BZM still remains the only
approved boron-containing drug. However, in recent years, a growing number of
boron-based therapeutics
9–14
are being investigated in clinical trials, including the
new proteasome inhibitor Delanzomib (CEP-18770)
15–17
(Figure 1-1, 2).
Figure 1-1. Structures of Boron-based Proteasome Inhibitors

2

The discovery
2,3
and clinical development
5–7
of BZM as an effective
therapeutic agent demonstrated for the first time that the selective inhibition of
the 26S proteasome
18
is a viable approach for the treatment of cancer.
4,19–24

Cellular proteasome is a key enzymatic complex in all cells that degrades and
recycles targeted and misfolded intracellular proteins. As a key component of the
ubiquitin proteasome pathway (UPP) and the unfolded protein response (UPR),
the proteasome plays a critical role in cell division and cell survival. Selective and
temporary inhibition of the 26S proteasome by BZM or other proteasome
inhibitors results in a selective apoptotic effect in cancer cells, which occurs via
several mechanisms.
25
By blocking the degradation of IκB, proteasome inhibition
partially prevents the activation of the transcription factor NF-κB, and results in
cell cycle arrest and inhibition of cell growth.
4,19–24
It also leads to the
accumulation of ubiquitinated unfolded or misfolded proteins resulting in the
induction of endoplasmic reticulum stress (ER stress).
26,27
Based on its novel
mechanisms of action, BZM has been shown to be effective as a single agent and
in combination therapies.
5,7,28

As part of the Petasis group’s efforts towards the development of novel
proapoptotic agents for the treatment of various types of cancer, including
multiple myeloma,
29
lung cancer,
30
lymphoma,
31
glioblastoma,
32,33
and breast
cancer,
34–36
certain molecules have been identified that selectively induce
apoptosis in cancer cells by increasing ER stress.
32,37–43
These ER-stress-
aggravating agents (ERSAs) are novel anticancer therapeutics and are effective
alone and in certain combinations with other therapies.
44,45
In this context,

3

previous work in the Petasis group investigated the combination of such
molecules with BZM.
32
That work has shown that BZM promotes enhanced
killing of glioblastoma cells when used in combination with the COX-2 inhibitor
Celecoxib, as well as 2,5-dimethyl-celecoxib (DMC) a structural analog that does
not inhibit COX-2.
32
This increased anticancer activity was attributed to a modest
increase in ER stress, evidenced by elevated levels of key ER stress markers, such
as the chaperone protein glucose regulated protein 78 kilodalton (GRP78) and
the pro-apoptotic transcription factor C/EBP homologous protein (CHOP).
Although cancer cells are able to survive under a hostile microenvironment by
adapting to chronic elevated ER stress conditions, even a modest additional ER
stress aggravation is not well tolerated, triggering cellular apoptosis.
These interesting findings prompted the investigation of the combination
of BZM with (−)-epigallocatechin-3-gallate, abbreviated as EGCG (3), the most
bioactive polyphenol component of green tea, a commonly used dietary
supplement (Figure 1-2). Extracts from green tea have attracted great interest
from the scientific and alternative medicine communities,
46
and its
chemopreventive
47
and epigenetic
48,49
properties have been shown in multiple
animal models of cancer.
50–52

Dietary polyphenols
53,54
(Figure 1-2) are present in large quantities in
many common plant-based food components (e.g. green tea, apples, grapes,
wine, cocoa, etc.) and recent research has pointed to their potential utility in the
prevention of cancer, cardiovascular diseases, osteoporosis, neurodegenerative

4

diseases and diabetes. For example, the bioactivity of (–)-epicatechin (6, EC),
and resveratrol (7, RSV),
55
which are key components of dark cocoa
55,56
and red
wine
55
respectively, have received increased interest for their chemopreventive
properties. These polyphenols have shown several mechanisms of action as
anticancer agents, including regulation of oxidative stress as well and inhibition
of cell proliferation.
56
Also, procyanidin B2 (8), a common component of apples
and grapes and a dimer of (–)-epicatechin (6, EC), was shown to block the
activity of NF-κB, by preventing its binding to DNA.
57

Figure 1-2. Structures of EGCG and Other Dietary Polyphenols

5

The therapeutic effects of green tea have been primarily attributed to its
component catechins (Figure 1-2), namely (–)-epigallocatechin-3-gallate (3,
EGCG), (–)-epicatechin gallate (4, ECG) (–)-epigallocatechin (5, EGC), and (–)
epicatechin (6, EC). These polyphenols were shown to interact with a number of
biological pathways including GRP78 a key chaperone protein involved in ER
stress.
40
Interestingly, it was also reported that EGCG and structural analogs
inhibit the proteasome, while their acetylated or methylated derivatives do not
show such activity.
58–66

In order to examine the potential for synergistic effects of BZM with green
tea components, our group had investigated the combination of EGCG and
related polyphenols with BZM for targeting multiple myeloma and
glioblastoma.
40
Given the presence of the boronic acid group in BZM, we
considered the possibility that a BZM and EGCG combination may result in the
formation of a boronate adduct. However, in the presence of water and other
hydroxylated molecules, these interactions could be formed reversibly, allowing
both BZM and EGCG to exert their actions, despite the formation of this type of
adduct.
Our group’s previous studies
40
revealed that EGCG and other polyphenols
instead of being synergistic; they effectively antagonized the apoptotic effects of
BZM and significantly reduced its ability to induce cancer cell death in vitro and
in vivo. The inactivation of BZM by green tea extracts was demonstrated in a
number of experiments using the multiple myeloma cell line RPMI/8226 and

6

U266, and the glioblastoma cell line LN229. These results illustrated a complete
abrogation of BZM-mediated antitumor properties, with EGCG showing the most
antagonistic effect. It was shown that this result was duplicated in vivo by
exhibiting BZM’s inactivation by EGCG in nude mice models implanted with
multiple myeloma cells as well as in experiments using bone marrow isolated
from multiple myeloma patients. Additional similar findings for BZM
inactivation were also reported by others,
64,67–71
while one study
72
showed that
under certain conditions EGCG potentiates the effect of BZM.
This work postulated
40
that the inactivation of BZM was the consequence
of a covalent interaction to form a boronate adduct between the polyphenols and
BZM. Some preliminary evidence for this transformation was obtained by
1
H
NMR and
13
C NMR, suggesting the formation of a new condensation product
between the two compounds, that prevents covalent binding of the boronic acid
group of BZM to the proteasome site.
40
Despite these findings, the significant
variability in the inhibitory potency among the various polyphenols studied, did
not fully account for the strong ability of EGCG to inactivate BZM. The
condensation adducts of boronic acids with diols have been extensively
investigated,
9,73,74
and are well known to be formed reversibly. In principle, the
boron adducts of polyphenols,
68,75,76
can also behave similarly, further
complicating the analysis of EGCG-mediated inactivation.
In order to gain additional insights regarding the effects of dietary
polyphenols on the efficacy of BZM and potentially the growing number of other

7

emerging boron-based drugs
9–14
and diol-based pro-drugs,
75,77
the work discussed
in this chapter focused on a detailed molecular characterization of the postulated
BZM/EGCG boron adduct. Towards this goal a number of spectroscopic
techniques were employed in an effort to identify the key factors involved in this
process.
EGCG contains three polyphenol rings (A,B,D), two of which contain three
adjacent hydroxyl groups, generating a number of possibilities for forming
adducts with the boronic acid group of BZM. Moreover, the BZM/EGCG adduct
could involve the formation of a neutral boronate adduct or an anionic borate
group involving the participation of a third oxygen substituent at the boron.
Additionally, the conversion of boronic acids to boronate or borate adducts with
diols is a reversible process, depending on the conditions and the relative stability
of the boron adducts. Thus, in order to fully compare the relative stability and
physiological relevance of adducts such as the one formed from BZM and EGCG,
it is important to determine the equilibrium constant of this transformation.
Therefore, in order to elucidate the key structural features of the
BZM/EGCG adduct we relied on several NMR techniques, modeling studies
(performed by my collegue Kevin Gaffney) as well as cell-based assays
(Performed by my collegue Marcos Sainz).

8

1.2 Results and Discussion
1.2.1 Structure elucidation of the BZM/EGCG adduct by
1
H NMR
The 400 MHz
1
H NMR spectra of BZM, EGCG, and the 1:2 BZM/EGCG
combination were analyzed and all of the C–H chemical shifts of EGCG and BZM
were fully assigned (A, B) (Figure 1-3, A, B). Interestingly, the
1
H NMR
spectrum of the BZM/EGCG combination revealed several new peaks (Figure 1-
3, C) that could be attributed to two isomeric adducts 9 and 10 (Figure 1-3, D).
The presence of new boron adducts such as 9, involving the condensation
of the boronic acid group of BZM with the gallic acid unit of EGCG (ring D), was
suggested by two new downfield peaks resulting from deshielding of EGCG
protons H 9 and H 2. This deshielding is consistent with the formation of a new O–
B bond with oxygen atom O a adjacent to H 9, resulting in the reduction of the
electron donating effect of this phenolic OH of EGCG. A similar deshielding
effect, involving a reduced electron donating effect by oxygen atom Ob towards
the ester carbonyl, can explain the new downfield peak for H 2. Presumably, the
O b–H group in EGCG is the most acidic due to its conjugation with the carboxyl
ester and would exist as an anion in solution, while in adduct 9 oxygen O b is more
electron deficient due to its bonding to the boron atom. Notably, integration of
the new peaks for both H 9 and H 2 revealed similar ratios in comparison with
unreacted EGCG, namely 31 : 69 and 30 : 70 respectively (Figure 1-3, C).
Comparable ratios of 30 : 70 and 35 : 65 were also observed for BZM at a new
upfield peak for methyl groups H19 and H20 and a new downfield peak for H14
respectively. The differentiation of these diastereotopic methyl groups in the

9

BZM/EGCG adduct is expected, due to the greater barrier of rotation adjacent to
the boron atom.
The
1
H NMR spectrum of the BZM/EGCG adduct also indicated two new
upfield peaks adjacent to protons H 6 and H 3 with a ratio of 83 : 17 and 81 : 19,
respectively. These peaks suggest the presence of a second type of BZM adduct
10, formed by the condensation of the boronic acid of BZM with the pyrogallol
moiety of EGCG (ring C). The shielding of both H 6 and H 3 in 10 is consistent with
the presence of an anionic oxygen atom O f, presumably due to bond formation
between oxygen atoms O d and O e and the boron atom.
Figure 1-3. 400 MHz
1
H NMR Analysis of BZM/EGCG Adduct. (A) 400 MHz
1
H
NMR of EGCG in MeCN; (B). 400 MHz
1
H NMR of Bortezomib (BZM) in MeCN; (C) 400
MHz
1
H NMR of 1 : 2 mixture of EGCG : BZM in MeCN; (D) postulated structures of boronate
adducts (9, 10) suggested by
1
H NMR analysis. Arrows indicate proton shifts corresponding to
adducts of EGCG and BZM. Percentages indicate relative ratios of designated peaks.

10

1.2.2 Quantification and structural analysis of the BZM/EGCG
adduct by
11
B NMR
Although the
1
H and
13
C NMR spectra of the BZM/EGCG combination
revealed evidence for the formation of a boron adduct, these techniques are not
adequate for determining the equilibrium constant of this interaction. The
1
H
NMR shows multiple overlapping and poorly resolved peaks, while the most
affected H-atoms are located at a distance, limiting the precise characterization of
the boron unit. Additionally, the limited sensitivity of
13
C NMR requires that the
experiments be carried out at higher concentrations. However, since equilibrium
constants are a function of concentration, these conditions do not accurately
portray the formation of the boron adduct in physiologic conditions. Moreover,
due to the high dilution and reversible nature of the BZM/EGCG adduct, liquid
chromatography mass spectrometer analysis results in the break-up of this
adduct.
In order to overcome the above challenges, we identified
11
B NMR as a
better technique that allows studies at relevant concentrations and simplifies the
analyses by focusing on the lone boron atom that connects BZM and EGCG. The
11
B NMR spectrum of BZM shows a broad downfield peak at 27.8 ppm for the
trigonal boronic acid group (Figure 1-4, A, peak a). When combined with
increasing concentrations of EGCG, the boronic acid group is condensed with a
diol moiety from EGCG resulting in a growing fraction of a new narrow upfield
boron peak at 19.5 ppm (Figure 1-4, A, peak b). Interestingly, given its shielded
position and narrow shape, peak b is unlikely to be from a simple trigonal cyclic

11

boronate adduct, such as 9 or 10 (Figure 1-3). Presumably, peak b provides
direct evidence of a BZM adduct between BZM and EGCG. Thus, unlike
1
H and
13
C NMR spectra (Figure 1-3),
11
B NMR provides a simple, direct, and more
accurate method to characterize the BZM/EGCG adduct and the nature of the
boron group.
Figure 1-4. 400 MHz
11
B NMR Analysis of BZM/EGCG Adduct. (A)
11
B NMR in 4 : 1
CD3CN/D2O of the combination of BZM and EGCG in molar ratios of 1:0, 1:0.5, 1:1, 1:2, and
1:4 BZM:EGCG. The free boronic acid of BZM has a broad peak a at 27.8 ppm, while this peak
is shifted to a narrow boronate peak b at 19.5 ppm. (B) Increased formation of the boronate
adduct with the ratio of EGCG:BZM, determined by integration of the two boron peaks in 11B
NMR.

12

Integration of the
11
B peaks allowed us to quantify the percentage of the
BZM/EGCG adduct relatively to free BZM in solution (Figure 1-4). As we had
shown by
1
H NMR (Figure 1-3) and had been shown in our lab’s previous work
by
13
C NMR,
40
EGCG readily creates a boron adduct with BZM in a concentration
dependent manner (Figure 1-4, B). At a 4 : 1 EGCG to BZM ratio the free BZM
was reduced significantly.
1.2.3 Comparison by
11
B NMR of selected polyphenols for their
ability to form boron adducts with BZM
While EGCG was shown to be the most potent among related natural
polyphenols for the inactivation of BZM’s antitumor properties, the basis of this
inactivation remains unclear. In order to determine the molecular characteristics
of EGCG responsible for its high potency, we employed the above
11
B NMR
approach to selected phenolic compounds with variable substitution patterns
(Figure 1-5). Thus, EGCG was divided into three fragments representing its
three phenolic moieties (rings A,C,D): the 1,3-diol resorcinol (11, RES), the 1,2,3-
triol pyrogallol (12, PYR), and the isopropyl gallate (13, IGA). The
11
B NMR of
BZM by doubling the ratio of RES, PYR, IGA are shown in (Figure 1-5, A).
PYR and IGA effectively formed the boronate adduct peaks b and c
(Figure 1-5, A) at a 4:1 ratio to BZM, where the two adducts combined reached
85% and 88%, respectively. RES showed a decreased capacity to form a boronate
adduct, with the majority of BZM remaining as the boronic acid. Since the m-
substitution pattern of RES does not allow the formation of a cyclic BZM

13

boronate, one of its hydroxyl groups forms a hydrolytically labile boronate
monoester or boronate diester.

Figure 1-5. 400 MHz
11
B NMR Analysis of BZM With Select Polyphenols. The
11
B
NMR of the adduct of BZM with increasing molar ratios of selected polyphenols was evaluated
by integrating the boron peaks of BZM in combination with each of the following compounds:
(A) Three polyphenols (RES, PYR, IGA) representing EGCG fragments. (B) The natural
polyphenols (–)-epicatechin (EC) and resveratrol (RSV). (C) Phenol (PHE), catechol (CAT)
and 4-nitro-catechol (NCT).

14

The above trends were further examined by comparing the
11
B NMR
spectra of two dietary polyphenols, namely (–)-epi-catechin (6, EC) and
resveratrol (7, RSV) that contain 1,3- and/or 1,2-diol moieties respectively
(Figure 1-5, B). Based on the 1,3-position of RSV’s hydroxyl groups it was
expected that this poly- phenol would not readily complex with BZM, while the
epicatechin (EC), which contains a 1,2-diol would be able to form a stable adduct
at the 1 : 4 molar ratio with BZM. This was indeed confirmed (Figure 1-5, B).
Although the
11
B NMR of all of the BZM/polyphenol adducts (Figure 1-5,
A, B) had the two boron peaks at 27.8 ppm (peak a) and 19.5 ppm (peak b)
observed for the BZM/EGCG adduct (Figure 1-4, A), in some cases (PYR, IGA,
EC) a third upfield and broad boron peak was observed at 15.8 ppm (peak c). To
further investigate the factors leading to the formation of this additional boron
adduct, we evaluated the
11
B NMR spectra of the BZM adducts of the parent
phenol (14, PHE), catechol (15, CAT), and 4-nitrocatechol (16, NCT). As shown
in (Figure 1-5, C) the 1,2 diols CAT and NCT did have this third peak (c) with
variable intensity, while PHE and RSV that lack a 1,2 diol did not. Taken
together, these data indicate that the third peak c is likely to reflect the formation
of a stable anionic cyclic borate species from a catechol (1,2-diol) moiety.
Interestingly, peak c was not observed in the
11
B NMR of the BZM/EGCG adduct,
even though EGCG has multiple 1,2-diol moieties in its structure. Presumably, as
discussed below, the more complex structural features of EGCG may prevent the
formation of this type of anionic cyclic borate adduct.

15

1.2.4 Determination of the equilibrium constant of the BZM boron
adduct by
11
B NMR and
19
F NMR
To further validate the applicability and accuracy of using
11
B NMR and to
evaluate the BZM/EGCG adduct, we sought to compare the
11
B data with related
data generated by
19
F NMR. The use of
19
F NMR for studying biological systems
78

has been shown to be very effective in drug discovery
79
and for investigating
protein function, including the calculation of binding constants in competition-
based high throughput screening,
80,81
for screening enzyme inhibitors,
82
and for
determining the binding affinity of fluorinated molecules to protein targets.
In order to evaluate the BZM adduct with polyphenols we chose 4-
fluorocatechol (Figure 1-6, 17, FCT) as the fluorine probe, where the single
fluorine atom simplifies the
19
F NMR analysis to a single peak. For recording the
NMR spectra, a 1 : 1 molar ratio of BZM and FCT were combined at a 2.6 mM
concentration in a 1 : 1 ratio, and the experiment was run in triplicate. This
experimental setup allowed the fraction of the unbound FCT to represent the
concentration of both the free BZM and free FCT, which further simplified the
calculation of the equilibrium constant. Moreover, both the
11
B and
19
F NMR
spectra could be run on the same sample, providing a direct comparison of the
two techniques.
Similarly to the
11
B NMR spectra of the BZM/CAT mixture, there were
three peaks in the
11
B NMR for the BZM/FCT combination. The
19
F NMR spectra
showed a narrow peak for FCT and a broad peak for the BZM/FCT boron adduct,

16

presumably reflecting the two forms of the adduct. From these data, we
calculated the binding constant of BZM/FCT at a 2.6 mM concentration to be
1.40 × 10−2 (±0.002) M
−1
by
19
F NMR, and 1.40 × 10−2 (±0.006) M
−1
by
11
B
NMR (Figure 1-6). The high degree of agreement between these methods
indicates that
11
B NMR data would be useful for the calculation of equilibrium
constants for BZM/polyphenol adducts.
1.2.5 NMR evaluation of polyphenol adducts of boron drugs
The use of NMR spectroscopy to elucidate the type of boron adduct being
formed (if any) provides a useful method for determining the potential ability of
dietary polyphenol compounds to form undesired adducts with boron-based
drugs and interfere with their actions. With the increasing interest in developing
Figure 1-6. Use of
11
B NMR and
19
F NMR to Calculate the Equilibrium Constant of
the Boron Adduct of 4-fluorocatechol (FCT) with BZM. These experiments were
performed in triplicate (1–3) at a 2.6 mM concentration of a 1 : 1 mixture of BZM and FCT.
The ratios of BZM/FCT were quantified by integrating the
11
B NMR peaks of BZM at 29 ppm
(a) and the adduct at 19.7 ppm (b) and 16.4 ppm (c), as well as the
19
F NMR peaks of FCT at
−124.9 ppm (d) and the adduct at −125.7 ppm (e).

17

new therapeutics containing boronic acid groups,
9–17
this comparative approach
may have a broader applicability.
1.3 Conclusion
In summary, this work has comprehensively characterized the covalent
boron adduct of BZM with the green tea extract polyphenol EGCG. Through the
use of
1
H NMR,
11
B NMR and
19
F NMR, we characterized the structures of the
BZM boron adducts of EGCG and related polyphenols, and investigated the
factors contributing to their formation. We were also able to quantify the adduct
formation and determine its equilibrium constant, which was found to be
biologically significant.
The observed adducts included both neutral boronate and anionic borate
derivatives, while their formation was preferably initiated at the most acidic
phenolic OH group. The type and amount of the boron adduct produced is
defined by both electronic and steric effects that can affect the reversibility of
these steps. These structural characteristics were correlated with cell-based
evaluation performed by my colleague Marcos Sainz to understand the ability of
EGCG and other phenols to suppress the anticancer activity of BZM.
In the case of EGCG, its ability to make a more stable adduct less
reversibly with BZM seems to be due to two key factors: (a) electronic effects that
drive the formation of boron adducts at the gallate ring D and the pyrogallol ring
B, and (b) steric effects that favor the conversion of anionic cyclic borate species
to intramolecularly stabilized neutral cyclic borate BZM/EGCG adducts, which

18

are less susceptible to hydrolysis as indicated in computational studies performed
by my colleague Kevin Gaffney.
The reported approach provides a useful method for determining the
potential ability of dietary polyphenol compounds to form undesired adducts
with boron-based therapeutics and interfere with their actions.
1.4 Experimental
Unless otherwise noted, all reactions were carried out in a flame-dried
flask with stir bar under argon routed through a three-necked valve. Reactions
were carried out at room temp using DriSolv solvents purchased commercially
from VWR. All reagents used were purchased without further purification from
Sigma Aldrich, LC Laboratories, Combi-Blocks and Alfa Aesar.
Characterization was carried out using LC-MS, NMR and UV-VIS
instrumentation. All
1
H,
13
C and gcosy spectra were procured on the Departments
Varian 400, 500 and 600 MHz NMR instruments in the solvent indicated.
1
H and
13
C chemical shifts, (δ), are recorded in parts per million, (ppm), and referenced
to the residual solvent converted by the TMS scale (CDCl 3,
1H
= 7.26 ppm).
Splitting patterns are denoted by s, d, t, dd, td, ddd, and m and refer to the
respective multiplicities; singlet, doublet, triplet, doublet of doublets, triplet of
doublets, doublet of doublet of doublet and multiplet. Mass spectra was recorded
on an Agilent 1260 LC-MS. UV-Vis spectra was obtained by a Hewlett- Packard
8350 instrument.

19

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25

71. Bannerman, B., Xu, L., Jones, M., Tsu, C., Yu, J., Hales, P.,
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proteasome chymotrypsin-like activity: an investigative tool for
studying allosteric regulation, Org. Biomol. Chem. 12, 4576.

26

Chapter 2. DHA Derived Lipid Mediators in
Inflammation Resolution
2.1 Introduction
The Resolvin D series and Maresins are two recently discovered subsets of
a growing class of specialized pro-resolving lipid mediators (SPM) derived from
the essential fatty acid docosahexanoic acid (DHA), a major Omega-3 fatty acid
found in fish oil. These lipid mediators play a crucial role in a host’s immune
response and each contains a unique biological role and singular chemical
structure. Recently several novel types of lipid mediators have been discovered,
acclaimed for their novel tissue regenerative properties. These new types of lipid
mediators include the Maresin Conjugate Tissue Regenerative Series (MCTR)
and the Protectin Conjugate Tissue Regenerative Series (PCTR). As part of an on
going project in our group in conjunction with several collaborators across
various fields have investigated numerous lipid mediators for their beneficial
role in inflammation. This thesis includes our efforts towards the design and total
synthesis of several individual compounds in the Resolvin D series, Maresin
series and the newly discovered conjugate tissue regenerative series. The
successful completion of synthetic materials has allowed for the matching of the
synthetic products with the biologically isolated counterparts to confirm chemical
structures of the metabolites, as well as investigation of the biological activity in
the inflammation pathway.

27

2.2 Inflammatory Response and the Resolution of Inflammation
Inflammation, which is initiated in response to infection of tissue injury,
was once considered to be resolved on its own in a passive manner. However
recent discoveries have established that the resolution of inflammation is a highly
coordinated active process creating new paradigms for understanding and
treating inflammation and related conditions.
1-4
The host’s initial pro-
inflammatory response to acute inflammation resulting from injury or infection
is a necessary and normal response, however if left unchecked it can progress to
encompass acute, chronic and systematic inflammatory disorders.
2

Lipid mediators such as prostaglandins (PGs) and leukotrienes (LTs) have
been widely investigated for their roles in initiating leukocyte trafficking to the
site of inflammation including neutrophil (PMN) influx to tissues the initial step
in host defense (Figure 2-1).
2,5
The superfluous collection of PMNs in the
inflammation site can lead to additional tissue damage, elaboration of the
inflammatory response, internal injury, and the progression towards chronic
inflammation.
4
As such the control of neutrophil infiltration is of great interest as
new anti-inflammatory agents are pursued to control and combat excess
neutrophil infiltration with the ultimate goal of a host’s return to homeostasis.
6

28

Just as pro-inflammatory lipid mediators such as PGs and LTs initiate the
neutrophil response, novel resolution-phase lipid mediators that possess potent
proresolving actions have been identified including resolvins, protectins and
maresins.
5
These families of lipid mediators endogeneously generated during
inflammation from docosahexanoic acid (DHA) an omega-3 fatty acid metabolite
that promote and stimulate active resolution.
4
These lipid mediators termed
specialized proresolving mediators (SPMs) compete with their inflammatory
counterparts; PGs and LTs at the site of inflammation. Demonstrating potent
anti-inflammatory and proresolving actions in regulating PMN tissue
accumulation, and the recruitment of monocytes resulting in the production of
Figure 2-1. Dual Anti-inflammatory and Pro-resolution Actions of Specialized
Pro-resolving Mediators. Reprinted (adapted) with permission from (Serhan, C. N., and
Petasis, N. A. (2011) Resolvins and protectins in inflammation resolution., Chem. Rev. 111,
5922–5943.). Copyright (2011) American Chemical Society.

29

macrophages to eliminate and remove apoptotic cells, these SPMs possess a
crucial responsibility in the host’s restoration process back to homeostasis
(Figure 2-1).
2,4
The discovery of the resolvins, protectins and maresins have not
only proved important for the investigation of the host’s biological response to
inflammation, they have also provided a molecular basis for the many health
benefits associated with omega-3 fatty acids.
2

2.3 Identification of DHA-Derived SPMs
Docosahexanoic acid (DHA), a fatty acid component of omega-3 fish oil
has been acclaimed as beneficial in several human diseases including
atherosclerosis, asthma, cardio-vascular, cancer and more recently mental
depression.
7
The isolation and investigation of lipid mediators biosynthetically
produced from DHA provides the first molecular evidence for many of these
health benefits.
2

Enzymatic oxygenation of arachodionic acid (AA), a component of omega-
6 fatty acid has been extensively studied and known produce both pro-
inflammatory (LTs and PGs) and proresolving mediators (lipoxin A 4 and aspirin-
triggered lipoxin A 4) (Figure 2-2).
2
The enzymatic breakdown of omega-3 fatty
acids eicosapentaenoic acid (EPA) and DHA generally lead to the formation of
anti-inflammatory, proresolving lipid mediators (resolvins, protectins and
maresins) (Figure 2-2).
2
This work resulted directly from the lipidomics and
systematic approach, investigating the enzymatice oxygenation of DHA to isolate
and identify the SPMs produced during the inflammation process.
2,7

30

This includes the aspirin-triggered DHA metabolome that biosynthesizes a
potent product in inflammatory exudates and human leukocytes, including
aspirin-triggered stereoisomers of the resolvin series and protectin series.
4
The
aspirin-triggered products are biosynthesized from DHA in the presence of
aspirin-acetylated COX-2, acting as a modified dioxygenase inserting a molecule
of oxygen with the opposite stereochemistry.
2

2.4 Biological Activity of DHA Derived SPMs
The resolvin D, maresin and protectin families of SPMs have all shown
potency at nanomolar concentrations, which can promote the resolution of
inflammation. Each individual SPM has a unique structure, biosynthetic pathway
and biological profile. The identification of the resolvin D series initially
produced reported the elucidation of several distinct bioactive metabolites that
Figure 2-2. Biosynthetic Derivation of Lipid Mediators From Omega-3 and
Omega-6 Fatty Acids. Reprinted (adapted) with permission from (Serhan, C. N., and
Petasis, N. A. (2011) Resolvins and protectins in inflammation resolution., Chem. Rev. 111,
5922–5943.). Copyright (2011) American Chemical Society.

31

blocked PMN influx and migration, designated resolvin D1 through resolvin D6.
5

The investigation of the biological activity of each of these molecules is of
continuous interest; the activity of RvD1,
8,9,10
RvD2,
11,12
RvD3
13
and their aspirin-
triggered stereoisomers has been previously reported. RvD4 and AT-RvD4 is
known to be an agonist playing a role in leukocyte trafficking in host defense as
with the rest of the resolvin D series.
14
However the precise mechanism
accounting for that activity is still unclear. Preliminary results indicate that
synthetic RvD4 displays potent actions in vivo by reducing infiltration of
neutrophils and subsequently reduces murine peritonitis and dermal
inflammation.
15
In addition to the D series resolvins, DHA also biosynthetically produces
the maresin family of lipid mediators.
6
However unlike the resolvin D series
maresins are produced from DHA by macrophages leading to the name maresins
(macrophage mediators in resolving inflammation) due to their means of
production and biological role in inflammation.
6
Maresin 1 (MaR1) exceeds the
activity of RvD1 where at 1nM it was found to be a potent stimulator of human
macrophage efferocytosis.
5
MaR1 also possesses tissue regenerative properties
accelerating surgical regeneration in planaria, increasing the rate of head
reappearance.
5
In neurons, MaR1 dose-dependently inhibits TRPV1 currents,
blocks capsaicin-induced inward currents with a low nanomolar IC50 and reduces
inflammatory and neuropathic pain in mice.
14
Maresin 2 (MaR2) also carries the
key defining bioactions of a pro-resolving mediator. Displaying similar potencies
to MaR1 in limiting PMN infiltration, however MaR1 biological capabilities

32

exceed MaR2 in human macrophage phagocytosis of zymosan and human
macrophage uptake of apoptotic PMN.
16
More recently a new family of sulfido-conjugated mediators was isolated,
17

initial isolated in the resolution of Escherichia coli infection. This DHA
metabalome shares a biosynthetic pathway with the maresins and were coined
maresin conjugate in tissue regeneration (MCTR).
17
In addition to demonstrated
anti-inflammatory, pro-resolving properties, these mediators have been shown to
promote repair and regeneration in planaria, mouse, and human tissues during
infection.
18
In addition to the MCTR series two more novel sulfido-conjugate
autocoid classes have been discovered; the protectin conjugate in tissue
regeneration (PCTR) family, and the resolvin conjugate in tissue regeneration
(RCTR) family. These mediators are produced enzymatically from DHA, sharing
biosynthetic pathways with the protectins and resolvins respectively.
17
These
compounds have also displayed extremely potent anti-inflammatory, pro-
resolving properties, and tissue regenerative activity in several models. Expressly
promoting phagocytosis of bacteria and efferocytosis of apoptotic cells by
macrophages, identified in human sepsis, and accelerated tissue regeneration in
planaria.
17
The MCTR, PCTR, and RCTR series have yet to be thoroughly
investigated since they were discovered very recently, however there potent tissue
regenerative capabilities have provoked significant interest.

33

2.5 The Chemistry of Lipid Mediators
The biosynthetic conversion of DHA to pro-resolving lipid mediators
occurs locally at the site of inflammation. Each of the lipid mediators focused on
in this work are biologically activity at nanomoalr concentrations, and can have
exceptionally short metabolic stability. As such biological isolation of these
compounds occurs in nanogram quantities and full characterization is
prohibitively difficult due to the low quantities obtained.
2
Using an unbiased
lipidomics and systematic approach towards the enzymatic oxygenation of DHA,
the lipid mediators have been isolated and identified by mass spectrometry. By
an understanding of their biosynthetic pathway, enzymatic degradation and
preliminary biological profiles a proposed structure of the lipid mediators can be
advanced.
2
To confirm the chemical structure and establish an exact
stereochemical assignment each compound must be characterized and confirmed
via total synthesis. This is completed by preparation of the biological compound
enzymatically and its structure is matched to the synthetic materials by its
chromatographic, physical, and biological properties.
2
The synthetic production of these lipid mediators is not only essentialfor
completing their stereochemical assignment; it is also necessary for biological
assessment. Due to the extremely small quantities garnered by enzymatic
production and isolation, often a synthetic stockpile of these lipid mediators is
essential for biological investigation. In addition to the synthesis of the defined
families of lipid mediators such as the maresins and resolvin D series total
synthesis has been used to produce compounds within the enzymatic pathway

34

such as 13S, 14S-epoxy maresin to investigate it’s formation, stereochemistry, and
precursor role in inflammation models.
19
Synthetic production of isotopically
substituted derivatives has also been used in lipidomic analysis,
20
synthetically
produce radiolabeled derivatives have also been used for receptor studies.
21
The synthesis of these compounds has proven to be a formidable challenge
and has attracted the efforts of many synthetic laboratories. Nobel Laureate E.J.
Corey and his lab first synthesized compounds from the prostaglandin family in
1968,
22
as well as compounds from leukotriene family in 1980.
23
Professor K.C.
Nicolaou published the first total synthesis of lipoxin A 4 in 1985.
24
For the past
several decades our lab has been at the forefront of this chemistry, some of our
recent efforts encompass the total synthesis of resolvin E1 (RvE1),
21,25,26
resolvin
D1 (RvD1),
3,9,27
resolvin D2 (RvD2),
12,
neuroprotectin D1/protectin D1
(NPD1/PD1),
28,29
maresin 1 (MaR1),
20
resolvin D3 (RvD3)
30
and 13S, 14S-epoxy-
maresin
19
while additional studies will be reported in due course.
Each of these molecules contains a unique biological role and singular
chemical structure and they typically possess several hydroxyl substituents with
precise R/S configuration and multiple C=C bonds with specific Z/E geometry.
Demanding a synthetic strategy relying on iterative cross-coupling of isomerically
pure building blocks and selective hydrogenations of alkyne intermediates.
Making them as synthetically demanding as they are biologically intriguing.

35

2.6 Conclusion
In conclusion the investigation of DHA derived lipid mediators and their
role in a host’s immune response is an on going project performed in conjunction
with several collab orators across various fields. This research has presented
challenges and breakthroughs in an array of academic disciplines including
chemistry and biology. Creating new paradigms that has shifted the once believed
notion that inflammation tapers off over time, and replaced it with the now
generally accepted view that inflammation resolution is a highly coordinated
active process creating new foundations for understanding and treating the
condition.
1-4

COOH
HO
O
HO
OH OH
COOH
OH
COOH
HO OH
OH OH
OH
COOH
HO
OH
COOH
HO
OH COOH
OH
Derived'from'omega-6'fa/y'acids:'
Derived'from'omega-3'fa/y'acids'(Fish'oil):'
Prostaglandin,E2,(PGE
2
),
Corey,,1968,
Leukotriene,B
4
,(LTB
4
),
Corey,,1980,
Lipoxin,A
4
,(LXA4),
Nicolaou,,1985,
Resolvin,E1,(RvE1),
Petasis,,2005,
Resolvin,D1,(RvD1),
Petasis,,,2007,
NeuroprotecLn,D1,(NPD1),
Petasis,,2012,
Figure 2-3. Timeline in the Synthesis of Lipid Mediators. Reprinted (adapted) with
permission from (Serhan, C. N., and Petasis, N. A. (2011) Resolvins and protectins in
inflammation resolution., Chem. Rev. 111, 5922–5943.). Copyright (2011) American Chemical
Society.

36

2.7 References
1. Dalli, J., Colas, R. A., and Serhan, C. N. (2013) Novel n-3
Immunoresolvents: Structures and Actions, Sci. Rep. 3.
2. Serhan, C. N., and Petasis, N. A. (2011) Resolvins and protectins in
inflammation resolution., Chem. Rev. 111, 5922–5943.
3. Kasuga, K., Yang, R., Porter, T. F., Agrawal, N., Petasis, N. A.,
Irimia, D., Toner, M., and Serhan, C. N. (2008) Rapid appearance
of resolvin precursors in inflammatory exudates: novel mechanisms
in resolution., J. Immunol. 181, 8677–8687.
4. Serhan, C. N., Fredman, G., Yang, R., Karamnov, S., Belayev, L. S.,
Bazan, N. G., Zhu, M., Winkler, J. W., and Petasis, N. A. (2011)
Novel Proresolving Aspirin-Triggered DHA Pathway, Chem. Biol.
18, 976–987.
5. Serhan, C. N., Chiang, N., Dalli, J., and Levy, B. D. (2015) Lipid
Mediators in the Resolution of Inflammation, Cold Spring Harb
Perspect Biol 7, a016311.
6. Serhan, C. N., Dalli, J., Colas, R. A., Winkler, J. W., and Chiang, N.
(2015) Protectins and maresins: New pro-resolving families of
mediators in acute inflammation and resolution bioactive
metabolome, BBA - Molecular and Cell Biology of Lipids, Elsevier
B.V. 1851, 397–413.
7. Serhan, C. N., Hong, S., Gronert, K., Colgan, S. P., Devchand, P. R.,
Mirick, G., and Moussignac, R. L. (2002) Resolvins: A Family of
Bioactive Products of Omega-3 Fatty Acid Transformation Circuits
Initiated by Aspirin Treatment that Counter Proinflammation
Signals, Journal of Experimental Medicine 196, 1025–1037.
8. Xu, Z.-Z., Zhang, L., Liu, T., Park, J. Y., Berta, T., Yang, R., Serhan,
C. N., and Ji, R.-R. (2010) Resolvins RvE1 and RvD1 attenuate
inflammatory pain via central and peripheral actions, Nat Med,
Nature Publishing Group 16, 592–597.
9. Krishnamoorthy, S., Recchiuti, A., Chiang, N., Yacoubian, S., Lee, C.
H., Yang, R., Petasis, N. A., and Serhan, C. N. (2010) Resolvin D1
binds human phagocytes with evidence for proresolving receptors,
Proc. Natl. Acad. Sci. U.S.A. 107, 1660–1665.
10. Sun, Y.-P., Oh, S. F., Uddin, J., Yang, R., Gotlinger, K., Campbell,
E., Colgan, S. P., Petasis, N. A., and Serhan, C. N. (2007) Resolvin
D1 and its aspirin-triggered 17R epimer. Stereochemical
assignments, anti-inflammatory properties, and enzymatic
inactivation., J. Biol. Chem. 282, 9323–9334.
11. Park, C. K., Xu, Z. Z., Liu, T., Lu, N., Serhan, C. N., and Ji, R. R.
(2011) Resolvin D2 Is a Potent Endogenous Inhibitor for Transient
Receptor Potential Subtype V1/A1, Inflammatory Pain, and Spinal
Cord Synaptic Plasticity in Mice: Distinct Roles of Resolvin D1, D2,
and E1, Journal of Neuroscience 31, 18433–18438.

37

12. Spite, M., Norling, L. V., Summers, L., Yang, R., Cooper, D., Petasis,
N. A., Flower, R. J., Perretti, M., and Serhan, C. N. (2009) Resolvin
D2 is a potent regulator of leukocytes and controls microbial sepsis,
Nature, Nature Publishing Group 461, 1287–1291.
13. Haeggström, J. Z., and Hamberg, M. (2013) Resolving Resolvins,
CHBIOL, Elsevier 20, 138–140.
14. Serhan, C. N., and Chiang, N. (2013) Resolution phase lipid
mediators of inflammation: agonists of resolution, Current Opinion
in Pharmacology, Elsevier Ltd 13, 632–640.
15. Winkler, J., Orr, S., Dalli, J., Chiang, N., Petasis, N.A., and Serhan
C.N. (2015) Resolvin D4 Potent Antiiinflammatory Proresolving
Actions Confirmed via Total Synthesis, FASEB J. 29, 285.10
16. Deng, B., Wang, C.-W., Arnardottir, H. H., Li, Y., Cheng, C.-Y. C.,
Dalli, J., and Serhan, C. N. (2014) Maresin Biosynthesis and
Identification of Maresin 2, a New Anti-Inflammatory and Pro-
Resolving Mediator from Human Macrophages, PLoS ONE
(Wallace, J., Ed.) 9, e102362.
17. Dalli, J., Ramon, S., Norris, P. C., Colas, R. A., and Serhan, C. N.
(2015) Novel proresolving and tissue-regenerative resolvin and
protectin sulfido-conjugated pathways, FASEB J. 29, 2120–2136.
18. Dalli, J., Chiang, N., and Serhan, C. N. (2014) Identification of 14-
series sulfido-conjugated mediators that promote resolution of
infection and organ protection, Proc. Natl. Acad. Sci. U.S.A.
19. Dalli, J., Zhu, M., Vlasenko, N. A., Deng, B., Haeggstrom, J. Z.,
Petasis, N. A., and Serhan, C. N. (2013) The novel 13S,14S-epoxy-
maresin is converted by human macrophages to maresin 1 (MaR1),
inhibits leukotriene A4 hydrolase (LTA4H), and shifts macrophage
phenotype, FASEB J. 27, 2573–2583.
20. Serhan, C. N., Dalli, J., Karamnov, S., Choi, A., Park, C. K., Xu, Z.
Z., Ji, R. R., Zhu, M., and Petasis, N. A. (2012) Macrophage
proresolving mediator maresin 1 stimulates tissue regeneration and
controls pain, FASEB J. 26, 1755–1765.
21. Arita, M. (2005) Stereochemical assignment, antiinflammatory
properties, and receptor for the omega-3 lipid mediator resolvin E1,
Journal of Experimental Medicine 201, 713–722.
22. Corey, E. J., Andersen, N. H., Carlson, R. M., Paust, J., Vedejs, E.,
Vlattas, I., and Winter, R. E. (1968) Total synthesis of
prostaglandins. Synthesis of the pure dl-E1, -F1-alpha-F1-beta, - A1,
and -B1 hormones., J. Am. Chem. Soc. 90, 3245–3247.
23. Corey, E. J., Marfat, A., Goto, G., and Brion, F. (1980) Leukotriene
B. Total synthesis and assignment of stereochemistry, ., J. Am.
Chem. Soc. 102, 784-785.
24. Nicolaou, K. C., Veale, C. A., and Webber, S. E. (1985)
Stereocontrolled total synthesis of lipoxins A, J. Am. Chem. Soc.
107, 7515-7518.

38

25. Petasis, N. A. (2013) Trihydroxy polyunsaturated eicosanoid
derivatives, US Patent Office.
26. Arita, M., Oh, S. F., Chonan, T., Hong, S., Elangovan, S., Sun, Y.-P.,
Uddin, J., Petasis, N. A., and Serhan, C. N. (2006) Metabolic
inactivation of resolvin E1 and stabilization of its anti-
inflammatory actions., J. Biol. Chem. 281, 22847–22854.
27. Sun, Y.-P., Oh, S. F., Uddin, J., Yang, R., Gotlinger, K., Campbell,
E., Colgan, S. P., Petasis, N. A., and Serhan, C. N. (2007) Resolvin
D1 and its aspirin-triggered 17R epimer. Stereochemical
assignments, anti-inflammatory properties, and enzymatic
inactivation., J. Biol. Chem. 282, 9323–9334.
28. Serhan, C. N., Gotlinger, K., Hong, S., Lu, Y., Siegelman, J., Baer,
T., Yang, R., Colgan, S. P., and Petasis, N. A. (2006) Anti-
Inflammatory Actions of Neuroprotectin D1/Protectin D1 and Its
Natural Stereoisomers: Assignments of Dihydroxy-Containing
Docosatrienes, J. Immunol. 176, 1848–1859.
29. Petasis, N. A., Yang, R., Winkler, J. W., Zhu, M., Uddin, J., Bazan,
N. G., and Serhan, C. N. (2012) Stereocontrolled total synthesis of
Neuroprotectin D1/Protectin D1 and its aspirin- triggered
stereoisomer, Tetrahedron Lett. 53, 1695–1698.
30. Winkler, J. W., Uddin, J., Serhan, C. N., and Petasis, N. A. (2013)
Stereocontrolled Total Synthesis of the Potent Anti-inflammatory
and Pro-resolving Lipid Mediator Resolvin D3 and Its Aspirin-
Triggered 17 R-Epimer, Org. Lett. 15, 1424–1427.

39

Chapter 3. Total Synthesis of Resolvin D4 and
Aspirin-triggered Resolvin D4
3.1 Introduction
The D-series resolvins were originally identified in resolving murine
exudates, this work resulted in isolation and structure elucidation of six distinct
biologically active structures denoted resolvin D1 through D6.
1
Here in we report
the stereocontrolled total synthesis of resolvin D4 and aspirin triggered resolvin
D4. As part of the D-series resolvins these molecules contain potent pro-resolving
properties such as governing neutrophil trafficking and clearance of apoptotic
polymorphonuclear neutrophils (PMN) and cellular debris.
2
To this extent
resolvin D4 showed the ability to promote the clearance of apoptotic PMN by
human macrophage and dermal fibroblasts at doses as low as 1 nM.
3

The structures of RvD4 and AT-RvD4 were originally inferred using mass
spectrometry.
2
The detailed Z/E aliphatic geometry and R/S stereochemistry was
speculated from the hypothesized biosynthesis. The convergent and
stereocontrolled total synthesis of these molecules outlined in scheme 2
confirmed the postulated structures of RvD4 and AT-RvD4.
3.2 Biosynthesis of RvD4 and AT-RvD4
The proposed biosynthesis of RvD4
2,4
is illustrated in scheme 3-1. The
biosynthesis is initiated by the conversion of DHA to (17S)-hydroperoxy DHA
(17S-HpDHA) catalyzed by a lipoxygenase enzyme (LOX), such as 15-LOX. This
intermediate can also be further reduced to the hydroxyl product (17S-HDHA).
Both 17S-HpDHA and 17S-HDHA experience a second lipoxygenation via 1,5-

40

lipoxygenase (15-LO) yielding a peroxide intermediate at the C4 position. This
peroxide is then converted enzymatically to the 4S, 5S epoxide, followed by
enzymatic hydrolysis to produce RvD4.
Aspirin triggered resolvin D4 has a related biosynthetic pathway.
1,2

Importantly the oxygenation of DHA is induced by the COX-2 enzyme in the
presence of aspirin to establish an R stereochemistry at the 17-peroxide
substituent to form the (17R)-hydroperoxy DHA (17RHpDHA). This is further
reduced to a hydroxyl group establishing the 17R-HDHA intermediate.
Subsequent enzymatic transformations generate AT-RvD4.
COOH COOH
(S)
17
(S)
17 H(O)O
(S)
17
(S)
17 HO
(S)
4
(S)
4
COOH HOO
(S)
17
(S)
17 HO
(S)
5
(S)
5
(S)
4
(S)
4
COOH
O
(S)
17
(S)
17
HO
5
(R)
5
(R)
4
(S)
4
(S)
HO
COOH
OH
COOH
17
(R)
17
(R)
H(O)O
17
(R)
17
(R)
HO
5
(R)
5
(R)
4
(S)
4
(S)
HO
COOH
OH
O
2
O
2
O
2
COX-2
Aspirin
LOX LOX
DHA
Enzymatic
Epoxidation
(4S)-Hydroperoxy-(17S)-Hydroxy-DHA 17S-H(p)DHA
17S-HDHA
17R-H(p)DHA
17R-HDHA
(4S,5S)-Epoxy-(17S)-hydroxy-DHA
Enzymatic
Hydrolysis
Resolvin D4
RvD4, 1A
AT-Resolvin D4
AT-RvD4, 1B
Scheme 3-1. Biosynthesis of Resolvin D4 and Aspirin-triggered Resolvin D4

41

3.3 Results and Discussion
3.3.1 Synthesis of RvD4 and AT-RvD4
The synthetic strategy required to produce RvD4 and AT-RvD4
established the diene and triene moieties separated by a methylene bridge in the
final steps due to the inherent sensitivity of Z/E isomerization. This required a
mild reduction of the bis-acetylenic precursor 3.3, or 3.4 to the 6E, 8E, 10Z,
triene and the 13Z, 15E diene while preserving the methylene bridge. The
presence of the alcohol substituent at the 4 position promoted the creation of a
lactone moiety with the carboxyl acid formed just three carbons away during the
silyl deprotection with tetrabutylammonium flouride. Demanding the reduction
of the bis-acetylenic precursor 3.3, or 3.4 take place prior to the deprotection
and subsequent hydrolysis. This was achieved by employing a mild Lindlar
reduction to produce the 6E, 8E, 10Z, triene and the 13Z, 15E diene units of RvD4
in a 76% yield.
5
The bis-acetylenic precursor 3.3, or 3.4 is attained via a copper
mediated coupling reaction from the propargyl bromide intermediate 3.5 and the
terminal alkyne 3.6 or 3.7.

42

3.3.2 Synthesis of RvD4 and AT-RvD4 Building Blocks
The synthesis of propargyl bromide intermediate 3.5 is detailed in
scheme 3-3. This synthesis utilizes the sugar D-erythrose as a chiral feedstock, a
Wittig reaction with commercially available methyl
(triphenylphosphoranylidene)acetate produces intermediate 3.14 in the precise
4(S), 5(R) diol stereochemistry of resolvin D4.
6,7
Complete reduction with
palladium on carbon followed by silylation produced the silyl protected
unsaturated intermediate 3.12.
8
Selective deprotection of the primary alchohol
HO
(R) (R)
(S) (S)
HO
COOH
OH
17
4
5
TBSO
(R) (R)
(S) (S)
TBSO
COOMe
OTBS
(R) (R) TBSO
(R) (R)
(S) (S)
TBSO
COOMe
OTBS
Br
(R) (R)
O
OTBS
(R) (R)
(S) (S)
TBSO
COOMe
OTBS
I
HO
(R) (R)
(S) (S)
TBSO
COOMe
OTBS
(R) (R)
O
(R) (R)
OH
HO OH
(S) (S) TBSO
(S) (S)
O
OTBS
3.1: 17(S)
3.2: 17(R)
3.3: 17(S)
3.4: 17(R)
+
OH
+
Selective
reduction
Copper mediated
Sonogashira
coupling
3.5
3.7 3.6
3.9 3.8
3.11 3.12 3.13
3.10
Scheme 3-2. Retrosynthesis of Resolvin D4 and Aspirin-triggered Resolvin D4

43

by treatment with camphorsulfonic acid (CSA)
9
with a subsequent Dess-Martin
oxidation achieved the aldehyde intermediate 3.15.
10
A homolongation with
commercially available activated phosphonium salt
(triphenylphosphoranylidene) acetaldehyde constructed intermediate 3.16, the
aldehyde was converted to a vinyl iodide via a Takai olefination to provide
intermediate 3.11.
11
Intermediate 3.11 was then subjected to a copper free
Sonogashira coupling with propargyl alcohol 3.10 to produce intermediate 3.17,
the propargyl alcohol was then replaced by bromine yielding intermediate 3.5.
12
Key intermediates 3.6 and 3.7 are stereoisomers and were produced via
the same synthetic steps from the chiral starting materials 3.8 and 3.9
respectively, depicted in scheme 3-4. The epoxide was opened by addition of the
lithiated 1-butyne in the presence of boron trifluoride to produce the alcohol
O
OH
HO OH
Methyl
(triphenylphosphoran
ylidene)acetate,
THF, reflux, 92%
HO
HO
COOMe
OH
TBSO
TBSO
COOMe
OTBS
1) Pd/C, H
2
, EtOAC
89%
2) TBS-Cl, imidazole,
DMAP, CH
2
Cl
2
O
TBSO
COOMe
OTBS
1) CSA, Et
3
N,
MeOH/CH
2
Cl
2
, 0°C
2) DMP, pyridine
CH
2
Cl
2
86%
TBSO
COOMe
OTBS
O
O
PPh
3
THF, 65°C, 75%
TBSO
COOMe
OTBS
I
CrCl
2
, CHI
3
,THF
0°C to rt, 77%
OH
Pd(OAc)
2
/PPh
3
,
piperidine, rt, 92%
TBSO
COOMe
OTBS
HO
TBSO
COOMe
OTBS
Br
NBS, PPh
3
,DCM
0°C, 87%
3.13 3.14
3.12
3.15
3.16 3.11
3.17 3.5
3.10
Scheme 3-3. Synthesis of Building Block 3.5

44

3.18, 3.22.
13
The secondary alcohol was then silyl protected
14
followed by
selective deprotection of the primary alcohol under mildly acidic conditions to
yield intermediate 3.19, 3.23.
9
Selective reduction of the alkyne to the cis-alkene
was achieved by use of the Lindlar catalyst,
15
with subsequent oxidation of the
primary alcohol to produce the aldehyde intermediate 3.20, 3.24.
10
A
homolongation with commercially available activated phosphonium salt
n-BuLi, BF3lEt2O,
THF, -78°C to rt, 94%
O
OTBS
TBSO
OH
1) TBDPS-Cl,
Imidazole,
DMAP, CH
2
Cl
2
2) CSA, Et
3
N,
MeOH/CH
2
Cl
2
87%
HO
OTBDPS
TBDPSO
O
1) Quinoline, lindlar
catalyst, EtOAc
2) DMP, pyridine,
CH
2
Cl
2
81%
O
PPh
3
THF, 65°C, 75%
TBDPSO
O
TBSO
1) CBr
4
, PPh
3
,
DCM, 0°C
2) LDA, THF, -78°C
73%
3.18
3) TBAF, THF
4) TBS-Cl, Imidazole,
DMAP, CH
2
Cl
2
3.8
n-BuLi, BF3lEt2O,
THF, -78°C to rt, 94%
O
OTBS
TBSO
OH
1) TBDPS-Cl,
Imidazole,
DMAP, CH
2
Cl
2
2) CSA, Et
3
N,
MeOH/CH
2
Cl
2
87%
HO
OTBDPS
TBDPSO
O
1) Quinoline, lindlar
catalyst, EtOAc
2) DMP, pyridine,
CH
2
Cl
2
81%
O
PPh
3
THF, 65°C, 75%
TBDPSO
O
TBSO
1) CBr
4
, PPh
3
,
DCM, 0°C
2) LDA, THF, -78°C
73%
3.22
3) TBAF, THF
4) TBS-Otf, 2,6-
lutidine, CH2Cl
2
3.9
3.19
3.20
3.21 3.6
3.23
3.24
3.25 3.7
Scheme 3-4. Synthesis of building block 3.6 and 3.7

45

(triphenylphosphoranylidene) acetaldehyde constructed intermediate 3.21, 3.25,
the aldehyde was converted to a terminal alkyne via the Corey-Fuchs reaction.
16, 17

Deprotection of the bulky tert-butydiphenyl silyl protecting group, reprotecting
the alcohol with a tert-butyl silyl protecting group produced intermediates 3.6,
3.7.
3.3.3 Construction of RvD4 and AT-RvD4
The synthesis of RvD4 and it’s stereoisomer AT-RvD4 was completed from
intermediates 3.5 and 3.6 or 3.7 and is outlined in scheme 3-5. A copper
mediated coupling of the two late stage intermediates produced the silyl-
protected bis-acetylenic intermediates 3.3, 3.4.
18
Reduction of the 10 and 13
position alkyne moities to cis alkenes required a mild Lindlar catalyzed
hydrogenation to create silyl-protected RvD4 or AT-RvD4.
5
The alcohols were
then deprotected by TBAF followed by treatment with freshly prepared diazo
methane to reinstall the methyl ester producing a mixture of the methyl ester
3.30, 3.31 and the lactone products 3.28, 3.29.
19
Both the methyl ester and
lactone intermediates were subjected to hydrolysis by lithium hydroxide and
HPLC purification to yield the single stereoisomer RvD4 3.1 or AT-RvD4 3.2.
LC/MS, UV, 1H NMR, 13C NMR and 2-D COSY confirmed the structures of
compounds 3.1 and 3.2.

46

3.4 Conclusion
Stereochemically pure synthetic RvD4 and AT-RvD4 have been obtained
via stereocontrolled total synthesis. These compounds were used to further
investigate the biological profile of these lipid metabolites. Preliminary results
indicate that synthetic RvD4 displays potent actions in vivo by reducing
infiltration of neutrophils and subsequently reduce murine peritonitis and
dermal inflammation.
20
TBSO
COOMe
OTBS
Br
3.5
TBSO
TBSO
COOMe
OTBS
3.3: 17(S)
3.4: 17(R)
TBSO
TBSO
COOMe
OTBS
3.26: 17(S)
3.27: 17(R)
HO
O
OH
3.6: 5(S)
3.7: 5(R)
K
2
CO
3
, CuI, NaI,
DMF, -20°C to rt
86%
Pyridine, 1-octene,
lindlar catalyst,
EtOAc, rt
72%
OTBS
1) TBAF, THF
2) CH
2
N
2
, Et
2
O
65%
HO
HO
COOMe
OH
O
+
+
HO
HO
COOH
OH
3.1: 17(S)
3.2: 17(R)
LiOH, MeOH, H
2
O
58%
3.28: 17(S)
3.29: 17(R)
3.30: 17(S)
3.31: 17(R)
Scheme 3-5. Construction of RvD4 and AT-RvD4 from Building Blocks 3.5 and
3.6 or 3.7

47

In summary, the first total synthesis of Resolvin D4 and Aspirin-Triggered
Resolvin D4 has been achieved, aiding in the complete assignment of the
stereochemistry and geometry of the lipid mediators. The synthetic strategy is
concise, convergent, and highly stereocontrolled forming these molecules in
stereochemically pure form by using enantiomerically pure commercially
available building blocks. The synthetic availability of these lipid mediators will
support the identification of their role during inflammation and confirm their
novel anti-inflammatory and pro-resolving properties. Overall, these data offer
new insights for the biological roles of aspirin and DHA.
3.5 Experimental
Unless otherwise noted, all reactions were carried out in a flame-dried
flask with stir bar under argon routed through a three-necked valve. Reactions
were carried out at room temp using DriSolv solvents purchased commercially
from VWR. All reagents used were purchased without further purification from
Sigma Aldrich, Strem, Combi-Blocks and Alfa Aesar.
Progress was monitored and recorded using EMD analytical thin layer
chromatography plates, Silica Gel 60 F254. TLC plates were visualized through
UV absorbance, (254 nm), or staining techniques including vanillin,
phosphomolybdic acid, potassium-permanganate, or ninhydrin followed by
heating. Unless otherwise stated, purification was carried out by flash column
chromatography manually using Silica Gel (100-200 mesh) or automatically
using the Biotage Isolera One.

48

Characterization was carried out using LC-MS, NMR and UV-VIS
instrumentation. All
1
H,
13
C and gcosy spectra were procured on the Departments
Varian 400, 500 and 600 MHz NMR instruments in the solvent indicated.
1
H and
13
C chemical shifts, (δ), are recorded in parts per million, (ppm), and referenced
to the residual solvent converted by the TMS scale (CDCl 3,
1H
= 7.26 ppm).
Splitting patterns are denoted by s, d, t, dd, td, ddd, and m and refer to the
respective multiplicities; singlet, doublet, triplet, doublet of doublets, triplet of
doublets, doublet of doublet of doublet and multiplet. Mass spectra was recorded
on an Agilent 1260 LC-MS. UV-Vis spectra was obtained by a Hewlett- Packard
8350 instrument.

49

(4S,5R,E)-methyl 4,5,6-trihydroxyhex-2-enoate (3.14) To a
solution of D-erythrose 3.13 (0.85 g, 7.1 mmol) in 10 mL of anhydrous THF was
added methyl (triphenylphosphoranylidene) acetate (2.4 g, 7.1 mmol). The
reaction mixture was stirred at 65°C overnight. Without workup the solvent was
removed in vacuo and the crude mixture was purified on silica gel using
MeOH/CH2Cl2 (10%) as the eluent to afford the triol ester (3.14) (1.1 g, 90%) as
a clear colorless oil.
1
H NMR (400 MHz, Methanol-d 4) 7.13 (dd, J = 15.7, 4.8 Hz,
1H), 6.19 (ddd, J = 5.8, 2.0, 0.4 Hz, 1H), 6.09 (dd, J = 15.7, 1.8 Hz, 1H), 4.16 (m,
1H), 3.72 (s, 3H), 3.52 (m, 4H).
13
C NMR (400 MHz, Methanol-d 4) 174.13, 167.20,
148.69, , 74.35, 71.25, 62.92, 50.66.
(4S,5R)-methyl 4,5,6-tris(tert-butyldimethylsilyloxy)hexanoate
(3.12) To a solution of triol (3.14) (740 mg, 4.20 mmol) in 12 mL of EtOAc was
added one scoop of 5% palladium on charcoal. The reaction was stirred under H2
overnight. The reaction mixture was filtered through celite and with no workup
the solvent evaporated. To a flask with imidazole (1.71 g, 25.2 mmol) and DMAP
(256 mg, 2.10 mmol) in 10 mL of anyhrous DMF was added TBS-Cl (3.78 g,
3.12
TBSO
TBSO
COOMe
OTBS
3.14
HO
HO
COOMe
OH

50

25.2mmol) dropwise at 0°C. The triol (220 mg, 1.25 mmol) was cannulated to the
flask and stirred overnight at room temperature. The reaction mixture was
quenched with saturated aqueous NH4Cl (7 mL) and extracted with Et2O (3 x 7
mL). The organic layer was dried with MgSO4, filtered and the solvent removed
in vacuo. The crude reaction mixture was purified on silica gel using EtOAc-
hexanes (2%) as the eluent to afford the protected triol ester (3.12) (1.86 g, 85%)
as a clear colorless oil.
1
H NMR (400 MHz, Chloroform-d) 3.75 (m, 1H), 3.66 (s,
3H), 3.55 (m, 1H), 3.45 (dd, J = 10.0, 5.9 Hz, 1H), 2.28 (m, 2H), 1.75 (m, 2H),
0.85 (m, 24H), 0.03 (m, 14H).
13
C NMR (400 MHz, cdcl 3) 174.38, 77.10, 72.74,
64.84, 51.42, 30.11, 27.19, 25.98, 25.97, 25.93, 25.89, 25.86, 25.64, -4.11, -4.37, -
4.68, -4.88, -5.38, -5.44.
(4S,5S)-methyl-4,5-bis((tert-butyldimethylsilyl)oxy)-6-
oxohexanoate (3.15) To a solution of protected triol (3.12) (525 mg, 1.00
mmol) in 14 mL of a 1:1 mixture of CH2Cl2/MeOH was added camphorsulfonic
acid (200 mg, 0.86 mmol) at 0°C. The reaction was quenched after 50 min with
Et3N (0.15 mL, 1.10 mmol) and the solvent was removed in vacuo. The crude
mixture was purified on silica gel using EtOAc-hexanes (12%) as the eluent to
afford the protected triol ester (285 mg, 70%) as a clear colorless oil.
1
H NMR
(400 MHz, Chloroform-d) 3.80 (q, J = 5.1 Hz, 1H), 3.67 (s, 3H), 3.59 (dd, J = 4.5,
1.6 Hz, 2H), 2.38 (dd, J = 8.0, 5.6 Hz, 2H), 1.99 (dd, J = 7.3, 4.6 Hz, 1H), 1.84 (m,
3.15
O
TBSO
COOMe
OTBS

51

2H), 0.90 (s, 14H), 0.03 (m, 9H).
13
C NMR (400 MHz, cdcl 3) 174.11, 74.98, 72.70,
63.71, 51.54, 29.18, 28.37, 25.89, 25.85, 18.10, 18.07, -4.45, -4.60. To a solution of
alcohol in 15 mL of CH 2Cl 2 was added 25 drops of pyridine and Dess-Martin
periodinane (445 mg, 1.05 mmols) the mixture was stirred at room temperature
for 20 minutes. The reaction mixture was quenched with 1:1 mixture of saturated
aqueous NaHCO 3 and saturated aqueous Na 2S 2O 3 (7 mL) and extracted with
Et2O (3 x 7 mL). The organic layer was dried with MgSO4, filtered and the
solvent removed in vacuo. The crude reaction mixture was purified on silica gel
using EtOAc-hexanes (10%) as the eluent to afford the aldehyde (3.15) (227 mg
80%) as clear colorless oil.
1
H NMR (500 MHz, Chloroform-d) 9.60 (d, J = 1.9
Hz, 1H), 3.96 (dd, J = 3.5, 2.1 Hz, 1H), 3.85 (m, 1H), 3.67 (s, 3H), 2.37 (t, J = 7.7
Hz, 2H), 1.76 (m, 2H), 0.89 (d, J = 22.7 Hz, 18H), 0.14 ? 0.00 (m, 12H).
13
C NMR
(500 MHz, cdcl 3) 203.06, 173.36, 80.59, 73.76, 51.34, 29.27, 28.14, 25.55, 25.54,
25.52, 17.99, 17.81, -4.66, -5.12.
(4S,5R,E)- methyl 4,5-bis((tert-butyldimethylsilyl)oxy)-8-
oxooct-6-enoate (3.16) To a flask with
(Triphenylphosphoranylidene)acetaldehyde (426 mg, 1.40 mmols) was
cannulated aldehyde (227mg, 0.56 mmols) in 6.5 mL of anhydrous THF. The
mixture was refluxed at 65°C overnight. The reaction mixture with no workup
was condensed in vacuo followed by purification of the crude mixture on silica gel
3.16
TBSO
COOMe
OTBS
O

52

using EtOAc-hexanes (5%) as the eluent to afford the extended aldehyde (3.16)
(180 mg, 75%) as a dark red colored oil.
1
H NMR (500 MHz, Chloroform-d) 9.57
(d, J = 7.9 Hz, 1H), 6.83 (dd, J = 15.7, 5.2 Hz, 1H), 6.25 (ddd, J = 15.7, 7.9, 1.4 Hz,
1H), 4.27 (t, J = 4.6 Hz, 1H), 3.75 (q, J = 5.5 Hz, 1H), 3.66 (s, 3H), 2.39 (td, J =
7.5, 2.3 Hz, 2H), 1.77 (m, 2H), 0.88 (d, J = 17.9 Hz, 18H), 0.04 (m, 12H).
13
C
NMR (500 MHz, cdcl 3) 193.26, 157.17, 132.42, 75.63, 75.07, 51.55, 29.42, 28.35,
25.86, 25.82, 18.09, -4.05, -4.48, -4.79, -4.81.
(4S,5R,6E,8E)- methyl 4,5-bis((tert-butyldimethylsilyl)oxy)-9-
iodonona-6,8-dienoate (3.11) To a solution of CrCl2 (5.16 g, 4.2 mmol)
dissolved in 8 mL of anhydrous THF was cannulated a mixture of aldehyde
(3.16) (180 mg, 0.42 mmol) and CHI3 (827 mg, 2.1 mmol) dissolved in 3 mL of
anhydrous THF at 0°C. The reaction was stirred at 0°C for 3 h and an additional 1
h at room temperature. The reaction mixture was quenched with brine (50 mL)
extracted with Et2O (3 x 50 mL) and dried over MgSO4. The organic phase was
filtered and the solvent was removed in vacuo to afford a crude oil which was
purified on silica gel using first pure pentanes and then EtOAc-hexanes (2%) as
the eluent to afford the vinyl iodide (3.11) (150 mg, 64%) as a yellow colored oil.
1
H NMR (400 MHz, Chloroform-d) 7.01 (dd, J = 14.3, 10.7 Hz, 1H), 6.30 (d, J =
14.4 Hz, 1H), 6.01 (m, 1H), 5.67 (dd, J = 15.3, 7.0 Hz, 1H), 3.94 (ddd, J = 6.9, 4.4,
1.1 Hz, 1H), 3.66 (s, 3H), 3.63 (t, J = 5.4 Hz, 1H), 2.38 (td, J = 7.4, 2.6 Hz, 2H),
3.11
TBSO
COOMe
OTBS
I

53

1.80 (dd, J = 7.3, 1.4 Hz, 2H), 0.87 (dd, J = 5.2, 0.5 Hz, 19H), 0.04 (m, 12H).
13
C
NMR (400 MHz, cdcl 3) 174.19, 144.63, 135.19, 131.16, 78.97, 74.97, 51.49, 29.53,
28.16, 25.97, 25.93, 25.90, 18.22, 18.13, -4.03, -4.72, -4.74.
(4S,5R,6E,8E)- methyl 4,5-bis((tert-butyldimethylsilyl)oxy)-12-
hydroxydodeca-6,8-dien-10-ynoate (3.17) To a solution of vinyl iodide
(3.11) (283 mg, 0.51 mmol) in 2 mL of Piperidine was added Pd(OAc)2/PPh3
(40 mg, 0.052 mmol) and propargyl alcohol (0.32 mL, 5.6 mmol). The reaction
was quenched after 1 h with NH4Cl (5 mL) and extracted with Et2O (3 x 5 mL).
The solvent was removed in vacuo and the crude mixture was purified on silica
gel using EtOAc-hexanes (12%) as the eluent to afford compound (3.17) (255mg
0.53 mmols) as a yellow colored oil.
1
H NMR (400 MHz, Chloroform-d) 6.58 (dd,
J = 15.6, 10.8 Hz, 1H), 6.12 (m, 1H), 5.75 (dd, J = 15.3, 6.6 Hz, 1H), 5.59 (d, J =
15.7 Hz, 1H), 4.41 (s, 2H), 3.96 (m, 1H), 3.66 (s, 3H), 3.63 (q, J = 5.2 Hz, 1H),
2.38 (dd, J = 9.1, 6.8 Hz, 2H), 1.78 (m, 2H), 0.88 (d, J = 6.7 Hz, 20H), 0.03 (dd, J
= 11.8, 7.7 Hz, 12H).
13
C NMR (400 MHz, cdcl 3) 174.23, 135.87, 127.54, 29.70,
27.02, 26.99, 25.93, 25.90, 20.55, 19.35, 18.21, 18.11, 14.07, -4.02, -4.09, -4.68, -
4.76.
3.17
TBSO
COOMe
OTBS
HO

54

(4S,5R,6E,8E)- methyl 12-bromo-4,5-bis((tert-
butyldimethylsilyl)oxy)dodeca-6,8-dien-10-ynoate (3.5) To a solution of
propargyl alcohol (3.17) (255 mg, 0.53 mmol) in 10 mL of anhydrous CH 2Cl 2 at
0°C was added PPh3 (125 mg, 0.48 mmol) and N-bromosuccinimide (85 mg,
0.48 mmol). After 30 mins the reaction was quenched with NaHCO 3 (10 mL) and
extracted with Et 2O (3 x 10 mL). The solvent was removed in vacuo and the crude
mixture was purified on silica gel using EtOAc-hexanes (2%) as the eluent to
afford compound (3.5) as a clear colorless oil (240 mg, 83%).
1
H NMR (400
MHz, Chloroform-d) 6.60 (dd, J = 15.5, 11.0 Hz, 1H), 6.17 (dd, J = 15.4, 10.9 Hz,
1H), 5.77 (dd, J = 15.2, 6.9 Hz, 1H), 5.59 (d, J = 15.6 Hz, 1H), 4.09 (d, J = 2.3 Hz,
2H), 3.96 (m, 1H), 3.65 (s, 3H), 3.59 (m, 1H), 2.33 (m, 2H), 1.81 (dd, J = 7.1, 1.7
Hz, 2H), 0.87 (d, J = 6.3 Hz, 19H), 0.04 (m, 12H).
13
C NMR (400 MHz, cdcl 3)
174.16, 142.60, 138.10, 130.37, 109.92, 86.49, 86.04, 76.47, 75.02, 51.48, 29.43,
28.23, 25.95, 25.91, 25.88, 18.20, 18.10, 15.55, -4.04, -4.15, -4.70, -4.77.
(S)-tert-butyldimethyl(oxiran-2-ylmethoxy)silane (3.8) To a
solution of TBS-Cl (15.3 g, 101.5 mmol), imidazole (6.9 g, 101.5 mmol) and DMAP
(412 mg, 3.4 mmol) dissolved in 125 mL of anhydrous CH 2Cl 2 at 0°C was added
R-glycidol (5.0 g, 67.5 mmol) at 0°C. The reaction was allowed to stir overnight at
3.5
TBSO
COOMe
OTBS
Br
3.8
O
OTBS

55

room temperature. It was then quenched with saturated aqueous NH 4Cl (125 mL)
and extracted with Et2O (3 x 125 mL). The combined extract was dried with
Na 2SO 4 and evaporated to give a crude clear oil which was then chromatographed
on silica gel using EtOAc-hexanes (0.5%) as the eluent to afford the (S)-protected
glycidol (3.8) as a viscous and colorless oil (12.2 g, 96%).
1
H NMR (400 MHz,
Chloroform-d) 3.84 (dd, J = 11.9, 3.2 Hz, 1H), 3.65 (dd, J = 11.9, 4.8 Hz, 1H), 3.05
(m, 1H), 2.76 (dd, J = 5.2, 4.1 Hz, 1H), 2.60 (m, 1H), 0.89 (s, 9H), 0.07 (d, J = 3.7
Hz, 6H).
13
C NMR (400 MHz, cdcl 3) 63.72, 52.38, 44.42, 25.84, 18.33, -5.34, -
5.38.
(R)-tert-butyldimethyl(oxiran-2-ylmethoxy)silane (3.9) This
compound was prepared from S-Glycidol similarly to its enantiomer, compound
(3.8).
1
H NMR (400 MHz, CDCl3) δ 3.84 (dd, J = 11.9, 3.2 Hz, 1H), 3.65 (dd, J =
11.9, 4.8 Hz, 1H), 3.13 – 3.01 (m, 1H), 2.76 (dd, J = 5.2, 4.0 Hz, 1H), 2.63 (dd, J =
5.2, 2.7 Hz, 1H), 0.90 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H).
13
C NMR (400 MHz,
CDCl3) 63.88, 52.56, 44.60, 26.01, 18.50, -5.17, -5.21.
2S, 1-(t-butyldiphenylsilyloxy)hept-4-yn-2-ol (3.18) To a flask at -
78°C was added 1- butyne (0.29 g, 5.3 mmol), and n-BuLi (2.12 mL, 5.3 mmol)
added dropwise at - 78°C. After 0.25 h BF 3Et 2O (0.64 mL, 5.3 mmol) was
3.9
O
OTBS
3.1
TBSO
OH

56

added drop wise at -78°C. To the reaction mixture was added protected glycidol
(3.8) (0.5 g, 2.65 mmol) and stirred for 3 h at -78°C. The reaction mixture was
warmed to room temperature, quenched with saturated aqueous NH4Cl (15 mL)
and extracted with Et2O (3 x 15 mL). The organic layer was dried with MgSO4,
filtered and the solvent removed in vacuo. The crude reaction mixture was
purified on silica gel using EtOAc-hexanes (4%) as the eluent to afford compound
(3.18) (600 mg, 94%) as a clear colorless oil.
1
H NMR (500 MHz, Chloroform-d)
3.73 (d, J = 6.2 Hz, 1H), 3.68 (dd, J = 9.9, 4.3 Hz, 1H), 3.60 (d, J = 5.8 Hz, 1H),
2.48 (d, J = 5.0 Hz, 1H), 2.36 (dd, J = 4.1, 2.0 Hz, 1H), 2.14 (d, J = 7.5 Hz, 1H),
1.10 (t, J = 7.5 Hz, 3H), 0.89 (s, 9H), 0.06 (s, 5H).
13
C NMR (500 MHz, cdcl 3)
83.91, 75.04, 70.44, 65.62, 25.83, 23.36, 18.26, 14.14, 12.36, -5.43.
2R, 1-(t-butyldiphenylsilyloxy)hept-4-yn-2-ol (3.22) This
compound was prepared from protected S-Glycidol (3.9) similarly to its
enantiomer, compound (3.18).
1
H NMR (400 MHz, CDCl3) δ 3.77 – 3.72 (m,
1H), 3.70 (dd, J = 9.9, 4.2 Hz, 1H), 3.60 (dd, J = 9.8, 5.8 Hz, 1H), 2.46 (d, J = 4.8
Hz, 1H), 2.42 – 2.33 (m, 2H), 2.22 – 2.10 (m, 2H), 1.11 (t, 3H), 0.90 (s, 9H), 0.08
(s, 6H).
13
C NMR (400 MHz, CDCl3) δ 84.15, 75.21, 70.63, 65.81, 26.03, 23.55,
18.46, 14.33, 12.55, -5.23, -5.26.
3.22
TBSO
OH

57

(S), 2-(t-butyldiphenylsilyloxy)hept-4-yn-1-ol (3.19) To a flask with
imidazole (95 mg, 1.39 mmol) and DMAP (8 mg, 0.06 mmol) in 3.5 mL of
anhydrous CH2Cl2 was added TBDPS-Cl (0.36 mL, 1.39 mmol) dropwise at 0°C.
The alcohol (3.18) (280 mg, 1.15 mmol) was cannulated to the flask in 1.5 mL of
anhydrous CH2Cl2 and stirred overnight at room temperature. The reaction
mixture was quenched with saturated aqueous NH4Cl (7 mL) and extracted with
Et2O (3 x 7 mL). The organic layer was dried with MgSO4, filtered and the
solvent removed in vacuo. The crude reaction mixture was purified on silica gel
using EtOAc-hexanes (1%) as the eluent to afford the protected diol (525 mg, 9%)
as a clear colorless oil.
1
H NMR (400 MHz, CDCl3) δ 7.85 – 7.65 (m, 4H), 7.55 –
7.29 (m, 6H), 3.83 (p, J = 5.5 Hz, 1H), 3.55 (dd, J = 5.4, 3.3 Hz, 2H), 2.44 – 2.34
(m, 1H), 2.32 – 2.23 (m, 1H), 2.12 (qt, J = 7.5, 2.4 Hz, 2H), 1.10 (t, 3H), 1.07 (s,
9H), 0.84 (s, 9H), -0.03 (s, 3H), -0.06 (s, 3H).
13
C NMR (400 MHz, CDCl3) δ
136.10, 136.03, 134.47, 134.34, 129.69, 129.66, 127.62, 83.29, 76.47, 72.78, 65.80,
27.09, 26.05, 24.12, 19.54, 18.46, 14.34, 12.62, -5.34, -5.35. To a solution of
protected diol (0.5 g, 1.04 mmol) in 5 mL of CH2Cl2/MeOH (1:1) was added
camphor sulfonic acid (144 mg, 0.62 mmol) at room temperature and monitored
by TLC until complete consumption of the starting material, approximately 1 h.
The reaction was quenched with Et3N (0.09 mL, 0.62 mmol) and the solvent was
evaporated in vacuo without workup. The crude reaction mixture was purified on
silica gel using EtOAc-hexanes (12%) as the eluent to afford alcohol (3.19) (370
3.19
HO
OTBDPS

58

mg, 97%) as a clear colorless oil.
1
H NMR (400 MHz, Chloroform-d) 7.70 (m,
4H), 7.38 (m, 6H), 3.90 (m, 1H), 3.64 (m, 2H), 2.39 (m, 1H), 2.28 (m, 1H), 2.05
(m, 2H), 1.12 (s, 9H), 1.08 (t, J = 7.5 Hz, 3H).
13
C NMR (400 MHz, cdcl 3) 135.77,
135.62, 133.52, 133.50, 129.82, 129.77, 127.73, 127.60, 75.26, 72.52, 65.54, 26.92,
23.81, 19.24, 13.99, 12.31
(R), 2-(t-butyldiphenylsilyloxy)hept-4-yn-1-ol (3.23). This
compound was prepared from alcohol (3.22) similarly to its enantiomer,
compound (3.19).
1
H NMR (400 MHz, CDCl3) δ 7.82 – 7.72 (m, 4H), 7.57 – 7.37
(m, 6H), 4.06 – 3.87 (m, 1H), 3.70 (d, J = 4.6 Hz, 2H), 2.47 (ddt, J = 16.4, 7.7, 2.5
Hz, 1H), 2.36 (ddt, J = 16.4, 4.9, 2.4 Hz, 1H), 2.18 – 2.06 (m, 3H), 1.16 (s, 9H),
1.10 (t, J = 7.5 Hz, 3H).
13
C NMR (400 MHz, CDCl3) δ 135.86, 135.71, 133.64,
133.62, 129.89, 129.85, 127.80, 127.68, 83.83, 75.43, 72.62, 65.56, 27.01, 23.91,
19.33, 14.10, 12.41.
2S, 4Z, 2-(t-butyldiphenylsilyloxy)-1-oxohept-4-enal (3.20). To a
solution of alcohol (3.19) (3.9 g, 10.2 mmol) in 200 mL of EtOAc was added
Lindlar catalyst (200 mg) and 5 drops of quinoline. The reaction mixture was
placed under a H2 atmosphere and stirred for 2 h. The reaction was filtered
HO
OTBDPS
3.23
TBDPSO
O
3.20

59

through celite and the solvent was removed in vacuo. To a solution of the crude
alcohol (0.5 g, 1.87 mmol) in 20 mL of CH 2Cl 2 was added 35 drops of pyridine
and Dess-Martin periodinane (1.189 g, 2.80 mmols) the mixture was stirred at
room temperature for 20 minutes. The reaction mixture was quenched with 1:1
mixture of saturated aqueous NaHCO 3 and saturated aqueous Na 2S 2O 3 (15 mL)
and extracted with Et2O (3 x 15 mL). The organic layer was dried with MgSO4,
filtered and the solvent removed in vacuo. The crude reaction mixture was
purified on silica gel using EtOAc-hexanes (10%) as the eluent to afford the
aldehyde (3.20) (0.37 g, 81%) as a colorless oil.
1
H NMR (400 MHz, Chloroform-
d) 9.59 (d, J = 1.7 Hz, 1H), 7.64 (m, 4H), 7.35 (m, 6H), 5.44 (m, 1H), 5.37 (dt, J =
9.7, 1.3 Hz, 1H), 4.09 (ddd, J = 6.4, 5.6, 1.6 Hz, 1H), 2.42 (m, 1H), 2.33 (m, 1H),
1.90 (m, 2H), 1.15 (s, 9H), 0.92 (t, J = 7.5 Hz, 3H).
13
C NMR (400 MHz, cdcl 3)
203.27, 135.77, 134.92, 133.06, 132.94, 130.00, 129.96, 127.77, 127.71, 122.00,
77.77, 30.97, 26.90, 20.56, 19.31, 13.97.
2R, 4Z, 2-(t-butyldiphenylsilyloxy)-1-oxohept-4-enal (3.24). This
compound was prepared from alcohol (3.23) similarly to its enantiomer,
compound (3.20).
1
H NMR (400 MHz, CDCl3) δ 9.57 (d, J = 1.7 Hz, 2H), 7.71 –
7.61 (m, 4H), 7.49 – 7.33 (m, 6H), 5.52 – 5.41 (m, 1H), 5.40 – 5.27 (m, 1H), 4.06
(td, J = 6.5, 1.7 Hz, 1H), 2.44 (dt, J = 14.1, 6.7 Hz, 1H), 2.34 (dt, J = 13.9, 6.5 Hz,
1H), 1.99 – 1.87 (m, 2H), 1.12 (s, 9H), 0.90 (t, J = 7.5 Hz, 3H).
13
C NMR (400
TBDPSO
O
3.24

60

MHz, CDCl3) δ 203.52, 135.95, 135.95, 135.11, 133.24, 133.12, 130.18, 130.13,
127.95, 127.89, 122.17, 77.94, 31.15, 27.08, 20.74, 19.49, 14.14.
(S,2E,6Z)-4-((tert-butyldiphenylsilyl)oxy)nona-2,6-dienal
(3.21). To a flask with (Triphenylphosphoranylidene)acetaldehyde (500 mg, 1.63
mmols) was cannulated aldehyde (3.20) (300mg, 0.82 mmols) in 7 mL of
anhydrous THF. The mixture was refluxed at 65°C overnight. The reaction
mixture with no workup was condensed in vacuo followed by purification of the
crude mixture on silica gel using EtOAc-hexanes (5%) as the eluent to afford the
extended aldehyde (3.21) (512 mg, 75%) as a dark red colored oil.
1
H NMR (400
MHz, Chloroform-d) 9.46 (d, J = 8.0 Hz, 1H), 7.57 (m, 4H), 7.32 (m, 6H), 6.72
(dd, J = 15.6, 4.9 Hz, 1H), 6.22 (ddd, J = 15.6, 8.0, 1.5 Hz, 1H), 5.37 (m, 1H), 5.17
(m, 1H), 4.42 (m, 1H), 2.27 (ddt, J = 29.4, 14.6, 7.6 Hz, 2H), 1.74 (m, 2H), 1.09 (s,
9H), 0.84 (t, J = 7.5 Hz, 3H).
13
C NMR (400 MHz, cdcl 3) 193.53, 158.92, 135.83,
135.75, 134.92, 133.49, 133.16, 130.93, 129.94, 129.92, 127.68, 127.65, 122.31,
72.34, 34.97, 26.96, 20.56, 19.30, 14.02.
3.21
TBDPSO
O

61

(R,2E,6Z)-4-((tert-butyldiphenylsilyl)oxy)nona-2,6-dienal
(3.25). This compound was prepared from alcohol (3.24) similarly to its
enantiomer, compound (3.21).
1
H NMR (400 MHz, CDCl3) 9.46 (d, J = 8.0 Hz,
1H), 7.57 (m, 4H), 7.32 (m, 6H), 6.72 (dd, J = 15.6, 4.9 Hz, 1H), 6.22 (ddd, J =
15.6, 8.0, 1.5 Hz, 1H), 5.37 (m, 1H), 5.17 (m, 1H), 4.42 (m, 1H), 2.27 (ddt, J = 29.4,
14.6, 7.6 Hz, 2H), 1.74 (m, 2H), 1.09 (s, 9H), 0.84 (t, J = 7.5 Hz, 3H).
13
C NMR
(400 MHz, CDCl3) 193.53, 158.92, 135.83, 135.75, 134.92, 133.49, 133.16, 130.93,
129.94, 129.92, 127.68, 127.65, 122.31, 72.34, 34.97, 26.96, 20.56, 19.30, 14.02.
tert-butyl(((S,3E,7Z)-deca-3,7-dien-1-yn-5-yl)oxy)diphenylsilane
(3.6). Triphenylphosphine (692 mg, 2.65 mmol) in 5 mL of anhydrous CH2Cl2
was cannulated in to a solution of CBr4 (440 mg, 1.33 mmol) at 0°C in 20 mL of
anhydrous CH2Cl2 . Aldehyde (3.21) (278 mg, 0.66 mmol) in 3 mL of anhydrous
CH2Cl2 was cannulated in to the reaction mixture. The reaction was stirred for 1
h at 0°C. Without workup the solvent was evaporated in vacuo and the crude
mixture was purified on silica gel using EtOAc-hexanes (1%) as the eluent to
3.25
TBDPSO
O
TBSO
3.6

62

afford the dibromo ester (351 mg, 97%) as a viscous and yellow colored oil. To a
solution of the dibromo ester (351 mg, 64 mmol) at -78°C in 15 mL of anhydrous
THF was added 2.0 M solution of LDA (1.92 mL, 3.84 mmol) drop wise and
stirred for 0.5 h. The reaction was quenched with water (30 mL) and extracted
with Et2O (3 x 30 mL), dried using MgSO4, filtered and concentrated. The crude
was then purified using silica gel with a EtOAc- hexanes eluent (2%) to afford the
alkyne product (206 mg, 83%) as a viscous and yellow colored oil.
1
H NMR (400
MHz, Chloroform-d) 7.63 (m, 4H), 7.39 (m, 6H), 6.26 (dd, J = 16.1, 5.7 Hz, 1H),
5.63 (dt, J = 15.9, 1.9 Hz, 1H), 5.36 (m, 1H), 5.18 (m, 1H), 4.26 (q, J = 5.1 Hz, 1H),
2.87 (d, J = 2.2 Hz, 1H), 2.13 (m, 2H), 1.83 (td, J = 7.5, 6.7, 3.2 Hz, 2H), 1.12 (s,
9H), 0.88 (t, J = 7.5 Hz, 3H).
13
C NMR (101 MHz, cdcl 3) 147.18, 135.85, 135.83,
134.24, 133.96, 133.50, 129.72, 129.69, 127.56, 123.04, 108.22, 82.04, 73.01,
35.36, 27.00, 20.54, 19.33, 14.06. To the terminal alkyne stirring in 4 mL of
anhydrous THF at was added 1.0 M solution of TBAF (1.6 mL, 1.60 mmol) at 0°C
and stirred for 2 h. The reaction mixture was quenched with saturated aqueous
NH4Cl (7 mL) and extracted with Et2O (3 x 7 mL). The organic layer was dried
with MgSO4, filtered and the solvent removed in vacuo. The crude reaction
mixture was dissolved in 3 mL of anhydrous CH2Cl2 and cooled to 0°C, 2,6-
lutidine (0.135 mL 1.16 mmols) and TBSDMS triflate (0.27 mL 1.16 mmols) were
added and the reaction mixture was allowed to stir overnight at room
temperature. It was then quenched with saturated aqueous NH 4Cl (125 mL) and
extracted with Et2O (3 x 125 mL). The combined extract was dried with Na 2SO 4
and evaporated to give a crude yellow oil which was then chromatographed on

63

silica gel using EtOAc-hexanes (2%) as the eluent to afford the protected terminal
alkyne (3.6) as a viscous and yellow oil (127 mg, 73%).
1
H NMR (400 MHz,
Chloroform-d) 6.25 (dd, J = 15.9, 5.1 Hz, 1H), 5.66 (d, J = 15.9 Hz, 1H), 5.43 (m,
1H), 5.28 (m, 1H), 4.14 (m, 1H), 2.86 (d, J = 2.3 Hz, 1H), 2.20 (m, 2H), 1.95 (m,
2H), 0.96 (t, J = 7.5 Hz, 3H), 0.90 (s, 9H), 0.05 (d, J = 6.0 Hz, 6H).
13
C NMR
(400 MHz, cdcl 3) 147.66, 133.76, 123.48, 107.31, 107.30, 81.76, 71.94, 35.41,
25.50, 20.41, 17.89, 13.81, -4.98, -5.14.
tert-butyl(((R,3E,7Z)-deca-3,7-dien-1-yn-5-
yl)oxy)diphenylsilane (3.7). This compound was prepared from alcohol
(3.25) similarly to its enantiomer, compound (3.6).
1
H NMR (400 MHz, CDCl3)
6.23 (dd, J = 15.7, 5.0 Hz, 1H), 5.66 (d, J = 15.7 Hz, 1H), 5.44 (m, 1H), 5.27 (m,
1H), 4.18 (m, 1H), 2.84 (d, J = 2.4 Hz, 1H), 2.20 (m, 2H), 1.98 (m, 2H), 0.99 (t, J
= 7.6 Hz, 3H), 0.88 (s, 9H), 0.03 (d, J = 6.1 Hz, 6H).
13
C NMR (400 MHz, CDCl3)
147.64, 133.83, 123.46, 107.31, 107.32, 81.76, 71.95, 35.39, 25.53, 20.40, 17.89,
13.78, -5.00, -5.14.
3.7
TBSO

64

Methyl-(4S, 5R, 17R, 6E, 8E, 15E, 19Z)-tris-(tert-
butyldimethylsilyloxy)-docosa- 6,8,15,19-tetraen-10,13-diynoate (3.3).
To a flame dried flask with CuI (42 mg, 0.22 mmol), NaI (33 mg, 0.22 mmol),
and K2CO3 (30 mg, 0.22 mmol) in 5 mL of anhydrous DMF was cannulated
alkyne (3.6) (60 mg, 0.22 mmol) and allylic bromide (3.5) (60 mg, 0.11 mmol).
The reaction was stirred for 18 h and quenched with saturated NH4Cl (5 mL).
The mixture was extracted with Et2O (3 x 5 mL), rinsed with water to remove any
DMF, and the organic layer was dried with MgSO4, filtered and the solvent
removed in vacuo. The crude reaction mixture was purified on silica gel using
EtOAc- hexanes (1%) as the eluent to afford compound (3.3) as a clear colorless
oil (71 mg, 89%).
1
H NMR (400 MHz, Chloroform-d) 6.55 (dd, J = 15.6, 10.8 Hz,
1H), 6.14 (m, 1H), 6.11 (dd, J = 10.5, 5.3 Hz, 1H), 5.65 (m, 1H), 5.51 (m, 2H), 5.40
(m, 1H), 5.33 (d, J = 7.4 Hz, 1H), 4.16 (q, J = 5.6, 5.0 Hz, 1H), 3.96 (dd, J = 7.0,
4.8 Hz, 1H), 3.65 (s, 3H), 3.62 (q, J = 5.0 Hz, 1H), 3.44 (s, 2H), 2.33 (m, 2H), 2.18
(m, 2H), 1.97 (m, 2H), 1.77 (m, 2H), 1.25 (s, 3H), 0.95 (t, J = 7.5 Hz, 3H), 0.88 (q,
J = 5.6, 5.2 Hz, 22H), -0.02 (m, 14H).
13
C NMR (400 MHz, cdcl 3) 174.20, 145.99,
141.09, 136.79, 133.89, 130.72, 124.01, 110.86, 108.44, 85.91, 83.29, 79.85, 79.06,
76.55, 75.01, 72.43, 35.84, 29.41, 28.24, 25.91, 20.72, 18.19, 18.09, 14.14, 11.29, -
4.04, -4.12, -4.61, -4.70, -4.78, -4.83.
TBSO
TBSO
COOMe
OTBS
3.3

65

Methyl-(4S, 5R, 17S, 6E, 8E, 15E, 19Z)-tris-(tert-
butyldimethylsilyloxy)-docosa- 6,8,15,19-tetraen-10,13-diynoate (3.4).
This compound was prepared from allylic bromide (3.5), and alkyne (3.7)
similarly to its enantiomer, compound (3.3).
1
H NMR (600 MHz, CDCl3) δ 6.55
(dd, J = 15.6, 10.8 Hz, 1H), 6.15 (dd, J = 15.3, 10.9 Hz, 1H), 6.17 – 6.07 (m, 2H),
5.71 (dd, J = 15.3, 7.2 Hz, 1H), 5.64 (dq, J = 15.8, 2.0 Hz, 1H), 5.55 (dt, J = 15.5,
2.4 Hz, 1H), 5.52 – 5.40 (m, 1H), 5.37 – 5.25 (m, 0H), 4.20 – 4.11 (m, 1H), 3.96
(dd, J = 7.1, 4.8 Hz, 1H), 3.66 (s, 3H), 3.62 (q, J = 5.1 Hz, 1H), 3.44 (t, J = 2.2 Hz,
2H), 2.42 – 2.33 (m, 2H), 2.31 – 2.17 (m, 2H), 2.02 (p, J = 7.4 Hz, 2H), 1.87 –
1.76 (m, 2H), 0.95 (t, J = 7.6 Hz, 3H), 0.89 (s, 9H), 0.88 (s, 9H), 0.86 (s, 9H),
0.05 (s, 3H), 0.04 (s, 3H), 0.03 (s, 3H), 0.03 (s, 3H), 0.02 (s, 3H), -0.01 (s, 3H).
13
C NMR (600 MHz, CDCl3) δ 174.40, 146.17, 141.27, 136.98, 134.07, 130.88,
124.18, 111.01, 108.60, 86.07, 83.46, 80.03, 79.24 , 76.71, 75.17, 72.60, 51.63,
36.00, 29.59, 28.40, 26.08, 26.05, 25.99, 20.89, 18.37, 18.36, 18.26, 14.30, 11.46,
-3.87, -3.95, -4.45, -4.54, -4.61, -4.66.

3.4
TBSO
TBSO
COOMe
OTBS

66

Methyl (4S,5R,6E,8E,10Z,13Z,15E,17R,19Z)-4,5,17-
trihydroxydocosa-6,8,10,13,15,19-hexaenoate (3.2). Part 1: To a solution
of compound (3.4) (71 mg, 0.097 mmol) in 4 mL of EtOAc, Octene, (0.4 mL),
and Pyridine (0.4 mL). Next was added Lindlar catalyst (20 mg) and the reaction
mixture was allowed to stir at room temperature in an H 2 atmosphere. After 6 h
the reaction mixture was filtered threw celite and the solvent removed in vacuo.
The crude reaction mixture was purified on silica gel using EtOAc- hexanes (1%)
as the eluent to afford compound (3.27) as a clear colorless oil (51 mg, 72%).
1
H
NMR (400 MHz, Methanol-d 4) 6.48 (m, 2H), 6.17 (m, 2H), 6.03 (dt, J = 23.0, 11.1
Hz, 2H), 5.62 (m, 2H), 5.33 (m, 4H), 4.26 (dd, J = 6.4, 1.0 Hz, 1H), 4.06 (dd, J =
7.7, 4.2 Hz, 1H), 3.69 (q, J = 5.3 Hz, 1H), 3.65 (s, 2H), 2.97 (m, 2H), 2.38 (m, 2H),
2.25 (t, J = 7.4 Hz, 2H), 2.00 (m, 3H), 1.83 (dd, J = 13.2, 7.7 Hz, 2H), 1.25 (m,
4H), 0.88 (m, 27H), 0.03 (m, 15H).
13
C NMR (101 MHz, cd 3od) 175.78, 141.83,
137.98, 134.41, 133.63, 130.85, 129.82, 129.40, 129.11, 125.84, 78.48, 76.33,
74.36, 54.78, 37.31, 32.75, 30.41, 29.44, 27.56, 26.57, 26.54, 26.52, 26.42, 26.21,
23.70, 21.72, 19.17, 19.15, 19.06, 14.45, -3.60, -4.04, -4.34, -4.37, -4.42. Part 2: To
the hydrogenated crude mixture dissolved in 1 mL of anhydrous THF was added
dropwise 6 equivalents of 1M TBAF (0.42 mL, 0.42 mmol) at 0°C. The reaction
HO
HO
COOH
OH
3.2

67

was monitored closely via thin layer chromatography and after 4 h the reaction
was quenched with saturated NH4Cl (15 mL) and extracted with Et2O (5 x 15
mL). The organic layer was rinsed with brine, dried over MgSO4 and filtered. The
solvent was then concentrated and freshly prepared CH2N2 was added to convert
any acid to the ester-lactone mixture. The solvent was completely removed in
vacuo and the compound was purified on silica gel using MeOH-CH2Cl2 (1%) as
the eluent to afford an ester/lactone mixture. The product was then suspended in
a H2O- MeOH mixture (1:1, 1 mL) and 10 equivalents of LiOH (17 mg, 0.7 mmol)
was added. After 3 h the reaction mixture was dried and purified via C-18
reversed Phase HPLC using H2O-MeOH mixture (37%) to afford compound
(3.2) (1.15 mg 58%) as colorless oil.
1
H NMR (500 MHz, Methanol-d 4) 6.68 (dd,
J = 15.0, 10.9 Hz, 1H), 6.57 (dd, J = 15.1, 11.1 Hz, 1H), 6.37 (dd, J = 15.2, 10.6 Hz,
1H), 6.22 (m, 1H), 5.92 (m, 2H), 5.84 (dd, J = 15.1, 6.6 Hz, 1H), 5.70 (td, J = 14.9,
6.9 Hz, 2H), 5.44 (m, 1H), 5.33 (m, 2H), 4.14 (q, J = 6.5 Hz, 1H), 3.97 (m, 1H),
3.49 (m, 1H), 3.10 (t, J = 7.6 Hz, 1H), 2.86 (m, 1H), 2.31 (dtt, J = 29.9, 14.3, 7.3
Hz, 4H), 2.06 (p, J = 7.4 Hz, 2H), 1.81 (m, 1H), 1.73 (m, 1H), 1.62 (m, 1H), 1.52
(m, 1H), 0.96 (t, J = 7.5 Hz, 3H).
13
C NMR (500 MHz, cd 3od) 182.79, 137.52,
134.62, 134.47, 134.18, 133.14, 132.34, 130.46, 130.24, 130.18, 129.57, 128.74,
126.27, 125.49, 76.52, 76.22, 73.18, 36.25, 32.75, 27.48, 23.70, 21.68, 14.43.

68

Methyl (4S,5R,6E,8E,10Z,13Z,15E,17R,19Z)-4,5,17-
trihydroxydocosa-6,8,10,13,15,19-hexaenoate (3.1). This compound was
prepared from (3.3) similarly to its enantiomer, compound (3.2).
1
H NMR (600
MHz, MeOD) δ 6.51 – 6.43 (dd, J = 15.0, 11.1 Hz, 2H), 6.27 (dd, J = 15.3, 10.7 Hz,
1H), 6.15 (dd, J = 14.7, 10.7 Hz, 1H), 5.94 (t, J = 10.9 Hz, 1H), 5.90 (t, J = 10.9 Hz,
1H), 5.74 (dd, J = 15.2, 6.8 Hz, 1H), 5.59 (dd, J = 15.0, 6.6 Hz, 2H), 5.41 – 5.34
(m, 1H), 5.33 – 5.23 (m, 3H), 4.04 (q, J = 6.5 Hz, 1H), 3.89 (t, J = 6.0 Hz, 1H),
3.45 – 3.39 (m, 1H), 3.00 (t, J = 7.7 Hz, 2H), 2.32 – 2.13 (m, 4H), 1.95 (q, J = 7.6
Hz, 2H), 1.75 (ddd, J = 14.7, 7.1, 3.2 Hz, 1H), 1.58 (dq, J = 15.1, 7.5 Hz, 1H), 0.86
(t, J = 7.5 Hz, 3H).
13
C NMR (600 MHz, MeOD)) 182.81, 137.49, 134.63, 134.44,
134.18, 133.12, 130.56, 130.43, 130.24, 130.18, 129.57, 128.73, 126.27, 125.47,
76.54, 76.21, 73.16, 49.87, 32.78, 27.50, 23.70, 21.71, 14.62.
3.6 References
1. Serhan, C. N., Fredman, G., Yang, R., Karamnov, S., Belayev, L. S.,
Bazan, N. G., Zhu, M., Winkler, J. W., and Petasis, N. A. (2011)
Novel Proresolving Aspirin-Triggered DHA Pathway, Chem. Biol.
18, 976–987.
2. Serhan, C. N., Hong, S., Gronert, K., Colgan, S. P., Devchand, P. R.,
Mirick, G., and Moussignac, R. L. (2002) Resolvins: A Family of
Bioactive Products of Omega-3 Fatty Acid Transformation Circuits
Initiated by Aspirin Treatment that Counter Proinflammation
Signals, Journal of Experimental Medicine 196, 1025–1037.
3. Dalli, J., Zhu, M., Vlasenko, N. A., Deng, B., Haeggstrom, J. Z.,
Petasis, N. A., and Serhan, C. N. (2013) The novel 13S,14S-epoxy-
maresin is converted by human macrophages to maresin 1 (MaR1),
3.1
HO
HO
COOH
OH

69

inhibits leukotriene A4 hydrolase (LTA4H), and shifts macrophage
phenotype, FASEB J. 27, 2573–2583.
4. Serhan, C. N., and Petasis, N. A. (2011) Resolvins and protectins in
inflammation resolution., Chem. Rev. 111, 5922–5943.
5. Naidu, S. V., Gupta, P., and Kumar, P. (2007) Enantioselective
syntheses of (−)-pinellic acid, α- and β-dimorphecolic acid,
Tetrahedron 63, 7624–7633.
6. Webb, T. H.; Thomasco, L. M.; Schlachter, S. T.; Gaudino, J. J.;
Wilcox, C. S. Insight into the unusual reactions of stabilized
phosphorus ylides with lactols. A specific intramolecular hydroxyl
group effect leads to high z-selectivity, Tetrahedron Letters 1988,
29, 6823. 89.
7. Gallos, J. K.; Koumbis, A. E.; Xiraphaki, V. P.; Dellios, C. C.;
Coutouli-Argyropoulou, E. Diastereocontrol in the intramolecular
cycloadditions of 2-substituted-erythro-3,4-
isopropylidenedioxyhex-5-enenitrile oxides, Tetrahedron 1999, 55,
15167.
8. Gollner, A., Altmann, K.-H., Gertsch, J., and Mulzer, J. (2009) The
Laulimalide Family: Total Synthesis and Biological Evaluation of
Neolaulimalide, Isolaulimalide, Laulimalide and a Nonnatural
Analogue, Chem. Eur. J. 15, 5979–5997.
9. Ramulu, U., Ramesh, D., Reddy, S. P., Rajaram, S., and Babu, K. S.
(2014) The stereoselective total syntheses of pectinolides A, B, and
C, Tetrahedron: Asymmetry, Elsevier Ltd 25, 1409–1417.
10. Meyer, S. D., and Schreiber, S. L. (1994) Acceleration of the Dess-
Martin oxidation by water, The Journal of Organic Chemistry.
11. Takai, K.; Nitta, K.; Utimoto, K. Simple and Selective Method for
RCHO-(E)-RCH=CHX Conversion by Means of a CHX3-CrCI2
System. Journal of the American Chemical Society 1986, 108,
7408.
12. Dasse, O., Mahadevan, A., Han, L., Martin, B. R., and Di Marzo, V.
(2000) The Synthesis of N-Vanillyl-arachidonoyl-amide (Arvanil)
and its Analogs: An Improved Procedure for the Synthesis of the
Key Synthon Methyl 14-Hydroxy-(all-cis)-5,8,11-tetradecatrienoate,
Tetrahedron, 56, 9195-9202
13. Petasis, N. A., Yang, R., Winkler, J. W., Zhu, M., Uddin, J., Bazan,
N. G., and Serhan, C. N. (2012) Stereocontrolled total synthesis of
Neuroprotectin D1/Protectin D1 and its aspirin- triggered
stereoisomer, Tetrahedron Lett., Elsevier Ltd 53, 1695–1698.
14. Allard, M., Barnes, K., Chen, X., Cheung, Y.-Y., Duffy, B., Heap, C.,
Inthavongsay, J., Johnson, M., Krishnamoorthy, R., Manley, C.,
Steffke, S., Varughese, D., Wang, R., Wang, Y., and Schwartz, C. E.
(2011) Total synthesis of Resolvin E1, Tetrahedron Lett., Elsevier
Ltd 52, 2623– 2626.
15. Mitra, R. B., and Reddy, G. B. (1989) Selective cleavage of
dimethylhydrazones to the carbonyl compounds using silica gel and

70

its application in the synthesis of (Z)-9-tetradecenyl acetate,
Synthesis.
16. Corey, E. J.; Fuchs, P. L. (1972) A synthetic method for formyl →
ethynyl conversion (RCHO → RC CH or RC CR ′ )
Tetrahedron Letters, 3769.
17. Lu, W.; Zheng, G. R.; Gao, D. X.; Cai, J. C. (1999) Syntheses of two
diastereoisomers of panaxytriol, a potent antitumor agent isolated
from panax ginseng, Tetrahedron, 55, 7157.
18. Winkler, J. W.; Uddin, J.; Serhan, C. N.; Petasis, N. A. (2013)
Stereocontrolled Total Synthesis of the Potent Anti-inflammatory
and Pro-resolving Lipid Mediator Resolvin D3 and Its Aspirin-
Triggered 17R-Epimer, Organic Letters, 15, 1424.
19. Tashiro, T., Nakagawa, R., Inoue, S., Shiozaki, M., Watarai, H.,
Taniguchi, M., and Mori, K. (2008) RCAI-61, the 6′-O-methylated
analog of KRN7000: its synthesis and potent bioactivity for mouse
lymphocytes to produce interferon-γ in vivo, Tetrahedron Lett.,
Elsevier Ltd 49, 6825–6828.
20. Winkler, J., Orr, S., Dalli, J., Chiang, N., Petasis, N.A., and Serhan
C.N. (2015) Resolvin D4 Potent Antiiinflammatory Proresolving
Actions Confirmed via Total Synthesis, FASEB J. 29, 285.10

71

Chapter 4. Total Synthesis of Maresin 1
4.1 Introduction
Maresin 1 (MaR1) is a potent anti-inflammatory, pro-resolving lipid
metabolite produced locally at the site of inflammation from the essential fatty
acid DHA via an enzymatic process triggered by macrophages.
1
MaR1 has been a
subject of extensive biological investigation
2-5
due to 1nM potency when
stimulating human macrophage efferocytosis, activity that exceeds that of the
resolvin D series.
6
As mentioned in Chapter 2 MaR1 also possesses tissue
regenerative properties accelerating surgical regeneration in planaria, increasing
the rate of head reappearance.
6
This high level of interest has created the demand
for synthetic stockpiles of MaR1 and acetylenic MaR1 as a precursor for use in
radiolabeled experimentation or deuterium labelled experimentation. Originally
synthesized by my colleague Min Zhu in the Petasis lab this work utilizes several
of his published synthetic steps, however by utilizing synthetic methodology
pioneered by my colleague Nikita Vlasenko as well as other synthetic advances
this work presents a shortened more efficient synthesis of MaR1 and acetylenic
MaR1.
4.2 Biosynthesis of MaR1
Maresin 1 was the first lipid mediator discovered in the macrophage-
derived pro-resolving family of compounds.
1
The biosynthesis of MaR1 (Scheme
4-1) is initiated by the production of a 13,14-epoxide intermediate from DHA by
human macrophage. This epoxide intermediate is then enzymatically hydrolyzed
via an acid-catalyzed nucleophilic attack of water at 7-position carbon,

72

introducing a hydroxyl group at that position and alkene rearrangement to form
the stereochemistry of bioactive MaR1.
7

4.3 Results and Discussion
4.3.1 Retrosynthesis of MaR1
The synthetic strategy required to produce MaR1 outlined in scheme 4-2
established the triene moiety in the final steps due to the inherent sensitivity of
Z/E isomerization. This required a mild reduction of the acetylenic precursor 4.2
to the 8E, 10E, 12Z, triene. The reduction was achieved by employing a mild
reduction utilizing a Zn/Cu/Ag catalyst.
8
The acetylenic precursor 4.2, obtained
after a deprotection of the silyl-protected intermediate 4.3, intermediate 4.3
wasproduced via a Sonogashira coupling of terminal alkyne building block 4.4
and vinyl iodide building block 4.5. Terminal alkyne building block 4.4 was
produced by my colleagues Nikita Vlasenko and Rong Yang following apreviously
COOH
DHA
Enzymatic
Epoxidation
Human Macrophage
12-LOX
COOH
14
(S)
14
(S)
7 (R) 7 (R) OH
OH
COOH
14 (S) 14 (S)
OOH
14S-HpDHA
12-LOX
COOH
(S)
13
(S)
13
(S)
14
(S)
14
O
13S, 14S-epoxy-maresin
Enzymatic
Hydrolysis
MaR1
Scheme 4-1. Biosynthesis of Maresin 1

73

reported procedure.
9
The vinyl iodide intermediate 4.5 was produced from the
commercially available starting material (S) glycidol.
4.3.2 Synthesis of MaR1 Building Block
The synthesis of vinyl iodide intermediate 4.5 was initiated with the
production of the methyl ester from commercially available 4-pentynoic acid,
Scheme 4-2. Retrosynthesis of Maresin 1

OTBDPS
OTBDPS
COOMe
OH
OH
COOH
OH
OH
COOMe
OTBDPS
COOMe
I
OTBDPS
OTBS
OTBDPS
Br
O
OH
TMS
OH
O
O
OH
+
+
Sonogashira
coupling
Selective
reduction
4.1 4.2
4.3
4.4
4.5
4.6 4.7
4.8
4.9
4.10
4.11

74

followed by addition of protected (S)-glycidol and treatment with sodium
bis(trimethylsilyl)amide and boron trifluoride diethyl etherate to produce
intermediate 4.12.
10
The secondary alcohol was then protected with a diphenyl
silyl-protecting group,
11
followed by selective deprotection of the tert-butyl silyl-
protecting group on the primary alcohol under mildly acidic conditions to yield
intermediate 4.13.
12
Selective reduction of the alkyne to the cis-alkene was
achieved by use of the Lindlar catalyst,
13
with subsequent oxidation of the
primary alcohol to produce the aldehyde intermediate 4.14.
14
A homolongation
with commercially available activated Wittig salt (triphenylphosphoranylidene)
acetaldehyde constructed intermediate 4.15, the aldehyde was converted to vinyl
iodide 4.5 using a takei olefination procedure.
15

4.3.3 Construction of MaR1
MaR1 was constructed from the coupling of building blocks 4.4 and 4.5,
coupled via an improved copper free Sonogashira producing silyl-protected MaR1
Scheme 4-3. Synthesis of MaR1 Building Block 4.5.

O
OTBS
OH
O
1) MeOH, H
2
SO
4
,
reflux 98%
TBSO
(R) (R)
OH
O
O
NaHMDS, BF
3
•Et
2
O
-78°C 36%
HO
(R) (R)
OTBDPS
O
O
i. Lindlar, quinoline,
EtOAC
ii. DMP, pyridine,
DCM, 2 steps 85%
(Formylmethylene)
triphenylphosphorane
THF, 65°C, 81%
(R) (R)
OTBDPS
COOMe
I
CrCl
2
, CH
3
I, THF,
0°C to rt 68%
(R) (R)
O
OTBDPS
COOMe
O
(R) (R)
OTBDPS
COOMe
2)
1) TBDPS-Cl,
imidazole, DMAP,
DCM, 0°C to rt 96%
2) CSA, MeOH,
DCM, rt 87%
4.5
4.6
4.12
4.13
4.14
4.15
3.8

75

acetylene 4.3 in a 93% yield.
16
Late stage intermediate 4.3 was deprotected using
TBAF followed by treatment with freshly prepared diazomethane to reinstate any
cleaved methyl ester.
17
MaR1 was prepared from the hydrogenation of the
corresponding 12-yne in a modest 60% yield via a mild Ag/Cu/Zn catalyst
produced in house.
8
Following the hydrogenation the methyl ester was
successfully cleaved via a sodium hydroxide hydrolysis.
4.4 Conclusion
In summary this synthetic strategy towards the production is concise,
convergent, and highly stereocontrolled forming MaR1 in stereochemically pure
form by using enantiomerically pure commercially available starting material.
The synthetic availability of these lipid mediators will support the further
identification of their role during inflammation and confirm their novel anti-
OTBDPS
OTBDPS
COOMe
I
OTBDPS
OTBDPS
COOMe
Butylamine,
Pd(PPh
3
)
4
, rt 93%
OH
OH
COOMe
i. TBAF, THF, rt
ii. Et
2
O, CH
2
N
2
81%
OH
OH
COOH
i.Zn(Cu/Ag), MeOH, rt
ii. NaOH, MeOH,
H
2
O, THF, rt 60%
+
4.5
4.3
4.2 4.1
4.4
Scheme 4-4. Construction of MaR1 From Building Blocks 4.4 and 4.5.

76

inflammatory and pro-resolving properties. Overall, these data offer new insights
for the biological roles of DHA.
4.5 Experimental
Unless otherwise noted, all reactions were carried out in a flame-dried
flask with stir bar under argon routed through a three-necked valve. Reactions
were carried out at room temp using DriSolv solvents purchased commercially
from VWR. All reagents used were purchased without further purification from
Sigma Aldrich, Strem, Combi-Blocks and Alfa Aesar.
Progress was monitored and recorded using EMD analytical thin layer
chromatography plates, Silica Gel 60 F254. TLC plates were visualized through
UV absorbance, (254 nm), or staining techniques including vanillin,
phosphomolybdic acid, potassium-permanganate, or ninhydrin followed by
heating. Unless otherwise stated, purification was carried out by flash column
chromatography manually using Silica Gel (100-200 mesh) or automatically
using the Biotage Isolera One.
Characterization was carried out using LC-MS, NMR and UV-VIS
instrumentation. All
1
H,
13
C and gcosy spectra were procured on the Departments
Varian 400, 500 and 600 MHz NMR instruments in the solvent indicated.
1
H and
13
C chemical shifts, (δ), are recorded in parts per million, (ppm), and referenced
to the residual solvent converted by the TMS scale (CDCl 3,
1H
= 7.26 ppm).
Splitting patterns are denoted by s, d, t, dd, td, ddd, and m and refer to the
respective multiplicities; singlet, doublet, triplet, doublet of doublets, triplet of
doublets, doublet of doublet of doublet and multiplet. Mass spectra was recorded

77

TBSO
(R) (R)
OH
O
O
4.12
on an Agilent 1260 LC-MS. UV-Vis spectra was obtained by a Hewlett- Packard
8350 instrument.
Methyl (R)-8-((tert-butyldimethylsilyl)oxy)-7-hydroxyoct-4-
ynoate (4.12) To a solution of 4-pentynoic acid (4 g, 40.8 mmol) in 40 mL of
MeOH was added 0.4 mL of sulfuric acid. The solution was stirred overnight at
60°C. The resulted mixture was evaporated to give a crude yellow oil which was
then chromatographed on silica gel using Ether-Pentanes (15%) as the eluent to
afford the methyl ester (4.12) as a colorless liquid (4.48 g, 98%)
1
H NMR (400
MHz, Chloroform-d) 3.70 (s, 3H), 2.53 (m, 2H), 2.45 (m, 2H), 1.96 (m, 1H).
13
C
NMR (101 MHz, cdcl 3) 172.34, 82.58, 69.14, 51.96, 33.29, 14.49. A solution of
protected glycidol (3.8) (706 mg, 3.75 mmols) and 4-pentynoic acid methyl ester
(300 mg, 2.68 mmols) in 10 mL of anhydrousTHF was cooled to -78°C.
Simultaneously boron trifluoride diethyl etherate (0.33 mL, 2.68 mmols) and a
1M sodium bis(trimethylsilyl)amide in THF solution (3.2 mL, 3.21 mmols) were
added to the reaction mixture. The reaction was stirred at -78°C for 4 hours,
without quenching the reaction mixture was evaporated to give a crude brown oil
which was then chromatographed on silica gel using EtOAc-hexanes (15%) as the
eluent to afford methyl (R)-8-((tert-butyldimethylsilyl)oxy)-7-hydroxyoct-4-
ynoate (4.12) as a yellow oil (290 mg, 36%).
1
H NMR (400 MHz, Chloroform-d)
3.71 (m, 1H), 3.69 (s, 3H), 3.56 (m, 1H), 3.48 (q, J = 7.0 Hz, 1H), 2.46 (m, 4H),

78

2.37 (ddd, J = 5.7, 3.1, 1.7 Hz, 2H), 0.90 (s, 9H), 0.08 (s, 6H).
13
C NMR (101 MHz,
cdcl 3) 172.65, 80.60, 77.68, 70.51, 66.01, 65.74, 51.91, 33.83, 26.02, 23.57, 18.45,
14.93, -5.25.
Methyl (R)-7-((tert-butyldiphenylsilyl)oxy)-8-hydroxyoct-4-
ynoate (4.13) To a solution of TBDPS-Cl (330 mg, 1.2 mmol), imidazole (82 mg,
1.2 mmol) and DMAP (50 mg, 0.4 mmol) dissolved in 4 mL of anhydrous DMF at
0°C was added alcohol (4.12) (240 mg, 0.8 mmol) at 0°C. The reaction was
allowed to stir overnight at room temperature. It was then quenched with
saturated aqueous NH 4Cl (125 mL) and extracted with Et2O (3 x 125 mL). The
combined extract was dried with Na 2SO 4 and evaporated to give a crude clear oil
which was then chromatographed on silica gel using EtOAc-hexanes (3%) as the
eluent to afford the silyl-phenyl protected alcohol as a viscous and yellow oil (414
mg, 96%).
1
H NMR (400 MHz, Chloroform-d) 7.70 (dddd, J = 7.4, 3.8, 1.5, 0.7
Hz, 4H), 7.32 (m, 6H), 3.81 (p, J = 5.3 Hz, 1H), 3.67 (s, 3H), 3.53 (dt, J = 5.4, 0.8
Hz, 2H), 2.38 (m, 4H), 2.27 (m, 1H), 1.06 (d, J = 0.7 Hz, 9H), 0.84 (t, J = 0.7 Hz,
9H), -0.05 (d, J = 10.5 Hz, 6H).
13
C NMR (101 MHz, cdcl 3) 172.72, 136.06, 136.00,
134.32, 129.73, 129.70, 127.65, 79.74, 78.05, 72.55, 65.69, 51.83, 33.85, 31.74,
27.07, 26.03, 24.06, 22.81, 19.51, -5.35. To a solution of protected diol (650 mg,
1.2 mmol) in a 1:1 mixture of CH2Cl2/MeOH (16 mL) was added
camphorsulfonic acid (280 mg, 1.2 mmol). The reaction was quenched after 40
HO
(R) (R)
OTBDPS
O
O
4.13

79

min with Et3N (0.25 mL, 1.8 mmol) and the solvent was removed in vacuo. The
crude mixture was purified on silica gel using EtOAc-hexanes (15%) as the eluent
to afford methyl (R)-7-((tert-butyldiphenylsilyl)oxy)-8-hydroxyoct-4-ynoate
(4.13) (443 mg, 87%) as a yellow oil.
1
H NMR (400 MHz, Chloroform-d) 7.67
(ddd, J = 8.0, 5.3, 1.5 Hz, 4H), 7.35 (m, 7H), 3.88 (ddt, J = 8.0, 4.8, 4.2 Hz, 1H),
3.66 (s, 3H), 3.62 (dd, J = 6.5, 4.2 Hz, 2H), 2.34 (m, 5H), 2.22 (m, 1H), 1.07 (s,
10H).
13
C NMR (101 MHz, cdcl 3) 172.63, 135.96, 135.82, 133.65, 133.63, 130.07,
130.03, 127.97, 127.84, 80.51, 77.10, 72.56, 65.67, 51.87, 33.70, 27.11, 23.93,
19.45, 14.85.

Methyl (R,Z)-7-((tert-butyldiphenylsilyl)oxy)-8-oxooct-4-enoate
(4.14) To a solution of alcohol (4.13) (280 mg, 0.66 mmol) in EtOAc (65 mL)
was added Lindlar catalyst (59 mg) and 5 drops of quinoline. The reaction
mixture was placed under a H2 atmosphere and stirred for 2 h. The reaction was
filtered through celite and the solvent was removed in vacuo. To a solution of the
crude alcohol (180 mg, 0.42 mmol) in 12 mL of CH 2Cl 2 was added 12 drops of
pyridine and Dess-Martin periodinane (322 mg, 0.76 mmols) the mixture was
stirred at room temperature for 20 minutes. The reaction mixture was quenched
with 1:1 mixture of saturated aqueous NaHCO 3 and saturated aqueous Na 2S 2O 3
O
(R) (R)
OTBDPS
COOMe
4.14

80

(15 mL) and extracted with Et2O (3 x 15 mL). The organic layer was dried with
MgSO4, filtered and the solvent removed in vacuo. The crude reaction mixture
was purified on silica gel using EtOAc-hexanes (11%) as the eluent to afford the
aldehyde (4.14) (152 mg, 85%) as a yellow oil.
1
H NMR (400 MHz, Chloroform-
d) 9.56 (d, J = 1.6 Hz, 1H), 7.64 (ddd, J = 8.8, 8.0, 1.5 Hz, 4H), 7.34 (m, 7H), 5.40
(m, 2H), 4.07 (ddd, J = 6.3, 5.7, 1.6 Hz, 1H), 3.65 (s, 3H), 2.33 (m, 2H), 2.21 (m,
4H), 1.11 (s, 10H).
13
C NMR (101 MHz, cdcl 3) 203.09, 177.46, 135.97, 135.97,
135.95, 135.88, 133.05, 130.22, 127.97, 127.95, 77.09, 51.63, 31.73, 31.06, 27.07,
19.48, 13.96.
Methyl (R,4Z,8E)-7-((tert-butyldiphenylsilyl)oxy)-10-oxodeca-
4,8-dienoate (4.15) To a flask with (Triphenylphosphoranylidene)
acetaldehyde (325 mg, 1.07 mmols) was cannulated aldehyde (4.14) (260mg,
0.612 mmols) in 5 mL of anhydrous THF. The mixture was refluxed at 65°C
overnight. The reaction mixture with no workup was condensed in vacuo
followed by purification of the crude mixture on silica gel using EtOAc-hexanes
(8%) as the eluent to afford the extended aldehyde (4.15) (223 mg, 81%) as a
dark red colored oil.
1
H NMR (400 MHz, Chloroform-d) 9.47 (d, J = 7.9 Hz, 1H),
7.57 (m, 5H), 7.32 (m, 10H), 6.71 (dd, J = 15.6, 4.9 Hz, 1H), 6.21 (ddd, J = 15.7,
(R) (R)
O
OTBDPS
COOMe
4.15

81

8.0, 1.5 Hz, 1H), 5.30 (m, 2H), 4.42 (m, 1H), 3.64 (s, 3H), 2.21 (m, 3H), 2.10 (m,
2H), 1.09 (s, 9H).
13
C NMR (101 MHz, cdcl 3) ? 193.64, 173.41, 158.72, 135.97,
135.94, 135.91, 133.58, 133.25, 131.22, 130.85, 130.11, 127.86, 127.83, 127.81,
124.85, 72.27, 53.56, 51.67, 35.03, 33.80, 27.13, 27.11, 22.90, 19.45.

Methyl (R,4Z,8E,10E)-7-((tert-butyldiphenylsilyl)oxy)-11-
iodoundeca-4,8,10-trienoate (4.5) To a solution of CrCl2 (290 mg, 2.4
mmol) dissolved in 5 mL of anhydrous THF was cannulated a mixture of
aldehyde (4.15) (108 mg, 0.24 mmol) and CHI3 (470 mg, 1.2 mmol) dissolved in
1.5 mL anhydrous THF under Argon at 0°C. The reaction was stirred at 0°C for 3
h and an additional 1 h at room temperature. The reaction mixture was quenched
with brine (50 mL) extracted with Et2O (3 x 50 mL) and dried over MgSO4. The
organic phase was filtered and the solvent was removed in vacuo to afford a crude
oil which was purified on silica gel using first pure pentanes and then EtOAc-
hexanes (2%) as the eluent to afford the vinyl iodide (4.5) (94 mg, 68%) as a
yellow colored oil.
1
H NMR (400 MHz, Chloroform-d) 7.58 (m, 5H), 7.31 (m, 8H),
6.86 (m, 1H), 6.17 (d, J = 14.4 Hz, 1H), 5.79 (m, 1H), 5.58 (m, 1H), 5.33 (d, J = 5.9
Hz, 1H), 4.12 (m, 1H), 3.65 (s, 3H), 2.21 (m, 3H), 2.13 (m, 2H), 1.07 (s, 9H).
13
C
(R) (R)
OTBDPS
COOMe
I
4.5

82

NMR (101 MHz, cdcl 3) 173.58, 144.77, 136.70, 136.05, 136.04, 136.01, 134.19,
133.92, 129.91, 129.81, 129.79, 129.75, 127.69, 127.66, 127.62, 126.05, 78.91,
73.16, 53.56, 33.98, 31.73, 27.15, 19.47, 14.26.
Methyl (4Z,7R,8E,10E,14S,16Z,19Z)-7,14-bis((tert-
butyldiphenylsilyl)oxy)docosa-4,8,10,16,19-pentaen-12-ynoate (4.3)
To a solution of vinyl iodide (4.5) (18 mg, 0.04 mmol) in 2 mL of butylamine was
added a catalytic amount of palladium tetrakis and the mixture was stirred for 10
minutes at room temperature. Late stage alkyne intermediate (4.4) (28 mg,
0.07) was cannulated into the reaction mixture in 1 mL of butylamine, the
reaction was allowed to stir overnight in the dark. The reaction mixture was
quenched with saturated ammonium chloride (25 mL) extracted with Et2O (3 x
15 mL) and dried over MgSO4. The organic phase was filtered and the solvent
was removed in vacuo to afford a crude oil which was purified on silica gel using
first pure pentanes and then EtOAc-hexanes (1%) as the eluent to afford silyl-
protected acetylated maresin 1 (4.3) (31 mg, 83%) as a yellow colored oil.
1
H
NMR (400 MHz, Chloroform-d) 7.64 (m, 9H), 7.32 (m, 13H), 6.29 (dd, J = 15.5, 10.9
Hz, 1H), 6.13 (ddd, J = 14.8, 10.9, 1.2 Hz, 1H), 5.71 (dd, J = 15.1, 6.0 Hz, 1H), 5.39 (m,
6H), 5.30 (m, 1H), 5.18 (m, 1H), 4.49 (td, J = 6.4, 1.8 Hz, 1H), 4.35 (td, J = 6.4, 2.1 Hz,
4.3
(S) (S)
(R) (R)
OTBDPS
OTBDPS
COOMe

83

1H), 3.67 (s, 3H), 2.63 (m, 4H), 2.40 (m, 4H), 2.02 (tdd, J = 7.5, 3.8, 1.2 Hz, 4H), 1.08
(d, J = 2.0 Hz, 16H), 0.04 (d, J = 7.8 Hz, 5H).
Methyl (4Z,7R,8E,10E,14S,16Z,19Z)-7,14-dihydroxydocosa-
4,8,10,16,19-pentaen-12-ynoate (4.2) To the silyl-protected acetylated
maresin 1 (4.3) (29 mg, 0.04 mmols) dissolved in 0.8 mL of THF was added
dropwise 4 equivalents of 1M TBAF (0.16 mL, 0.16 mmol) at 0°C. The reaction
was allowed to stir overnight at room temperature, then was quenched with
saturated NH
4
Cl (15 mL) and extracted with Et
2
O (5 x 15 mL). The organic layer
was rinsed with brine, dried over MgSO4 and filtered. The solvent was then
concentrated and freshly prepared CH
2
N
2
was added to convert any acid to the
ester-lactone mixture. The solvent was completely removed in vacuo and the
compound was purified on silica gel using MeOH-CH
2
Cl
2
(3%) as the eluent to
afford acetylated maresin 1 (4.2) (12 mg, 81%).

1
H NMR (400 MHz, Methanol-
d 4) 7.06 (ddd, J = 14.4, 10.6, 0.8 Hz, 1H), 6.41 (m, 2H), 6.14 (m, 1H), 5.61 (m,
2H), 5.26 (m, 7H), 4.43 (td, J = 6.6, 1.9 Hz, 1H), 4.09 (m, 1H), 3.65 (s, 3H), 2.73
(m, 2H), 2.42 (m, 1H), 2.37 (d, J = 2.8 Hz, 3H), 2.26 (m, 4H), 1.96 (m, 2H), 0.97
(t, J = 7.5 Hz, 2H).
13
C NMR (101 MHz, cd 3od) 175.31, 146.25, 142.52, 138.12,
133.79, 133.13, 132.85, 132.00, 130.92, 127.51, 125.53, 93.78, 84.35, 79.32, 72.37,
63.20, 52.08, 49.64, 48.36, 36.97, 35.99, 34.69, 30.75, 23.94, 21.48, 14.64.
(S) (S)
(R) (R)
OH
OH
COOMe
4.2

84

(4Z,7R,8E,10E,12Z,14S,16Z,19Z)-7,14-dihydroxydocosa-
4,8,10,12,16,19-hexaenoic acid (4.1) 400 mg Zn dust was weighed in N
2
glove box and 6 mL degassed water was added. Argon was bubbled through the
mixture for 15 min. Then 100 mg Cu(OAc)
2
was added and stirred for 20 min.
After that, 100 mg AgNO
3
was added and stirred for another 30 min. The
precipitate was rinsed by water (2 x 3 mL), MeOH (2 x 3 mL), acetone (2 x 3 mL),
and ether (2 x 3 mL) and dried under in vacuo. Weigh 250 mg powder prepared
in this way and transferred into a 25 ml flask. 1 ml of degassed water was added
in, then added in acetylene (4.2) (1.0 mg, 0.005 mmols) dissolved in 1 ml MeOH.
The suspension was stirred at room temperature for 4 h and the progress of
reaction was monitored by LCMS. Filtrate off the solids and concentrate the
solution in vacuo. The product was purified on a HPLC preparatory column using
28% water/MeOH as eluent (1.24 mg, 55%).
1
H NMR (500 MHz, Methanol-d 4)
6.48 (m, 1H), 6.20 (m, 2H), 6.08 (td, J = 11.4, 1.1 Hz, 1H), 5.71 (m, 1H), 5.34 (m,
7H), 5.26 (m, 1H), 4.53 (m, 1H), 3.65 (d, J = 0.7 Hz, 3H), 2.77 (m, 2H), 2.20 (m,
1H), 2.05 (m, 2H), 1.85 (m, 2H), 1.71 (m, 3H), 1.61 (m, 2H), 0.86 (t, J = 0.7 Hz,
3H).
13
C NMR (151 MHz, cd 3od) 182.26, 138.17, 135.04, 134.74, 132.78, 132.61,
131.29, 131.28, 130.59, 128.76, 128.17, 126.30, 126.11, 73.10, 68.48, 39.00, 32.74,
25.80, 23.69, 21.50, 14.43.
(S) (S)
(R) (R)
OH
OH
COOH
4.1

85

4.6 References
1. Serhan, C. N., Dalli, J., Colas, R. A., Winkler, J. W., and Chiang, N.
(2015) Protectins and maresins: New pro-resolving families of
mediators in acute inflammation and resolution bioactive
metabolome, BBA - Molecular and Cell Biology of Lipids, Elsevier
B.V. 1851, 397–413.
2. Dalli, J. P., Krishnamoorthy, N., and Lever, B. B. (2014) Maresin-1
Is Produced In Temporally And Spatially Distinct Compartments To
Counter-Regulate Acute Lung Injury, Am J Respir Crit .d
3. Krishnamoorthy, N., Burkett, P. R., Dalli, J., Abdulnour, R. E. E.,
Colas, R., Ramon, S., Phipps, R. P., Petasis, N. A., Kuchroo, V. K.,
Serhan, C. N., and Levy, B. D. (2015) Cutting Edge: Maresin-1
Engages Regulatory T Cells To Limit Type 2 Innate Lymphoid Cell
Activation and Promote Resolution of Lung Inflammation, J.
Immunol. 194, 863–867.
4. Abdulnour, R.-E. E., Dalli, J., Colby, J. K., Krishnamoorthy, N.,
Timmons, J. Y., Tan, S. H., Colas, R. A., Petasis, N. A., Serhan, C.
N., and Levy, B. D. (2014) Maresin 1 biosynthesis during platelet–
neutrophil interactions is organ-protective, Proc. Natl. Acad. Sci.
U.S.A. 111, 16526– 16531.
5. Serhan, C. N., Dalli, J., Karamnov, S., Choi, A., Park, C. K., Xu, Z.
Z., Ji, R. R., Zhu, M., and Petasis, N. A. (2012) Macrophage
proresolving mediator maresin 1 stimulates tissue regeneration and
controls pain, FASEB J. 26, 1755–1765.
6. Serhan, C. N., Chiang, N., Dalli, J., and Levy, B. D. (2015) Lipid
Mediators in the Resolution of Inflammation, Cold Spring Harb
Perspect Biol 7, a016311.
7. Serhan, C. N.; Yang, R.; Martinod, K.; Kasuga, K.; Pillai, P. S.;
Porter, T. F.; Oh, S. F.; Spite, M. Maresins: novel macrophage
mediators with potent antiinflammatory and proresolving actions.
J. Exper. Med. 2008, 206, 15-23.
8. Petasis, N. A. (2013) Trihydroxy polyunsaturated eicosanoid
derivatives, US Patent Office.
9. Zhu, M. (2013) Total Synthesis of Specialized Pro-resolving Lipid
Mediators and Their Analogs. Ph.D. dissertation
10. Vlasenko, N. (2016) Total Synthesis of Specialized Pro-resolving
Lipid Mediators and Their Analogs. Ph.D. dissertation
11. Allard, M., Barnes, K., Chen, X., Cheung, Y.-Y., Duffy, B., Heap, C.,
Inthavongsay, J., Johnson, M., Krishnamoorthy, R., Manley, C.,
Steffke, S., Varughese, D., Wang, R., Wang, Y., and Schwartz, C. E.
(2011) Total synthesis of Resolvin E1, Tetrahedron Lett., Elsevier
Ltd 52, 2623– 2626.
12. Ramulu, U., Ramesh, D., Reddy, S. P., Rajaram, S., and Babu, K. S.
(2014) The stereoselective total syntheses of pectinolides A, B, and
C, Tetrahedron: Asymmetry, Elsevier Ltd 25, 1409–1417.

86

13. Mitra, R. B., and Reddy, G. B. (1989) Selective cleavage of
dimethylhydrazones to the carbonyl compounds using silica gel and
its application in the synthesis of (Z)-9-tetradecenyl acetate,
Synthesis.
14. Meyer, S. D., and Schreiber, S. L. (1994) Acceleration of the Dess-
Martin oxidation by water, The Journal of Organic Chemistry.
15. Takai, K.; Nitta, K.; Utimoto, K. Simple and Selective Method for
RCHO-(E)-RCH=CHX Conversion by Means of a CHX3-CrCI2
System. Journal of the American Chemical Society 1986, 108,
7408.
16. Alami, M., Ferri, F., and Linstrumelle, G. (1993) An efficient
palladium-catalysed reaction of vinyl and aryl halides or triflates
with terminal alkynes, Tetrahedron Lett.
17. Tashiro, T., Nakagawa, R., Inoue, S., Shiozaki, M., Watarai, H.,
Taniguchi, M., and Mori, K. (2008) RCAI-61, the 6′-O-methylated
analog of KRN7000: its synthesis and potent bioactivity for mouse
lymphocytes to produce interferon-γ in vivo, Tetrahedron Lett.,
Elsevier Ltd 49, 6825–6828.

87

Chapter 5. Total Synthesis of Maresin 2, 13S, 14S-
epoxy-maresin, and Maresin sulfido-conjugates:
MCTR1-methyl ester, and MCTR2-methyl ester
5.1 Introduction
The macrophage-derived pro-resolving family of compounds has grown
significantly in the past year with the discovery or maresin 2,
1
and the maresin
conjugate tissue regenerative series of compounds.
2
Maresin 1, maresin 2 and
maresin conjugate tissue regenerative series all are biosynthesized from the 13S,
14S-epoxy-maresin enzymatic intermediate.
3
While maresin 1 has received
extensive investigation into it’s anti-inflammatory, pro-resolving and tissue
regenerative properties
4-7
the other compounds of this family require further
investigation. Preliminary results have shown that MaR2 shows the hallmarks of
a pro-resolving lipid mediator however the compound does not possess the
potency of MaR1.
1
The MCTR series has demonstrated activity as anit-
inflammatory, pro-resolving, and tissue regenerative properties these mediators
have been shown to promote repair and regeneration in planaria, mouse, and
human tissues during infection.
2
The encouraging preliminary biological
evaluation of the MCTR series created a need for further investigation and
complete chemical assignment of structure and stereochemistry. However due to
the inherently small quantities available by enzymatic production and isolation a
synthetic means of producing these compounds was necessary.

88

5.2 Biosynthesis of the Macrophage-derived Pro-resolving Family
The proposed biosynthesis of the macrophage-derived pro-resolving
family scheme 5-1 is centered on the 13S, 14S-epoxide intermediate
3
produced
from the oxidation of DHA by human macrophages, to produce the 14-peroxide
the 13S, 14S-epoxide is then produced by enzymatic epoxidation. From this
epoxide intermediate MaR1
8
or MaR2
1
is produced via enzymatic hydrolysis. The
13S, 14S-epoxide intermediate also produces the MCTR series via the glutathione
sulfur transferase enzymatic process.
2

COOH
Enzymatic
Epoxidation
Human
Macrophage
12-LOX
COOH
(S) 14 (S) 14
OOH
12-LOX
COOH
(S)
13
(S)
13
14
(S)
14
(S)
O
13S, 14S-epoxy-maresin
Enzymatic
Hydrolysis
COOH
(R) (R)
(S) (S)
OH
HO
MaR2
MaR1
DHA
16S-HpDHA
14
13
Hydrolase
Enzymatic
Hydrolysis
Soluble epoxide
hydrolase
GST
Enzyme
COOH
(R) (R)
(S) (S)
OH
S
NH
O
HOOC
NH
2
NH
O
HOOC
COOH
(R) (R)
(S) (S)
OH
S
NH
2
NH
O
HOOC
MCTR1
MCTR2
OH
OH
COOH
14
13
14
13
Scheme 5-1. Biosynthesis of the Macrophage-derived Pro-resolving Family

89

5.3 Results and Discussion
5.3.1 Retrosynthesis of MaR2
The synthetic strategy required to produce MaR2 outlined in scheme 5-2
established the triene moiety in the final steps however unlike previously
reported molecules in this thesis, the Z/E geometry is set by a cis-selective Wittig
reaction. The development of cis-selective reaction conditions was useful for the
synthesis of MaR2, but even more essential for the synthesis of the MCTR series.
As such MaR2 was targeted to establish reaction conditions for the production of
the MCTR series and the 13S, 14S-epoxy maresin. Leading to the key cis-selective
O
O
O O
O
O
O IPh
3
P
+
OH
O O
O
O
O
OH
IPh
3
P
+
O
HO
HO
OH
HO
O
OH
O
+
Wittig
coupling
Wittig
coupling
O
OH
5.1
OH
HO
O O
5.2
5.3
5.4
5.5
5.6 4.6
5.9 5.11
5.8 5.10
Scheme 5-2. Retrosynthesis of Maresin 2

90

Wittig reaction of MaR2 the aldehyde building block 5.3 is produced in precise
stereochemistry from the chiral feedstock 2-deoxy-d-ribose. The construction of
this building block also requires a cis-selective Wittig with the protected sugar
and a synthesized unactivated ylide 5.8 a similar procedure to what was used in
the production of RvD1.
9
The phosphonium salt building block 5.4 is produced
from commercially available 4-pentynoic acid and the epoxide ethylene oxide.
5.3.2 Synthesis of MaR2 building blocks
The synthesis of aldehyde building block 5.3 (scheme 5-3) started with
the hemi-acetal protection of the sugar 2-deoxy-d-ribose producing the protected
sugar 5.9.
9
The Wittig salt 5.8 was produced from cis-3-hexene-1-ol by a
displacement of the alcohol with iodine to produce an alkyl iodide,
10
followed by
the production of the phosphonium salt from the alkyl iodide.
9
Following
reaction conditions developed in the Petasis lab for the production of RvD1 the
protected sugar 5.9 was coupled with the phosphonium salt 5.8 in a cis-selective
Wittig reaction to produce intermediate 5.5.
9
The primary alcohol was then
subjected to a Dess-Martin oxidation to produce the aldehyde. A one-step Wittig
reaction afforded the E, E-diene-aldehyde where the E-selective Wittig
homologation occurred twice.
11

91

The synthesis of the phosphonium salt 5.4 (scheme 5-4) was produced
from commercially available starting material 4-pentynoic acid. The dianion of 4-
pentynoic acid was prepared by treatment with excess n-BuLi in HMPA and was
reacted with ethylene oxide, after work-up and without purification theafforded
7-hydroxy-hept-4-ynoic acid was directly subjected to an esterification to produce
the methyl ester 5.13.
12
The alkyne was then reduced to a cis-alkene 5.14 by
treatment with the Lindlar catalyst.
13
The primary alcohol was then displaced by
iodine to produce the alkyl iodide, followed by treatment with
triphenylphosphine in toluene to produce the phosphonium salt 5.4.
14

O
HO
HO
OH
5.11
O
O
O
OH
5.9
HO
5.10
IPh
3
P
5.8
1) PPh
3
, Imidazole,
I
2
, DCM, 0°C, 92%
2) PPh
3
, CH
3
CN,
reflux, 55%
NaHMDS, THF,
-78°C to rt, 63%
OH
O O
5.5
O
O O
5.12
O O
O
5.3
O
O
O
OH
5.9
(Formylmethylene)
triphenylphosphorane
Toluene, 90°C, 16%
2-methoxypropene,
p-TSA, DMF, 0°C, 78%
DMP, pyridine,
NaHCO
3
,
DCM, 85%
Scheme 5-3. Synthesis of Building Block 5.3

O
OH
1) Ethylene Oxide,
HMPA, n-BuLi, 0°C to rt
2) MeOH, H
2
SO
4
HO
O
O
22%
Quinoline,
lindlar catalyst,
EtOAc, 81%
O
O
PPh
3
, Imidazole,
I
2
, THF, 0°C, 87%
O
O IPh
3
P
I
O
O HO
PPh
3
, Toluene,
112°C, 78%
5.13
5.14
5.15
5.4
4.6
Scheme 5-4. Synthesis of Building Block 5.4

92

5.3.3 Construction of MaR2
MaR2 was constructed from the coupling of building blocks 5.4 and 5.3
(scheme 5-4), coupled via a cis-selective Wittig reaction to produce the correct
7Z, 9E, 11E geometry of the MaR2 triene moiety. The hemi-acetal was then
deprotected by treatment with p-TSA at 0°C followed by a hydrolysis to cleave the
methyl ester producing the MaR2 free acid 5.1.
15

5.3.4 Retrosynthesis of MCTR Series
The synthetic strategy for the production of the MCTR series is extremely
similar to that used for the production of MaR2. Depending on a cis-selective
Wittig to establish the appropriate Z/E geometry of the final product. The Wittig
reaction was used to produce the maresin epoxide biosynthetic intermediate 13S,
14S-epoxy maresin (5.18) coupled the phosphonium salt (5.4) and the aldehyde
O
O
O O
O
O
O IPh
3
P
+
O
OH
5.1
OH
HO
O O
5.2
5.3
5.4
KHMDS, THF, -
78°C to rt, 54%
i.p-TSA, MeOH, 0°C
ii. NaOH, MeOH,
H
2
O, THF, rt 42%
Scheme 5-5. Construction of MaR2 From Building Blocks 5.3 and 5.4.

93

building block (5.19). My colleague Nikita Vlasenko following previously a
reported synthesis produced the aldehyde building block.
14,16
From this epoxide
intermediate MCTR1 methyl ester (5.16) and MCTR2 methyl ester (5.17) can be
produced by selectively opening the epoxide at the conjugated 13-position
carbon, under basic conditions with the appropriate peptide.
5.3.5 Construction of MCTR1 and MCTR2 Methyl Esters
Having worked out the reaction conditions for the cis-selective Wittig in
the production of MaR2 the same procedure was applied to coupling
phosphonium salt (5.4) with aldehyde (5.19) to produce the 13S, 14S-epoxy
O
O
OH
S
NH
O
HOOC
NH
2
NH
O
HOOC
O
O
OH
S
NH
2
NH
O
HOOC
O
O
O
O
O IPh
3
P
+
O
O
Wittig
Coupling
HO
Br Br
TMS
OH
HO
O
O
O
OH
O
5.16
5.17
5.18
5.19
5.4
5.20
5.21 5.22 5.23
5.13
4.6 5.6
Scheme 5-6. Retrosynthesis of MCTR1 and MCTR2 Methyl Esters

94

maresin with a modest 67% yield, however 100% Z geometry selectivity. From the
epoxide intermediate the production of MCTR1 methyl ester and MCTR2 methyl
ester required a selective opening of the epoxide at the more reactive 13-position
carbon with the appropriate peptide under basic conditions.
17,18

5.4 Conclusion
In summary this synthetic strategy is a concise, convergent, and highly
stereocontrolled process for the production of MaR2, MCTR1 methyl ester, and
MCTR2 methyl ester in stereochemically pure form by using enantiomerically
pure commercially available starting material. The synthetic availability of these
lipid mediators will support the further identification of their role during
O
O
PPh
3
I O
O
KHMDS, THF,
-78°C, 67%
O
O
O
O
O
OH
S
R
5.16 = R
1
=
HN
O
COOH
NH
2
HN
O
COOH
5.17 = R
2
=
H
2
N
HN
O
COOH
Amino acid, MeOH,
Et
3
NH, H
2
O, 62%
5.18
5.4
5.19
Scheme 5-7. Construction of MCTR1 and MCTR2 Methyl Esters From Building
Blocks 5.19 and 5.4.

95

inflammation and confirm their novel anti-inflammatory, pro-resolving, and
tissue regenerative properties. Overall, these data offer new insights for the
biological roles of DHA.
5.5 Experimental
Unless otherwise noted, all reactions were carried out in a flame-dried
flask with stir bar under argon routed through a three-necked valve. Reactions
were carried out at room temp using DriSolv solvents purchased commercially
from VWR. All reagents used were purchased without further purification from
Sigma Aldrich, Strem, Combi-Blocks and Alfa Aesar.
Progress was monitored and recorded using EMD analytical thin layer
chromatography plates, Silica Gel 60 F254. TLC plates were visualized through
UV absorbance, (254 nm), or staining techniques including vanillin,
phosphomolybdic acid, potassium-permanganate, or ninhydrin followed by
heating. Unless otherwise stated, purification was carried out by flash column
chromatography manually using Silica Gel (100-200 mesh) or automatically
using the Biotage Isolera One.
Characterization was carried out using LC-MS, NMR and UV-VIS
instrumentation. All
1
H,
13
C and gcosy spectra were procured on the Departments
Varian 400, 500 and 600 MHz NMR instruments in the solvent indicated.
1
H and
13
C chemical shifts, (δ), are recorded in parts per million, (ppm), and referenced
to the residual solvent converted by the TMS scale (CDCl 3,
1H
= 7.26 ppm).
Splitting patterns are denoted by s, d, t, dd, td, ddd, and m and refer to the
respective multiplicities; singlet, doublet, triplet, doublet of doublets, triplet of

96

doublets, doublet of doublet of doublet and multiplet. Mass spectra was recorded
on an Agilent 1260 LC-MS. UV-Vis spectra was obtained by a Hewlett- Packard
8350 instrument.
(3aS,7aR)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-
6-ol (5.9) To a solution of 2-deoxy-D-ribose (4.o g, 30 mmols) in 50 mL
anhydrous DMF at 0°C was added drierite
TM
(1.0 g), and 2-methoxypropene
(4.32 g 60 mmol), followed by p-toluenesulfonic acid (20 mg). The reaction
mixture was stirred for 3 h at 0°C. Sodium carbonate (5 g) was added to the
reaction mixture, and stirred for an addition 1 h at rt. The solids were filtered
through celite and the filtrate was removed in vacuo. The crude mixture was
purified on silica gel using EtOAc-hexanes (40%) as the eluent to afford the
protected sugar (5.9) (4 g, 78%) as a colorless liquid.
1
H NMR (400 MHz,
DMSO-d 6) 4.65 (dd, J = 6.3, 4.5 Hz, 1H), 4.36 (m, 0H), 4.29 (m, 1H), 4.22 (dt, J =
8.1, 6.1 Hz, 0H), 4.09 (dt, J = 6.6, 2.6 Hz, 1H), 4.04 (td, J = 5.5, 4.6 Hz, 0H), 3.81
(dd, J = 12.4, 5.3 Hz, 0H), 3.61 (m, 1H), 3.56 (dd, J = 12.9, 2.3 Hz, 1H), 1.92 (m,
1H), 1.68 (ddd, J = 14.6, 6.3, 4.5 Hz, 1H), 1.48 (m, 0H), 1.37 (dd, J = 2.0, 0.8 Hz,
3H), 1.25 (dd, J = 1.8, 0.7 Hz, 3H).
O
O
O
OH
5.9

97

(Z)-hex-3-en-1-yltriphenylphosphonium iodide (5.8) To a solution
of alcohol (5.10) (4 g, 40 mmol) in 40 mL of anhydrous CH2Cl2 at 0°C was
added triphenylphosphine (15.7 g, 60 mmols), and imidazole (8.2 g, 120 mmols).
Then molecular iodine (15.2 g, 60 mmol) was added slowly the reaction mixture
was stirred at rt for 1 h the solvent was removed in vacuo. The crude mixture was
purified on silica gel using pure petroleum ether as the eluent to afford the alkyl
iodine (7.74 g, 92%).
1
H NMR (400 MHz, Chloroform-d) 5.49 (m, 1H), 5.32 (dtt,
J = 10.7, 7.4, 1.6 Hz, 1H), 3.63 (t, J = 6.5 Hz, 2H), 2.32 (ddddd, J = 7.3, 6.5, 5.8,
1.6, 0.8 Hz, 2H), 1.98 (m, 2H), 0.96 (t, J = 7.5 Hz, 3H).
13
C NMR (101 MHz, cdcl 3)
135.16, 124.55, 62.41, 30.80, 20.77, 14.43. A solution of alkyl iodine (13.8 g, 66
mmols) and triphenylphosphine (17.23 g, 66 mmols) in 65 mL of anhydrous
acetonitrile was refluxed overnight. The reaction mixture was cooled to rt and
triterated with cold ethyl ether. The crude product was filtered; the solid was then
purified by recrystallization from CH3CN/Et 2O to afford the Wittig salt (5.8)
(16.92 g, 55%) as clear crystals.
1
H NMR (400 MHz, Acetonitrile-d) 7.42 (m,
16H), 5.09 (m, 2H), 3.03 (m, 2H), 2.05 (m, 2H), 1.57 (m, 2H), 0.63 (t, J = 7.5 Hz,
3H).
13
C NMR (101 MHz, cd 3cn) 136.01, 135.98, 134.66, 134.57, 131.20, 131.08,
119.51, 118.65, 118.17, 23.15, 22.66, 21.07, 14.18.

IPh
3
P
5.8

98

((4S,5R)-2,2-dimethyl-5-((2Z,5Z)-octa-2,5-dien-1-yl)-1,3-
dioxolan-4-yl)methanol (5.5) The Phosphonium salt (5.8) (16.92 g, 35.8
mmols) was added to a flame dried round bottom flask and stored in a vacuum
oven overnight at 70°C. The reaction flask was allowed to cool to rt, and 30 mL of
anhydrous THF was added and the mixture was then cooled to -78°C. To the
reaction mixture was added a 1M NaHMDS solution (6.568 g, 35.8 mmols)
dropwise via cannula the reaction was then allowed to stir at 0°C for 30 min. The
reaction mixture was then cooled to -78°C and the protected sugar (5.9) (3.11 g,
17.9 mmols) was added dropwise via cannula, the reaction was allowed to warm
to rt and stir overnight. It was then quenched with saturated aqueous NH 4Cl (125
mL) and extracted with Et2O (3 x 125 mL). The combined extract was dried with
Na 2SO 4 and evaporated to give a crude brown oil which was then
chromatographed on silica gel using EtOAc-hexanes (25%) as the eluent to afford
the alcohol (5.5) (2.706 g, 63%).
1
H NMR (400 MHz, Chloroform-d) 5.36 (m,
3H), 5.25 (m, 1H), 4.14 (m, 2H), 3.65 (d, J = 6.0 Hz, 2H), 2.80 (dddd, J = 8.0,
6.3, 1.7, 0.9 Hz, 2H), 2.28 (m, 2H), 2.02 (m, 2H), 1.48 (d, J = 0.9 Hz, 3H), 1.35
(m, 3H), 0.97 (td, J = 7.5, 0.9 Hz, 3H).
13
C NMR (101 MHz, cdcl 3) 132.42, 131.04,
126.67, 124.85, 108.35, 77.91, 76.79, 61.82, 28.26, 27.53, 25.92, 25.55, 20.71,
14.38.
5.5
OH
O O

99

(4R,5R)-2,2-dimethyl-5-((2Z,5Z)-octa-2,5-dien-1-yl)-1,3-
dioxolane-4-carbaldehyde (5.12) To a solution of the alcohol (5.5) (2.7 g,
11.3 mmol) in 100 mL of CH 2Cl 2 was added 10 mL of pyridine and Dess-Martin
periodinane (3.22 g, 0.76 mmols) the mixture was stirred at room temperature
for 20 minutes. The reaction mixture was quenched with 1:1 mixture of saturated
aqueous NaHCO 3 and saturated aqueous Na 2S 2O 3 (125 mL) and extracted with
Et2O (3 x 125 mL). The organic layer was dried with MgSO4, filtered and the
solvent removed in vacuo. The crude reaction mixture was then
chromatographed on silica gel using EtOAc-hexanes (9%) as the eluent to afford
the aldehyde (5.12) (2.3 g, 85%) as a yellow colored oil.
1
H NMR (500 MHz,
Chloroform-d) 9.63 (d, J = 3.1 Hz, 1H), 5.34 (m, 3H), 5.20 (m, 1H), 4.32 (m, 1H),
4.28 (dd, J = 7.2, 3.2 Hz, 1H), 2.72 (dddd, J = 8.3, 7.1, 2.2, 1.0 Hz, 2H), 2.28 (m,
2H), 2.02 (td, J = 7.5, 1.6 Hz, 2H), 1.56 (s, 3H), 1.37 (d, J = 1.0 Hz, 3H), 0.93 (t, J
= 7.6 Hz, 3H).
13
C NMR (126 MHz, cdcl 3) 201.66, 132.37, 131.59, 126.44, 126.44,
123.93, 110.60, 81.95, 78.36, 27.93, 27.49, 25.80, 25.25, 20.60, 14.27.
(2E,4E)-5-((4S,5R)-2,2-dimethyl-5-((2Z,5Z)-octa-2,5-dien-1-yl)-
1,3-dioxolan-4-yl)penta-2,4-dienal (5.3) To a solution of
(formylmethylene)triphenylphosphorane (955 mg, 3.14 mmols) in 6 mL of
5.12
O
O O
O O
O
5.3

100

anhydrous toluene was cannulated in aldehyde (5.12) (444 mg, 1.57 mmols) in 2
mL of anhydrous toluene the reaction mixture was allowed to stir at 90°C and
monitored closely by TLC until the appearance of over homolongation
approximately 2 h. At this point the reaction mixture with no workup was
condensed in vacuo followed by purification of the crude mixture on silica gel
using EtOAc-hexanes (7%) as the eluent to afford the extended aldehyde (5.3)
(72 mg, 16%) as dark red colored oil.
1
H NMR (400 MHz, Chloroform-d) 9.56 (d,
J = 8.0 Hz, 1H), 7.10 (dd, J = 15.3, 11.0 Hz, 1H), 6.53 (dd, J = 15.1, 11.1 Hz, 1H),
6.11 (m, 2H), 5.18 (m, 4H), 4.59 (m, 1H), 4.18 (m, 1H), 2.73 (q, J = 6.6, 6.1 Hz,
2H), 2.29 (td, J = 8.0, 3.9 Hz, 1H), 2.12 (m, 1H), 2.01 (qd, J = 7.3, 2.0 Hz, 2H),
1.50 (d, J = 3.3 Hz, 3H), 1.36 (d, J = 2.4 Hz, 3H), 0.93 (t, J = 7.6 Hz, 3H).
13
C
NMR (101 MHz, cdcl 3) 193.61, 150.57, 140.18, 132.33, 131.00, 130.28, 126.63,
124.57, 108.96, 78.31, 78.11, 28.92, 28.10, 25.90, 25.51, 20.66, 14.31.
Methyl 7-hydroxyhept-4-ynoate (5.13) To a solution of 4-pentynoic
acid (5.7) (5 g, 51 mmols) in 100 mL of anhydrous HMPA cooled to 0°C was
added n-BuLi (44.9 mL, 112 mmols) slowly. Ethylene oxide solution 2.5-3.3M in
THF (25 mL) was added dropwise via cannula at 0°C the reaction was allowed to
stir to rt overnight. The reaction mixture was then cooled to 0°C, quenched with
150 mL H 2O and acidified to a pH of 6 with concentrated HCL, the solvent was
then removed in vacuo. The crude product containing HMPA was dissolved in
250 mL of MeOH, to the reaction mixture was added 0.8 mL of H 2SO 4 and the
HO
O
O
5.13

101

mixture was refluxed overnight. The reaction mixture was cooled to rt and
quenched with 500 mg of NaHCO3 and 15 mL of saturated aqueous NaHCO 3. The
solvent was removed in vacuo and saturated aqueous NaHCO 3 (50 mL) was
added to the crude reaction mixture the crude product was then extracted with
Et2O (3 x 125 mL) followed by purification of the crude mixture on silica gel
using EtOAc-hexanes (30%) as the eluent to afford the alcohol (5.13) (1.757 g,
22%) as a colorless liquid.
1
H NMR (400 MHz, Chloroform-d) 3.67 (s, 3H), 3.64
(t, J = 6.2 Hz, 2H), 2.40 (m, 4H), 2.37 (tt, J = 6.2, 2.2 Hz, 2H).
13
C NMR (101
MHz, cdcl 3) 172.76, 80.46, 77.85, 61.29, 51.90, 33.77, 23.19, 14.88.
Methyl (Z)-7-hydroxyhept-4-enoate (5.14) To a solution of alcohol
(5.13) (1.757 g, 156 mmol) in 100 mL of EtOAc was added Lindlar catalyst (900
mg) and 18 drops of quinoline. The reaction mixture was placed under a H2
atmosphere and stirred for 1 h. The reaction was filtered through celite and the
solvent was removed in vacuo followed by purification of the crude mixture on
silica gel using EtOAc-hexanes (30%) as the eluent to afford the alcohol (5.14)
(1.435 g, 81%) as a colorless liquid.
1
H NMR (400 MHz, Chloroform-d) 5.39 (m,
2H), 3.66 (d, J = 0.6 Hz, 3H), 3.64 (d, J = 6.4 Hz, 2H), 2.39 (d, J = 3.0 Hz, 3H),
2.32 (m, 2H).
13
C NMR (101 MHz, cdcl 3) 173.82, 130.90, 127.31, 62.29, 51.77,
33.96, 30.94, 22.92.
5.14
O
O HO

102

Methyl (Z)-7-iodohept-4-enoate (5.15) To a mixture of PPh 3 (1.37 g,
5.23 mmols), imidazole (712 mg, 10.5 mmols), and I 2 (1.33 g, 5.23 mmols) in 42
mL of anhydrous THF at 0°C was cannulated in alcohol (5.14) (551 mg, 3.5
mmols). The reaction was allowed to stir for 1 h at rt, the solvent was removed in
vacuo followed by purification of the crude mixture on silica gel using EtOAc-
hexanes (5%) as the eluent to afford the alkyl iodine (5.15) (816 mg, 87%) as a
yellow colored liquid.
1
H NMR (400 MHz, Chloroform-d) 5.43 (m, 1H), 5.31 (m,
1H), 3.65 (d, J = 0.5 Hz, 3H), 3.12 (t, J = 7.2 Hz, 2H), 2.59 (m, 2H), 2.29 (m, 4H).

13
C NMR (400 MHz, cdcl 3) 173.41, 130.12, 129.48, 51.68, 51.67, 33.86, 31.38,
23.02, 5.31.
(Z)-(7-methoxy-7-oxohept-3-en-1-yl)triphenylphosphonium
iodide (5.4) A solution of alkyl iodine (130 mg, 0.49 mmols) and
triphenylphosphine (127 mg, 0.49 mmols) in 1 mL of anhydrous toluene was
refluxed overnight. The reaction mixture was cooled to rt and the solvent was
removed in vacuo. The residue was dissolved in 0.5 ml MeOH and transferred to
a 15 ml plastic centrifuge tube. 5 ml of pentane was added and the mixture was
shaken violently and then centrifuged for 5 minutes. The supernatant solution
O
O I
5.15
O
O IPh
3
P
5.4

103

was taken up using a Pasteur pipet and another 5 ml of pentane was added.
Repeat this cycle 4 times until no PPh
3
can be detected in the supernatant by
TLC. Remove the solvent and the product was afforded as a very thick colorless
oil (203 mg, 78%).
1
H NMR (400 MHz, Chloroform-d) 7.72 (m, 10H), 7.63 (m,
7H), 5.54 (m, 1H), 5.21 (m, 1H), 3.56 (m, 2H), 3.54 (s, 3H), 2.33 (m, 2H), 2.24 (t,
J = 7.1 Hz, 2H), 2.04 (m, 2H).
13
C NMR (400 MHz, cdcl 3) 173.16, 135.12, 135.09,
133.54, 133.44, 130.53, 130.41, 130.18, 127.33, 127.17, 118.05, 117.20, 51.44, 33.10,
23.17, 22.43, 20.16.
Methyl (4Z,7Z,9E,11E)-12-((4R,5S)-2,2-dimethyl-5-((2Z,5Z)-
octa-2,5-dien-1-yl)-1,3-dioxolan-4-yl)dodeca-4,7,9,11-tetraenoate (5.2)
The phosphonium salt (5.4) (77 mg, 0.145 mmols) was dried under vacuum and
P2O5 in the reaction flask overnight. 0.75 mL of still dried THF was added and
the mixture was then cooled to -78°C. To the reaction mixture was added a 1M
KHMDS solution (26 mg, 0.13 mmols) was added dropwise via syringe over 10
min the reaction was then allowed to stir at 0°C for 20 min, then rt for 10 min
until the reaction became an red/orange/brown color. The reaction mixture was
then cooled to -78°C and the aldehyde (5.3) (21 mg, 0.072 mmols) was added
dropwise via syringe over 10 min, the reaction was allowed to slowly warm to rt
and stirred for 1 h. The reaction mixture with no workup was condensed in vacuo
O
O
O O
5.2

104

followed by purification of the crude mixture on silica gel using EtOAc-hexanes
(10%) as the eluent to afford the hemiacetal protected maresin 2 (5.2) (16 mg,
54%) as yellow colored oil.
1
H NMR (500 MHz, Chloroform-d) 6.44 (m, 1H), 6.17
(m, 2H), 6.05 (dt, J = 19.0, 12.9 Hz, 1H), 5.66 (ddt, J = 24.3, 15.4, 5.8 Hz, 1H),
5.34 (m, 6H), 5.24 (m, 2H), 4.58 (ddd, J = 13.9, 7.9, 5.1 Hz, 1H), 4.17 (ddd, J =
10.3, 5.9, 4.2 Hz, 1H), 3.67 (d, J = 3.1 Hz, 3H), 2.85 (m, 2H), 2.76 (t, J = 7.3 Hz,
2H), 2.35 (m, 4H), 2.32 (dd, J = 14.6, 7.2 Hz, 1H), 2.14 (m, 1H), 2.05 (p, J = 7.5
Hz, 2H), 1.50 (d, J = 3.6 Hz, 3H), 1.37 (d, J = 2.8 Hz, 3H), 0.96 (t, J = 7.5 Hz,
3H).
13
C NMR (500 MHz, cdcl 3) 173.67, 133.91, 132.25, 132.09, 130.96, 130.67,
128.99, 128.90, 128.86, 128.78, 128.42, 126.97, 125.23, 108.41, 79.26, 78.54,
51.73, 34.11, 29.01, 28.35, 26.31, 25.95, 25.72, 22.97, 20.71, 14.41.
(4Z,7Z,9E,11E,13R,14S,16Z,19Z)-13,14-dihydroxydocosa-
4,7,9,11,16,19-hexaenoic acid (5.1) To a solution of hemiacetal protected
maresin 2 (5.2) (8 mg, 0.017 mmols) in 1.5 mL of MeOH at 0°C was added p-
toluenesulfonic acid (38 mg, 0.020 mmols). The reaction mixture was stirred for
4 h at 0°C the reaction was then quenched with saturated aqueous NaHCO 3 (15
mL) and extracted with Et2O (3 x 15 mL). The product was then suspended in a
H2O- MeOH mixture (1:1, 1 mL) and 10 equivalents of NaOH (6.8 mg, 0.17

mmols) was added. After 12 h the reaction mixture was dried and purified via C-
O
OH
OH
HO
5.1

105

18 reversed Phase HPLC using H2O-MeOH mixture (30%) to afford compound
(5.1) (2.57 mg 42%) as colorless oil.
methyl (4Z,7Z,9E,11E)-12-((2S,3S)-3-((2Z,5Z)-octa-2,5-dien-1-
yl)oxiran-2-yl)dodeca-4,7,9,11-tetraenoate (5.18) The phosphonium salt
(5.4) (91mg, 0.172 mmols) was dried under vacuum and P2O5 in the reaction
flask overnight. 0.75 mL of still dried THF was added and the mixture was then
cooled to -78°C. To the reaction mixture a 1M KHMDS solution (31 mg, 0.155
mmols) was added dropwise via syringe over 10 min the reaction was then
allowed to stir at 0°C for 20 min, then rt for 10 min until the reaction became an
red/orange/brown color. The reaction mixture was then cooled to -78°C and the
aldehyde (5.3) (20 mg, 0.086 mmols) was added dropwise via syringe over 10
min, the reaction was allowed to slowly warm to rt and stirred for 1 h. The
reaction mixture was directly subjected to purification without workup or
removal of solvent. The crude reaction mixture was purified on silica gel using
NEt 3-EtOAc-hexanes (5% - 5% - 90%) as the eluent to afford the cis-selective
Wittig product (5.18) (21 mg, 67%) as yellow colored oil store in NEt 3-benzene
(1%).
1
H NMR (400 MHz, Benzene-d 6) 6.53 (dd, J = 15.0, 11.3 Hz, 1H), 6.38 (dd, J
= 15.3, 10.8 Hz, 1H), 5.95 (m, 2H), 5.27 (m, 8H), 3.34 (d, J = 0.9 Hz, 3H), 3.05
(dd, J = 7.9, 2.1 Hz, 1H), 2.88 (t, J = 7.4 Hz, 2H), 2.69 (m, 3H), 2.18 (m, 4H), 2.12
5.18
O
O
O

106

(t, J = 7.4 Hz, 2H), 1.94 (m, 2H), 0.91 (td, J = 7.6, 0.9 Hz, 3H).
13
C NMR (101
MHz, c 6d6) 172.73, 134.25, 132.36, 132.32, 131.38, 131.30, 131.09, 129.17, 128.93,
128.85, 128.81, 127.22, 124.02, 60.13, 57.73, 51.07, 33.97, 30.19, 26.52, 26.05,
23.13, 20.93, 14.45.
S-((3Z,6Z,9S,10R,11E,13E,15Z,18Z)-9-hydroxy-22-methoxy-22-
oxodocosa-3,6,11,13,15,18-hexaen-10-yl)cysteinylglycine (5.17) To a
solution of epoxide (5.18) (2 mg, 0.0056 mmols) in 0.8 mL of MeOH/H 2O/NEt 3
(1/1/1) at 0°C was added L-cys-gly (4 mg, 0.022 mmols) and the reaction mixture
was allowed to stir overnight at rt. The reaction mixture with no workup was
condensed in vacuo followed and purified via C-18 reversed Phase HPLC using
H2O-MeOH mixture (5% to 80% over 40 min) to afford MCTR 2 methyl ester
(5.17) (2.57 mg 62%) as colorless oil.
1
H NMR (400 MHz, Methanol-d 4) 6.60 (s,
1H), 6.26 (s, 2H), 6.05 (t, J = 10.9 Hz, 1H), 5.72 (s, 1H), 5.43 (d, J = 9.4 Hz, 4H),
5.37 (m, 3H), 5.29 (d, J = 8.5 Hz, 1H), 3.79 (d, J = 23.9 Hz, 3H), 3.65 (d, J = 2.0
Hz, 3H), 3.64 (d, J = 1.7 Hz, 1H), 3.48 (ddt, J = 3.3, 2.2, 1.1 Hz, 1H), 3.10 (m, 1H),
3.00 (d, J = 6.7 Hz, 2H), 2.88 (s, 1H), 2.78 (s, 3H), 2.32 (m, 6H), 2.06 (p, J = 7.5
Hz, 3H), 1.25 (m, 4H), 0.92 (m, 4H).
O
O
OH
S
NH
2
NH
O
HOOC
5.17

107

N
5
-(1-((carboxymethyl)amino)-3-
(((3Z,6Z,9S,10R,11E,13E,15Z,18Z)-9-hydroxy-22-methoxy-22-
oxodocosa-3,6,11,13,15,18-hexaen-10-yl)thio)-1-oxopropan-2-
yl)glutamine (5.16) To a solution of epoxide (5.18) (2 mg, 0.0056 mmols) in
0.8 mL of MeOH/H 2O/NEt 3 (1/1/1) at 0°C was added glutathione (7 mg, 0.022
mmols) and the reaction mixture was allowed to stir overnight at rt. The reaction
mixture with no workup was condensed in vacuo followed and purified via C-18
reversed Phase HPLC using H2O-MeOH mixture (5% to 80% over 40 min) to
afford MCTR 1 methyl ester (5.16) (2.30 mg 62%) as colorless oil.
1
H NMR (400
MHz, Methanol-d 4) 7.68 (m, 1H), 7.45 (ddt, J = 16.5, 8.7, 4.5 Hz, 1H), 6.60 (dd, J
= 15.4, 9.4 Hz, 1H), 6.21 (m, 2H), 6.04 (t, J = 11.1 Hz, 1H), 5.71 (dd, J = 14.0, 9.7
Hz, 1H), 5.32 (m, 6H), 5.23 (m, 1H), 4.49 (m, 1H), 3.74 (d, J = 12.0 Hz, 2H), 3.63
(m, 3H), 3.63 (s, 1H), 2.91 (m, 3H), 2.73 (m, 3H), 2.46 (m, 2H), 2.29 (m, 4H),
2.02 (m, 3H), 1.24 (m, 4H), 1.07 (d, J = 6.4 Hz, 1H), 0.95 (td, J = 7.4, 0.7 Hz, 3H).

5.16
O
O
OH
S
NH
O
HOOC
NH
2
NH
O
HOOC

108

5.6 References
1. Deng, B., Wang, C.-W., Arnardottir, H. H., Li, Y., Cheng, C.-Y. C.,
Dalli, J., and Serhan, C. N. (2014) Maresin Biosynthesis and
Identification of Maresin 2, a New Anti-Inflammatory and Pro-
Resolving Mediator from Human Macrophages, PLoS ONE
(Wallace, J., Ed.) 9, e102362.
2. Dalli, J., Chiang, N., and Serhan, C. N. (2014) Identification of 14-
series sulfido-conjugated mediators that promote resolution of
infection and organ protection, Proc. Natl. Acad. Sci. U.S.A.
3. Dalli, J., Zhu, M., Vlasenko, N. A., Deng, B., Haeggstrom, J. Z.,
Petasis, N. A., and Serhan, C. N. (2013) The novel 13S,14S-epoxy-
maresin is converted by human macrophages to maresin 1 (MaR1),
inhibits leukotriene A4 hydrolase (LTA4H), and shifts macrophage
phenotype, FASEB J. 27, 2573–2583.
4. Dalli, J. P., Krishnamoorthy, N., and Lever, B. B. (2014) Maresin-1
Is Produced In Temporally And Spatially Distinct Compartments To
Counter-Regulate Acute Lung Injury, Am J Respir Crit .d
5. Krishnamoorthy, N., Burkett, P. R., Dalli, J., Abdulnour, R. E. E.,
Colas, R., Ramon, S., Phipps, R. P., Petasis, N. A., Kuchroo, V. K.,
Serhan, C. N., and Levy, B. D. (2015) Cutting Edge: Maresin-1
Engages Regulatory T Cells To Limit Type 2 Innate Lymphoid Cell
Activation and Promote Resolution of Lung Inflammation, J.
Immunol. 194, 863–867.
6. Abdulnour, R.-E. E., Dalli, J., Colby, J. K., Krishnamoorthy, N.,
Timmons, J. Y., Tan, S. H., Colas, R. A., Petasis, N. A., Serhan, C.
N., and Levy, B. D. (2014) Maresin 1 biosynthesis during platelet–
neutrophil interactions is organ-protective, Proc. Natl. Acad. Sci.
U.S.A. 111, 16526– 16531.
7. Serhan, C. N., Dalli, J., Karamnov, S., Choi, A., Park, C. K., Xu, Z.
Z., Ji, R. R., Zhu, M., and Petasis, N. A. (2012) Macrophage
proresolving mediator maresin 1 stimulates tissue regeneration and
controls pain, FASEB J. 26, 1755–1765.
8. Serhan, C. N.; Yang, R.; Martinod, K.; Kasuga, K.; Pillai, P. S.;
Porter, T. F.; Oh, S. F.; Spite, M. (2008) Maresins: novel
macrophage mediators with potent anti-inflammatory and
proresolving actions. J. Exper. Med., 206, 15-23.
9. Sun, Y.-P., Oh, S. F., Uddin, J., Yang, R., Gotlinger, K., Campbell,
E., Colgan, S. P., Petasis, N. A., and Serhan, C. N. (2007) Resolvin
D1 and its aspirin-triggered 17R epimer. Stereochemical
assignments, anti-inflammatory properties, and enzymatic
inactivation., J. Biol. Chem. 282, 9323–9334.

109

10. Detterbeck, R., Guggisberg, A., and Popaj, K. (2002) (−)-(3S)-3-
(Tosylamino) butano-4- lactone, a Versatile Chiral Synthon for the
Enantioselective Synthesis of Different Types of Polyamine
Macrocycles: Determination Determination of the Absolute
Configuration of (-)-(R)-Budmunchiamine A., Helvetica Chimica
Acta. 85, 1742-1758
11. Yang, R. (2006) Total synthesis of novel anti-inflammatory lipid
mediators. Ph.D. dissertation
12. Aursnes, M., Tungen, J. E., Vik, A., Dalli, J., and Hansen, T. V.
(2014) Stereoselective synthesis of protectin D1: a potent anti-
inflammatory and proresolving lipid mediator, Org. Biomol. Chem.
12, 432–437.
13. Mitra, R. B., and Reddy, G. B. (1989) Selective cleavage of
dimethylhydrazones to the carbonyl compounds using silica gel and
its application in the synthesis of (Z)-9-tetradecenyl acetate,
Synthesis.
14. Zhu, M. (2013) Total Synthesis of Specialized Pro-resolving Lipid
Mediators and Their Analogs. Ph.D. dissertation
15. Kumar, D., Chirumarry, S., and Vijaykumar, B. (2015) Practical
Synthesis of (9S, 10R)-9, 10- epoxy-(3Z, 6Z)-henicosadiene: The
Major Pheromone of the Saltmarsh Caterpillar moth Estigmene
acrea, Bulletin of the Korean Chemical Society. 36, 1245-1249
16. Vlasenko, N. (2016) Total Synthesis of Specialized Pro-resolving
Lipid Mediators and Their Analogs. Ph.D. dissertation
17. Corey, E. J., Clark, D. A., Marfat, A., and Goto, G. (1980) Total
synthesis of slow reacting substances (SRS).“Leukotriene C-2”(11-
trans-leukotriene C)(3) and leukotriene D (4), Tetrahedron Lett. 21,
3143-3146
18. Corey, A. E., Clark, D. A., Goto, G., Marfat, A., Mioskowski, C.,
Samuelsson, B., and Hammarstroem, S. (1980) Stereospecific total
synthesis of a“ slow reacting substance” of anaphylaxis, leukotriene
C-1, J. Am. Chem. Soc., ACS Publications 102, 1436–1439.

110

Chapter 6. Total Synthesis of 16S-17S-epoxy-
neuroprotectin D1/protectin D1
6.1 Introduction
The DHA derived docosatriene lipid mediator named neuroprotectin D1
(NPD1) due to its potent actions in protecting the retina and the brain during
oxidative stress.
1
However, the same compound NPD1 is also found in non-
neuronal tissues and has been identified to possess a broad range of anti-
inflammatory and pro-resolving activities being termed protectin D1 (PD1).
1-3
The
synthesis,
1,2
biosynthetic pathway
2-4
and biological activity
2-10
of NPD1/PD1 has
been extensively investigated. Although the biosynthetic pathways towards the
production of NPD1/PD1 is widely accepted to proceed threw a 16S, 17S-epoxy-
protectin intermediate to this point there has been no molecular evidence of this
neither compound nor stereochemical assignment of this intermediate. By the
use of a convergent, highly stereocontrolled synthetic strategy 16R, 17R-epoxy-
protectin synthetic material was achieved.
6.2 Biosynthesis of the Protectin Family of Lipid Mediators
The proposed biosynthesis of NPD1/PD1 represented in scheme 6-1
initiates with the production of the peroxide at the 17-position carbon (17S-
HpDHA) from DHA via the 15-lipoxygenase enzyme. The 16S, 17S-epoxy
protectin intermediate is then produced from this epoxide intermediate
NPD1/PD1 can be enzymatically produced or via the glutathione sulfur
transferase enzymatic pathway the newly identified PCTR series can be
produced.
4,11

111

6.3 Results and Discussion
6.3.1 Retrosynthesis of 16S, 17S-epoxy Protectin
The synthesis of the 16S, 17S-epoxy-protectin intermediate required the
production of the appropriate Z/E aliphatic geometry without the use of a late
stage selective hydrogenation step present in the synthetic strategy for the
synthetic production of many of these lipid mediators.
1,2,12-14
Due to the inherent
instability of the conjugated epoxide a synthetic strategy requiring a late stage
hydrogenation was not an option. Thus, the synthetic strategy (scheme 6-2)
established the Z/E geometry of the 16S, 17S-epoxy-protectin via a cis-selective
Wittig coupling reaction between aldehyde building block (6.2) and
phosphonium salt building block (6.3). The aldehyde building block (6.2) was
synthesized by my colleague Nikita Vlasenko as reported in his dissertation.
15

COOH COOH
(R) (R)
H(O)O
O
2
O
2
LOX LOX
DHA 17R-H(p)DHA
17
(S) (S)
(S) (S)
COOH
O
(R)
17
(R)
17
OH
10
(R)
10
(R)
OH
COOH
AT-NPD1/PD1
enzymatic
hydrolysis
PCTR epoxide
S
NH
O
HOOC
NH
2
NH
O
HOOC
PCTR1
(R) (R)
(S) (S)
OH COOH
S
NH
2
NH
O
HOOC
PCTR2
(R) (R)
(S) (S)
OH COOH
S
HOOC
NH
2
(R) (R)
(S) (S)
OH COOH
PCTR3
17
16
17
16
17
16
17
16
Scheme 6-1. Biosynthesis of the Protectin Family of Lipid Mediators

112

6.3.2 Synthesis of 16S, 17S-epoxy Protectin Building Block
The synthesis of phosphonium salt building block (6.3) (scheme 6-2)
used commercially available 4-pentynoic acid as the starting material. In a two
step-one-pot reaction the propargyl alcohol moiety and methyl ester of
intermediate (6.11) were established.
16
The propargyl alcohol was displaced by
bromide to produce the propargyl bromide via the appeal reaction yielding
intermediate (6.8).
17
The propargyl bromide intermediate (6.8) was then
coupled with but-3-yn-1-ol in copper catalysed conditions to produce the bis-
acetylenic alcohol (6.5).
12
The bis-acetylene was then selectively reduced to the
4Z, 7Z alkenes using Lindlars catalyst.
18
Following the selective reduction the
primary alcohol was the displaced by iodine to produce the alkyl iodide precursor
O
O
IPh
3
P
OH
O
O
O
O
O
O
O
OH
Br
O
O
HO
O
O
Br
OH
OH
O
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
6.10
4.6
Scheme 6-2. Retrosynthesis of 16S, 17S-epoxy Protectin

113

to the phosphonium salt intermediate (6.13).
19
The phosphonium salt building
block is then produced by addition of triphenylphosphine and reflux in toluene
(6.3).
18

6.3.3 Construction of 16S, 17S-epoxy Protectin
The final construction of the 16S, 17S-epoxy protectin utilized a cis-
selective Wittig reaction to couple building blocks (6.2) and (6.3) and establish
the Z/E geometry of the compound.
OH
O
O
O
HO
O
O
Br
O
O
HO
O
O
HO
O
O
I
O
O
IPh
3
P
OH
i) Ethylmagnesium
bromide,
paraformaldehyde,
THF, reflux
ii) H
2
SO
4
, MeOH,
reflux, 38%
PPh
3
, CBr
4
, DCM,
0°C to rt, 73%
NaI, CuI, K
2
CO
3
,
DMF, -20°C to rt,
98%
Lindlar catalyst,
quinoline,
EtOAc, rt, 81%
Iodine, Imidazole,
PPh
3
, Et
2
O/ACN,
0°C to rt, 84%
PPh
3
, Toluene,
reflux, 65%
4.6 6.11 6.8
6.7
6.5
6.12
6.13
6.3
Scheme 6-3. Synthesis of Building Block 6.3

O
O
IPh
3
P
KHMDS, THF,
-78°C to rt
88%
+
O
O
O
O
O
6.1
6.2 6.3
Scheme 6-4. Construction of the 16S, 17S-epoxy Protectin From Building
Blocks 6.2 and 6.3

114

6.4 Conclusion
In summary this synthetic strategy towards the production of the 16S, 17S-
epoxy protectin biosynthetic intermediate is concise, convergent, and highly
stereocontrolled forming the lipid metabolite in stereochemically pure form. The
synthetic availability of this compound will support the further identification of
it’s role during inflammation and confirm the novel anti-inflammatory, pro-
resolving, and tissue regenerative properties of this lipid metabolite. Overall,
these data offer new insights for the biological roles of DHA.
6.5 Experimental
Unless otherwise noted, all reactions were carried out in a flame-dried
flask with stir bar under argon routed through a three-necked valve. Reactions
were carried out at room temp using DriSolv solvents purchased commercially
from VWR. All reagents used were purchased without further purification from
Sigma Aldrich, Strem, Combi-Blocks and Alfa Aesar.
Progress was monitored and recorded using EMD analytical thin layer
chromatography plates, Silica Gel 60 F254. TLC plates were visualized through
UV absorbance, (254 nm), or staining techniques including vanillin,
phosphomolybdic acid, potassium-permanganate, or ninhydrin followed by
heating. Unless otherwise stated, purification was carried out by flash column
chromatography manually using Silica Gel (100-200 mesh) or automatically
using the Biotage Isolera One.
Characterization was carried out using LC-MS, NMR and UV-VIS
instrumentation. All
1
H,
13
C and gcosy spectra were procured on the Departments

115

Varian 400, 500 and 600 MHz NMR instruments in the solvent indicated.
1
H and
13
C chemical shifts, (δ), are recorded in parts per million, (ppm), and referenced
to the residual solvent converted by the TMS scale (CDCl 3,
1H
= 7.26 ppm).
Splitting patterns are denoted by s, d, t, dd, td, ddd, and m and refer to the
respective multiplicities; singlet, doublet, triplet, doublet of doublets, triplet of
doublets, doublet of doublet of doublet and multiplet. Mass spectra was recorded
on an Agilent 1260 LC-MS. UV-Vis spectra was obtained by a Hewlett- Packard
8350 instrument.
Methyl 6-hydroxyhex-4-ynoate (6.11) To a solution of 4-pentynoic
acid (4.6) (3.83 g, 39 mmols) in 19 mL of anhydrous THF at 0°C was added 1M
ethylmagnesium bromide in THF (10.4 g, 78 mmols) and the reaction was stirred
for 1 h. Paraformaldehyde (2.4 g) was added and the reaction mixture was
refluxed for 4 h. The reaction mixture was then cooled to 0°C, quenched with 50
mL 1M HCl the crude product was then extracted with EtOAC (3 x 50 mL), the
solvent was then removed in vacuo. The crude product was dissolved in 200 mL
of MeOH, to the reaction mixture was added 0.8 mL of H 2SO 4 and the mixture
was refluxed overnight. The reaction mixture was cooled to rt and quenched with
500 mg of NaHCO3 and 15 mL of saturated aqueous NaHCO 3. The solvent was
removed in vacuo and saturated aqueous NaHCO 3 (50 mL) was added to the
crude reaction mixture the crude product was then extracted with Et2O (3 x 125
6.11
O
O
HO

116

mL) followed by purification of the crude mixture on silica gel using EtOAc-
hexanes (32%) as the eluent to afford the alcohol (6.11) (2.11 g, 38%) as a
colorless oil.
1
H NMR (500 MHz, Chloroform-d) 4.07 (m, 2H), 3.63 (s, 3H), 2.47
(q, J = 2.0 Hz, 4H).
13
C NMR (500 MHz, cdcl 3) 172.59, 84.17, 79.32, 51.95, 51.21,
33.31, 14.74.
Methyl 6-bromohex-4-ynoate (6.8) To a solution of alcohol (6.11)
(2.11 g, 15 mmols) in 40 mL of anhydrous DCM at 0°C was added
tripenylphosphine (4.67 g, 18 mmols) and CBr 4 (5.91 g, 18 mmols) the reaction
was allowed to warm to rt and stir 1 h. It was then quenched with saturated
aqueous NH 4Cl (125 mL) and extracted with Et2O (3 x 125 mL). The combined
extract was dried with Na 2SO 4 and evaporated to give crude brown oil which was
then chromatographed on silica gel using EtOAc-hexanes (10%) as the eluent to
afford the propargyl bromide (6.8) (3.83 g, 73%).
1
H NMR (400 MHz,
Chloroform-d) 3.78 (m, 2H), 3.62 (s, 3H), 2.40 (m, 4H).
13
C NMR (400MHz,
cdcl 3) 171.99, 85.72, 76.00, 51.75, 32.86, 15.15, 14.79.
Methyl 10-hydroxydeca-4,7-diynoate (6.5) To a flame dried flask
with CuI (4.10 g, 22 mmols), NaI (3.24 g, 22 mmols), and K
2
CO
3
(3 g, 22 mmols)
in 35 mL of anhydrous DMF was cannulated alkyne (6.7) (1.53 g, 22 mmols) and
O
O
Br
6.8
6.5
O
O
HO

117

allylic bromide (6.8) (2 g, 10 mmol). The reaction was stirred for 18 h and
quenched with saturated NH
4
Cl (125 mL). The mixture was extracted with Et
2
O
(3 x 125 mL), rinsed with water to remove any DMF, and the organic layer was
dried with MgSO4, filtered and the solvent removed in vacuo. The crude reaction
mixture was purified on silica gel using EtOAc- hexanes (40%) as the eluent to
afford compound (6.5) (2.182 g, 98%) as red colorled oil.
1
H NMR (400 MHz,
Chloroform-d) 3.48 (m, 5H), 3.03 (dq, J = 4.6, 2.6 Hz, 2H), 2.26 (m, 6H).
13
C
NMR (400 MHz, cdcl 3) 172.47, 78.47, 75.08, 60.95, 51.72, 33.28, 22.93, 14.54,
9.63.
Methyl (4Z,7Z)-10-hydroxydeca-4,7-dienoate (6.12) To a solution
of alcohol (6.5) (500 mg, 2.57 mmol) in 7 mL of EtOAc was added Lindlar
catalyst (140 mg) and 1 drop of quinoline. The reaction mixture was placed under
a H2 atmosphere and stirred. The reaction mixture was closely monitored by TLC
until complete conversion of the starting material and single hydrogenated
intermediate, approximately 5 h. The reaction was filtered through celite and the
solvent was removed in vacuo followed by purification of the crude mixture on
silica gel using EtOAc-hexanes (30%) as the eluent to afford the alcohol (6.12)
(4.13 mg, 81%) as red colored oil.
1
H NMR (400 MHz, Chloroform-d) 5.29 (m,
4H), 3.63 (d, J = 0.9 Hz, 3H), 3.57 (m, 2H), 2.80 (dddt, J = 7.1, 6.2, 1.6, 0.8 Hz,
O
O
HO
6.12

118

2H), 2.28 (m, 4H).
13
C NMR (400 MHz, cdcl 3) 173.74, 130.60, 129.29, 127.93,
125.99, 62.13, 51.63, 34.01, 30.94, 25.76, 22.85.
Methyl (4Z,7Z)-10-iododeca-4,7-dienoate (6.13) To a solution of
triphenylphosphine (819 mg, 3.125 mmols), and imidazole (213 mg, 3.125
mmols) in 6 mL anhydrous Et 2O and 2 mL anhydrous CH 3CN at 0°C was added
I 2 (794 mg, 3.125 mmols) in 3 portions. The reaction mixture was then allowed to
stir at room temperature for 20 min. The reaction mixture was cooled to 0°C and
the alcohol (6.12) was added via cannula in 2 mL anhydrous Et 2O and 1 mL
anhydrous CH 3CN. The reaction was stirred for 1 h at rt and quenched with
saturated NH
4
Cl (125 mL). The mixture was extracted with Et
2
O (3 x 125 mL),
and the organic layer was dried with MgSO4, filtered and the solvent removed in
vacuo. The crude reaction mixture was purified on silica gel using ether-pentanes
(20%) as the eluent to afford compound (6.13) (647 mg, 84%) as yellow colorled
oil.
1
H NMR (400 MHz, Chloroform-d) 5.21 (m, 4H), 3.59 (s, 3H), 3.07 (t, J = 7.3
Hz, 2H), 2.73 (ddd, J = 7.5, 5.7, 1.8 Hz, 2H), 2.54 (m, 2H), 2.25 (m, 4H).
13
C NMR
(400 MHz, cdcl 3) 173.20, 130.12, 128.72, 128.28, 128.07, 51.46, 33.83, 31.34,
25.68, 22.72, 5.18.

O
O
I
6.13

119

((3Z,6Z)-10-methoxy-10-oxodeca-3,6-dien-
1yl)triphenylphosphonium iodide (6.3) A solution of alkyl iodine (6.13)
(157 mg, 0.51 mmols) and triphenylphosphine (134 mg, 0.51 mmols) in 1 mL of
anhydrous toluene was refluxed overnight. The reaction mixture was cooled to rt
and the solvent was removed in vacuo. The residue was dissolved in 0.5 ml
MeOH and transferred to a 15 ml plastic centrifuge tube. 5 ml of pentane was
added and the mixture was shaken violently and then centrifuged for 5 minutes.
The supernatant solution was taken up using a Pasteur pipet and another 5 ml of
pentane was added. Repeat this cycle 4 times until no PPh
3
can be detected in the
supernatant by TLC. Remove the solvent and the product was afforded as a very
thick colorless oil (189 mg, 65%).
1
H NMR (400 MHz, Methanol-d 4) 7.71 (m,
15H), 5.44 (dd, J = 2.9, 1.0 Hz, 1H), 5.24 (m, 2H), 3.57 (s, 3H), 2.63 (m, 2H), 2.38
(m, 3H), 2.24 (m, 3H), 2.15 (m, 2H).
13
C NMR (400 MHz, cd 3od) 175.18, 136.37,
136.34, 136.31, 136.28, 136.26, 136.23, 134.94, 134.92, 134.87, 134.84, 134.82,
134.77, 134.49, 134.38, 131.82, 131.80, 131.63, 131.63, 131.59, 131.51, 131.50,
131.46, 131.37, 129.45, 129.43, 127.52, 127.35, 120.20, 119.35, 54.82, 52.05, 49.85,
34.55, 26.49, 23.78.
O
O
IPh
3
P
6.3

120

Methyl (4Z,7Z,10Z,12E,14E)-15-((2R,3R)-3-((Z)-pent-2-en-1-
yl)oxiran-2-yl)pentadeca-4,7,10,12,14-pentaenoate (6.1) The
phosphonium salt (6.3) (178mg, 0.312 mmols) was dried under vacuum and
P2O5 in the reaction flask overnight. 1.5 mL of still dried THF was added and the
mixture was then cooled to -78°C. To the reaction mixture a 1M KHMDS solution
(58 mg, 0.288 mmols) was added dropwise via syringe over 10 min the reaction
was then allowed to stir at 0°C for 20 min, then rt for 10 min until the reaction
became an red/orange/brown color. The reaction mixture was then cooled to -
78°C and the aldehyde (6.2) (30 mg, 0.16 mmols) was added dropwise via
syringe over 10 min, the reaction was allowed to slowly warm to rt and stirred for
1 h. The reaction mixture was directly subjected to purification without workup or
removal of solvent. The crude reaction mixture was purified on silica gel using
NEt 3-EtOAc-hexanes (5% - 5% - 90%) as the eluent to afford the cis-selective
Wittig product (6.1) (50 mg, 88%) as yellow colored oil store in NEt 3-benzene
(1%).
1
H NMR (400 MHz, Benzene-d 6) 6.38 (m, 2H), 6.06 (m, 2H), 5.34 (m, 8H),
3.41 (m, 3H), 3.14 (tt, J = 7.7, 2.6 Hz, 1H), 3.01 (ddd, J = 7.3, 5.4, 1.7 Hz, 1H),
2.89 (dddt, J = 7.3, 4.1, 1.7, 0.8 Hz, 1H), 2.80 (dtt, J = 9.5, 4.3, 1.7 Hz, 1H), 2.34
(m, 1H), 2.11 (m, 5H), 1.95 (m, 2H), 1.22 (m, 2H), 0.98 (t, J = 7.5 Hz, 3H).
13
C
NMR (400 MHz, c 6d6) 172.76, 134.67, 134.18, 132.38, 131.42, 131.09, 129.33,
O
O
O
6.1

121

129.16, 128.95, 128.83, 128.54, 127.92, 123.22, 60.26, 57.75, 51.08, 34.02, 30.11,
26.62, 25.99, 23.17, 21.01, 14.41.
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docosahexaenoic acid-derived products: Analysis via
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122

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and Pro-resolving Lipid Mediator Resolvin D3 and Its Aspirin-
Triggered 17 R-Epimer, Org. Lett. 15, 1424–1427.
13. Serhan, C. N., Dalli, J., Karamnov, S., Choi, A., Park, C. K., Xu, Z.
Z., Ji, R. R., Zhu, M., and Petasis, N. A. (2012) Macrophage
proresolving mediator maresin 1 stimulates tissue regeneration and
controls pain, FASEB J. 26, 1755–1765.
14. Sun, Y.-P., Oh, S. F., Uddin, J., Yang, R., Gotlinger, K., Campbell,
E., Colgan, S. P., Petasis, N. A., and Serhan, C. N. (2007) Resolvin
D1 and its aspirin-triggered 17R epimer. Stereochemical
assignments, anti-inflammatory properties, and enzymatic
inactivation., J. Biol. Chem. 282, 9323–9334.
15. Vlasenko, N. (2016) Total Synthesis of Specialized Pro-resolving
Lipid Mediators and Their Analogs. Ph.D. dissertation
16. Aursnes, M., Tungen, J. E., Vik, A., Dalli, J., and Hansen, T. V.
(2014) Stereoselective synthesis of protectin D1: a potent anti-
inflammatory and proresolving lipid mediator, Org. Biomol. Chem.
12, 432–437.
17. Dasse, O., Mahadevan, A., Han, L., Martin, B. R., and Di Marzo, V.
(2000) The Synthesis of N-Vanillyl-arachidonoyl-amide (Arvanil)
and its Analogs: An Improved Procedure for the Synthesis of the
Key Synthon Methyl 14-Hydroxy-(all-cis)-5,8,11-tetradecatrienoate,
Tetrahedron, 56, 9195-9202
18. Zhu, M. (2013) Total Synthesis of Specialized Pro-resolving Lipid
Mediators and Their Analogs. Ph.D. dissertation
19. Detterbeck, R., Guggisberg, A., and Popaj, K. (2002) (−)-(3S)-3-
(Tosylamino) butano-4- lactone, a Versatile Chiral Synthon for the
Enantioselective Synthesis of Different Types of Polyamine
Macrocycles: Determination Determination of the Absolute
Configuration of (-)-(R)-Budmunchiamine A., Helvetica Chimica
Acta. 85, 1742-1758

123

Chapter 7. Total Synthesis of Benzo-SPM
Analogues
7.1 Introduction
Inflammation resolution has been established as a biosynthetically active
process,
1
regulated by a variety of biochemical mediators and a complex network
of receptor signaling pathways and directed by specialized pro-resolving
mediators (SPMs).
2
These SPM molecules are produced locally at the site of
inflammation from essential fatty acids including arachidonic acid,
eicosapentaenoic acid, and docosahexaenoic acid and often possess nano-molar
or pico-molar potency.
2
Each of these lipid metabolites is characterized by
distinct positioning of carbon-carbon double bonds with specific Z/E geometry,
and contains substituted hydroxyl groups with specific R/S configurations.
In addition to these pro-resolving SPM compounds the inflammatory
response is initiated by pro-inflammatory lipid metabolites including
leukotrienes and prostaglandins.
2
Previously the typical approach towards the
development of anti-inflammatory therapeutics has been aimed at inhibiting the
production of these pro-inflammatory mediators.
3,4
As an alternative to this
therapeutic approach we have investigated the use of novel probe molecules
based on the structures of anti-inflammatory and pro-resolving SPMs lipoxin A4
(LXA 4) and resolvin D1 (RvD1). The selection of LXA 4 and RvD1 as compounds to
mimic was due to a number of reasons. The first being both of these molecules
have been extensively investigated in inflammation models and demonstrated
extremely potent activity.
2,5-13
In addition to their activity each has been

124

identified as potent agonists of several distinct receptors allowing for specific
biological characterization of the produced analogs.
14-16
In addition to the
appealing biological characteristics of RvD1 and LXA 4 both of these compounds
contain a centralized triene moiety possessing a E-Z-E geometry that can be
substituted with a phenyl ring to produce an analogue with enhanced stability
while changing the geometric shape little.
Each of these analogs takes advantage of similar design features replacing
the triene moieties of RvD1 and LXA 4 with a phenyl ring, and inverting the
stereochemistry of the third hydroxyl group from (S) to (R). Both of these
structural features are aimed at increasing the metabolic stability of these
compounds. In addition to those structural pieces each of these compounds were
designed and synthesized using the same synthetic strategies, a convergent
synthesis utilizing a series of coupling reactions to produce a library (figure 7-1)
of unique analogs in a completely stereocontrolled manner.

125

Figure 7-1. LXA 4, RvD1 and the Library of Benzo-SPM Analogs
HO
OH
COOMe
HO
OH
COOMe
HO
OH COOMe
OH
HO
HO
HO
OH
COOMe
HO
F
OH
OH
OH
COOH
HO
OH
COOMe
OH
HO
OH
COOMe
HO
HO
OH
COOMe
OH
HO
OH
COOMe
N
N
N
HO
OH
OH
COOMe
N
N
N
HO
HO
OH
COOH
N
N
N
HO
OH
HO
COOH
HO
COOH
OH
HO OH
RvD1
LXA
4
7.46
7.47
7.48
7.54
7.55
7.49
7.58
7.57
7.29
7.63
7.64

126

7.2 Results and Discussion
7.2.1 Retrosynthesis of Benzo-lipoxin A4 Analogs
The synthesis of a library of benzo-lipoxin A 4 methyl ester analogs requires
a seires of palladium-mediated couplings pairing the two-alkenyl substituents to
a phenyl ring centerpiece in a convergent manner (scheme 7-1). Building blocks
with precise stereochemistry are produced from commercially available chiral
feedstock to ensure exact stereochemistry of the analog library. These synthetic
building blocks can then be coupled with different commercially available phenyl
rings possessing the necessary Suzuki coupling partner at varying positions along
the ring. The result is a set of benzo-lipoxin A 4 methyl esters that probe the
substitution pattern along the phenyl ring.
(R) (R)
(S) (S)
HO
OH
COOMe
(R) (R)
HO
(R) (R)
(S) (S)
TBSO
OTBS
COOMe
Br
O
B
O
(R) (R)
OTBS
(R) (R)
OH
O
HB
O
(R) (R)
(S) (S)
TBSO
OTBS
COOMe
I
(HO)
2
B
Br
O
(R) (R)
(S) (S)
OH
OH
HO
Ph
3
P
O
O
+
+
+
Hydroboration
Suzuki
coupling
Suzuki
coupling
7.1
7.3
7.4
7.2
7.5
7.6
7.7
7.8
+
5.11
Scheme 7-1. Retrosynthesis of Benzo-lipoxin A 4 Analogs

127

7.2.2 Synthesis of Building Blocks for Benzo-lipoxin A4 Analogs
The vinyl iodide building block (7.7) was synthesized from the chiral
feedstock 2-deoxy-d-ribose in a series of synthetic steps outlined in scheme 7-2.
The sugar was opened by a Wittig reaction using the commercially available
activated ylide methyl (triphenylphosphoranylidene)acetate to produce the triol
intermediate (7.9).
17,18
The triol is then protected with a tert-butyl-silyl
protecting group
19
, followed by a palladium catalyzed hydrogenation to reduce
the aliphatic bond produced during the Wittig coupling yielding intermediate
(7.10). The primary alcohol can then be selectively deprotected,
20
and oxidized
by treatment with Dess-Martin periodinane
21
to give the aldehyde intermediate
(7.11). The aldehyde is then transformed to the vinyl aldehyde via the Takai
olefination to produce building block (7.7).
22

Methyl
(triphenylphosphoran
ylidene)acetate,
THF, reflux, 92%
HO
HO
OH
1) Pd/C, H
2
, EtOAC
89%
2) TBS-Cl, imidazole,
DMAP, CH
2
Cl
2
O
TBSO
OTBS
1) CSA, Et
3
N,
MeOH/CH
2
Cl
2
, 0°C
2) DMP, pyridine
CH
2
Cl
2
62%
TBSO
OTBS
I
CrCl
2
, CHI
3
,THF
0°C to rt, 55%
7.9
7.10
7.11
O
OH
OH
HO
COOMe
TBSO
TBSO
OTBS
COOMe
COOMe
COOMe
5.11
7.7
Scheme 7-2. Synthesis of Building Block 7.7

128

The boronic ester intermediate is produced from commercially available
(R)-oct-1-yn-ol in a series of synthetic steps outlined in scheme 7-3. Initially the
hydroxyl group of (R)-oct-1-yn-ol is silyl-protected to produce intermediate
(7.12).
19
The terminal alkyne then undergoes a hydroboration with
catecholborane yielding the boronic ester intermediate (7.2) without the need for
purification.
23

7.2.3 Construction of Benzo-lipoxin A4 Analogs
The final construction of benzo-lipoxin A 4 methyl ester analogs requires a
series of palladium mediated Suzuki coupling reactions between commercially
available phenyl ring building blocks and synthetic building blocks 7.2 and 7.7
(scheme 7-4). The vinyl iodide intermediate 7.7 was coupled with the boronic
acid moiety of commercially available bromophenylboronic acids to produce
intermediates 7.3 containing aryl bromides at varying substitution positions
(compounds 7.38, 7.39, 7.40 and 7.41).
3
The intermediates then go threw a
second consecutive Suzuki coupling between the aryl bromide, and the boronic
acid that is produced from intermediate 7.2 in the presence of water to produce
silyl-protected benzo-lipoxin A 4 methyl ester analog intermediates (7.13)
(compounds 7.42, 7.43, 7.44 and 7.45).
3
The silyl groups are then deprotected
O
B
O
(R) (R)
OTBS
OH
O
HB
O
7.4
7.2
7.5
TBS-Cl, DMAP,
Imidazole,
CH
2
Cl
2
, 92%
OTBS
7.12
70°C
Scheme 7-3. Synthesis of Building Block 7.2

129

in the presence of TBAF and any cleaved methyl ester is reinstalled by treatment
with freshly prepared diazomethane.
24
The final products are then purified via
reverse phase HPLC in a mobile phase of 38% methanol, 62% water to yield
protected benzo-lipoxin A 4 methyl ester analogs 7.46, 7.47, 7.48 and 7.49,
represented in figure 7-1.
7.2.4 Retrosynthesis of Benzo-RvD1 Analogs
The synthesis of a library of benzo-RvD1 methyl ester analogs requires a
seires of palladium-mediated couplings pairing the two-alkenyl substituents to a
phenyl ring centerpiece in a convergent manner (scheme 7-5). Building blocks
with precise stereochemistry are produced from commercially available chiral
feedstock to ensure exact stereochemistry of the analog library. These synthetic
(R) (R)
(S) (S)
TBSO
OTBS
COOMe
I
7.7
PdP(Ph
3
)
4
,
K
2
CO
3
, DMF,
65°C, 38%
O
B
O
OTBS
1) TBAF, THF
2) CH
2
N
2
, Et
2
O
(R) (R)
(S) (S)
TBSO
OTBS
COOMe
Br 7.3
HO
OH
COOMe
HO
7.1
TBSO
OTBS
COOMe
TBSO
7.13
(HO)
2
B
Br 7.6
7.2
65%
PdP(Ph
3
)
4
,
K
2
CO
3
, H
2
O,
Dioxane, 80°C,
95%
Scheme 7-4. Construction of Benzo-lipoxin A 4 Analogs from Building Blocks 7.2
and 7.7

130

building blocks were coupled with different commercially available phenyl rings
possessing the necessary Suzuki coupling partners at varying positions along the
ring. The result is a set of benzo-RvD1 methyl ester analogs that probe the
substitution pattern along the phenyl ring as well as the use of an RvD1 diol
mimic moiety.
7.2.5 Synthesis of Building Blocks for Benzo-RvD1 Analogs
The vinyl idodie building block (7.15) was synthesized from the hemi-
acteal protected 2-deoxy-d-ribose intermediate 5.9 outlined in scheme 7-2. The
sugar was opened by a cis-selective Wittig reaction using the phosphonium salt
intermediate (7.16).
13
The phosphonium salt 7.16 was synthesized from methyl
4-bromobutanoate.
13
The cis-selective Wittig coupling produced the primary
O
B
O
(R) (R)
OTBS
(R) (R)
OH
O
HB
O
(HO)
2
B
Br
+
+
+
Hydroboration
Suzuki
coupling
Suzuki
coupling
7.4
7.2
7.5
7.6
+
7.14
TBSO
OTBS
(S) (S)
(R) (R)
Br
COOMe
HO
OH
HO
COOMe
7.13
TBSO
OTBS
(S) (S)
(R) (R)
COOMe
I
7.15
O
(R) (R)
(S) (S)
O
O
OH
5.9
COOMe BrPh
3
P
7.16
COOMe Br
7.17
Scheme 7-5. Retrosynthesis of Benzo-RvD1 Analogs

131

alcohol intermediate (7.18) the primary alcohol was then oxidized to the
aldehyde using Dess-Martin periodinane.
21
The aldehyde was then converted to
the vinyl iodide by a Takai olefination.
22
The hemi-acetal protecting group was
then cleaved by treatment with acid, followed by a re-protection of the diols with
tert-butyl silyl protecting groups producing building block 7.15.
19

7.2.6 Construction of Benzo-RvD1 Analogs
The construction of the benzo-RvD1 methyl ester analogs 7.54 and 7.55
followed them same synthetic steps applied to the construction of benzo-lipoxin
A 4 methyl ester analogs 7.46, 7.47, 7.48 and 7.49, from intermediates 7.2 and
7.15.

COOMe Br COOMe BrPh
3
P
HO
COOMe
O O
O
COOMe
O O
TBSO
OTBS
COOMe
I
7.15
COOMe
O O
I
PPh3, Toluene,
reflux, 93%
KHMDS, THF,
-78°C, 75%
O
O
O
OH
5.9
DMP, pyridine,
NaHCO
3
, DCM, 85%
CrCl
2
, CH
3
I, THF,
0°C to rt 56%
1) HCl,
MeOH, rt,
2) TBS-Otf,
2,6-lutidine,
CH2Cl
2
, 94%
7.17 7.16
7.18
7.19
7.20
Scheme 7-6. Synthesis of Building Block 7.15

132

7.2.7 Retrosynthesis of Alkyne-benzo-lipoxin A4 Analogs
The synthesis of a library of benzo-alkyne-lipoxin A 4 methyl ester analogs
utilizes an alternative coupling strategy then the previous sets of analogs shown
in scheme 7-8. The benzo-alkyne-lipoxin A 4 methyl ester analogs utilize a
Sonogashira coupling to attach the alkyne intermediate 7.4 to the phenyl ring
centerpiece. This results in a preservation of the alkyne moiety of 7.4 giving the
benzo-alkyne-lipoxin A 4 methyl ester analogs a unique shape and rigidity not
seen in the other benzo-SPM analogs. The diol piece is attached to the phenyl
ring via a Suzuki coupling mimicking the synthetic procedures of the benzo-
alipoxin A 4 methyl ester analogs and benzo-RvD1 methyl ester analogs.

PdP(Ph
3
)
4
,
K
2
CO
3
, DMF,
65°C, 39%
O
B
O
OTBS
1) TBAF, THF
2) CH
2
N
2
, Et
2
O
(HO)
2
B
Br 7.6
7.2
52%
PdP(Ph
3
)
4
,
K
2
CO
3
, H
2
O,
Dioxane, 80°C,
57%
TBSO
OTBS
COOMe
I
7.15
7.14
TBSO
OTBS
Br
COOMe
HO
OH
HO
COOMe
7.13
TBSO
OTBS
TBSO
COOMe
7.21
Scheme 7-7. Construction of Benzo-RvD1 Analogs from Building Blocks 7.2 and
7.15

133

7.2.8 Synthesis of Building Blocks for Benzo-alkyne-lipoxin A4
Analogs
The diol building block 7.23 was synthesized from the aldehyde precursor
7.11. From the aldehyde the terminal alkyne (7.26) can be produced via the
Corey-Fuchs procedure.
25,26
Following the production of the terminal alkyne the
boronic ester can be produced via a hydroboration with catecholborane.
23

The commercially available enantiomerically pure (R)-oct-1-yn-ol can be
coupled directly with diiodobenzene via a Sonogashira coupling to produce
intermediate 7.27.
27
The alcohol group was then protected to produce building
block 7.24.
19
B
(R) (R)
(S) (S)
TBSO
OTBS
COOMe
(R) (R)
(S) (S)
TBSO
OTBS
COOMe
+
+
Suzuki
coupling
7.23
O
O
(R) (R)
OH
(R) (R)
(S) (S)
HO
OH
COOMe
(R) (R)
OTBS
I
O
HB
O
7.5
Hydroboration
I
I
(R) (R)
OH
Sonogashira coupling
+
7.22
7.24
7.26
7.25
7.4
Scheme 7-8. Retrosynthesis of Benzo-alkyne-lipoxin A 4 Analogs

Scheme 7-9. Synthesis of Building Block 7. 23

O
TBSO
OTBS
7.11
COOMe
TBSO
OTBS
7.26
COOMe
1) CBr
4
, PPh
3
,
DCM, 0°C
2) LDA, THF,
-78°C, 83%
B
TBSO
OTBS
COOMe
7.23
O
O
O
HB
O
7.5
70°C

134

7.2.9 Construction of Benzo-alkyne-lipoxin A4 Analogs
To the aryl iodide intermediate 7.24 the diol boronic ester building block
7.23 was attached via a Suzuki coupling to produce the silyl protected benzo-
alkyne-lipoxin A 4 methyl ester 7.56.
3
The late stage intermediate was then
deprotected by treatment with TBAF, and any cleaved methyl ester was
reinstated by treatment with freshly prepared diazomethane producing benzo-
alkyne-lipoxin A 4 methyl ester 7.57.
24

OH
7.4
I
7.25
OH
7.27
PdP(Ph
3
)
4
, CuI,
Et
3
N, Benzene, 67%
OTBS
7.24
TBS-Cl, DMAP,
imidazole,
CH
2
Cl
2
, 93%
I
I I
Scheme 7-10. Synthesis of Building Block 7.24

B
TBSO
OTBS
COOMe
7.23
O
O
OTBS
TBSO
OTBS
COOMe
7.28
OH
HO
OH
COOMe
7.22
1) TBAF, THF
2) CH
2
N
2
, Et
2
O
65%
PdP(Ph
3
)
4
, K
2
CO
3
,
H
2
O, Dioxane, 80°C,
95% OTBS
I
7.24
Scheme 7-11. Construction of Benzo-alkyne-lipoxin A 4 Analogs from Building
Blocks 7.2 and 7.15

135

7.2.10 Retrosynthesis of Click-SPM Analogs
The synthesis of SPM analogs defined by click chemistry took advantage of
a “click” coupling reaction or a copper catalysed azide alkyne cycloaddition.
28

This reaction is appealing due to it’s high yields and mild reaction conditions. In
addition to the attractive synthetic characteristics of click coupling reactions the
resulting products possess a triazole moiety probing the electronic and lipophilic
properties of these SPM analogs. Following the click coupling of the diol building
blocks to the 1-azido-3-bromobenzene centerpiece a Suzuki coupling was needed
to attach building block 7.2, completing the synthesis of click-SPM analogs.

O
B
O
(R) (R)
OTBS
Suzuki
coupling
7.2
N
N
N
Br
(R) (R)
(S) (S)
TBSO
OTBS
COOMe
(R) (R)
(S) (S)
HO
OH
COOMe
N
N
N
(R) (R)
HO
COOMe
OTBS
TBSO
(S) (S)
(R) (R)
O
(R) (R)
(S) (S)
OH
OH
HO
Ph
3
P
O
O
7.8
+
5.11
Br I
N
3
Br
Click
coupling
7.31
7.32
7.26
7.30
7.29
+
+
Scheme 7-12. Retrosynthesis of Click-SPM Analogs

136

7.2.11 Synthesis of Building Blocks for Click-SPM Analogs
The terminal alkyne intermediate 7.35 was produced from the aldehyde
intermediate 7.11. Starting with a homolongation utilizing commercially
available ylide (triphenylphosphoranylidene)acetaldehyde constructing
intermediate 7.33. The terminal alkyne (7.35) was then constructed from the
aldehyde via the Corey-Fuchs reaction.
25,26
The terminal alkyne intermediate
7.36 was produced from the aldehyde intermediate 7.19. The aldehyde was first
converted to the terminal alkyne by the Corey-Fuchs procedure,
25,26
the hemi-
acetal protecting group was then cleaved by acid and the resulting alcohols were
reprotected with tert-butyl silyl protecting groups
19
providing intermediate 7.36.
7.2.12 Construction of Click-SPM Analogs
The construction of the Click-SPM analogs from building blocks 7.32, 7.2
and 7.26, 7.35 or 7.36 is shown in scheme The aryl azide intermediate 7.31 can
be produced in a single step from 1-iodo-3-bromobenzene. The azide selectively
displaces the iodide substituent of 1-iodo-3-bromobenzene producing 1-azido-3-
TBSO
OTBS
O
O
PPh
3
THF, 65°C, 75%
7.33
COOMe
O
TBSO
OTBS
7.11
COOMe TBSO
OTBS
7.35
COOMe
1) CBr
4
, PPh
3
,
DCM, 0°C
2) LDA, THF, -
78°C, 83%
O
COOMe
O O
COOMe
OTBS TBSO
1) CBr
4
, PPh
3
,
DCM, 0°C
2) LDA, THF, -78°C
63%
3) HCl, MeOH
4) TBS-Otf, 2,6-
lutidine, CH2Cl
2
7.19
7.36
Scheme 7-13. Synthesis of Building Blocks 7.35 and 7.36

137

bromobenzene 7.31.
29
The terminal alkyne intermediate 7.26, 7.35 or 7.36 were
then subjected to a click coupling with the aryl azide of 7.31 producing
intermediates 7.30, 7.59 or 7.60 respectively.
28
The aryl bromide was then
coupled with building block 7.2 using the same Suzuki coupling procedure
previously reported.
3
The silyl protecting groups of intermediates 7.37, 7.61 or
7.62 were then removed by treatment with TBAF to produce the click-SPM
analogs 7.29, 7.63 or 7.64.
24

7.3 Conclusion
We have completed the synthesis of a library of 11 unique SPM-based
chemical probes to further understand the chemistry and biological activity of
SPM metabolites. This library of analogs examines the relationship of the
Br I N
3
Br
7.31 7.32
N
N
N
Br
TBSO
OTBS
COOMe
7.30
TBSO
OTBS
COOMe
N
N
N
TBSO
7.37
HO
OH
COOMe
N
N
N
HO
7.29
COOMe
OTBS
TBSO
(S) (S)
(R) (R)
7.26
CuI, NaN
3
,
L-proline, NaOH,
DMSO, 60°C, 82%
Sodium ascorbate,
CuSO
4
, t-BuOH,
H
2
O, 20%
O
B
O
TBSO
7.2
PdP(Ph
3
)
4
,
K
2
CO
3
, H
2
O,
Dioxane, 80°C,
79%
1) TBAF, THF
2) CH
2
N
2
, Et
2
O
62%
Scheme 7-14. Construction of Click-SPM Analogs from Building Blocks 7.32, 7.2
and 7.26, 7.35 or 7.36

138

molecule’s shape, structure, electronic, and lipophilic properties to expand the
acumen of the chemical properties of these naturally occurring metabolites.
7.4 Experimental
Unless otherwise noted, all reactions were carried out in a flame-dried
flask with stir bar under argon routed through a three-necked valve. Reactions
were carried out at room temp using DriSolv solvents purchased commercially
from VWR. All reagents used were purchased without further purification from
Sigma Aldrich, Strem, Combi-Blocks and Alfa Aesar.
Progress was monitored and recorded using EMD analytical thin layer
chromatography plates, Silica Gel 60 F254. TLC plates were visualized through
UV absorbance, (254 nm), or staining techniques including vanillin,
phosphomolybdic acid, potassium-permanganate, or ninhydrin followed by
heating. Unless otherwise stated, purification was carried out by flash column
chromatography manually using Silica Gel (100-200 mesh) or automatically
using the Biotage Isolera One.
Characterization was carried out using LC-MS, NMR and UV-VIS
instrumentation. All
1
H,
13
C and gcosy spectra were procured on the Departments
Varian 400, 500 and 600 MHz NMR instruments in the solvent indicated.
1
H and
13
C chemical shifts, (δ), are recorded in parts per million, (ppm), and referenced
to the residual solvent converted by the TMS scale (CDCl 3,
1H
= 7.26 ppm).
Splitting patterns are denoted by s, d, t, dd, td, ddd, and m and refer to the
respective multiplicities; singlet, doublet, triplet, doublet of doublets, triplet of
doublets, doublet of doublet of doublet and multiplet. Mass spectra was recorded

139

on an Agilent 1260 LC-MS. UV-Vis spectra was obtained by a Hewlett- Packard
8350 instrument.
methyl (5S,6R,E)-5,6,7-trihydroxyhept-2-enoate (7.9) To a
solution of 2-deoxy-d-ribose 5.11 (5 g, 37.3 mmol) in 45 mL of anhydrous THF
was added methyl (triphenylphosphoranylidene) acetate (13.7 g, 41 mmol). The
reaction mixture was stirred at 65°C overnight. Without workup the solvent was
removed in vacuo and the crude mixture was purified on silica gel using
MeOH/CH2Cl2 (10%) as the eluent to afford the triol ester (7.9) (6.5 g, 92%) as
clear colorless oil.
1
H NMR (400 MHz, Chloroform-d) 7.01 (dt, J = 15.6, 7.3 Hz, 1H),
5.95 (dt, J = 15.7, 1.5 Hz, 1H), 3.87 (ddd, J = 8.9, 5.3, 4.0 Hz, 1H), 3.77 (m, 2H), 3.73 (s,
3H), 3.63 (d, J = 4.8 Hz, 1H), 2.40 (m, 2H).
(4S,5R)-methyl 4,5,6-tris(tert-butyldimethylsilyloxy)hexanoate
(7.10) To a solution of triol (7.9) (4.7 g, 24.7 mmol) in 100 mL of EtOAc was
added one scoop of 5% palladium on charcoal. The reaction was stirred under H2
overnight. The reaction mixture was filtered through celite and with no workup
the solvent evaporated. To a flask with imidazole (10.1 g, 148 mmol) and DMAP
7.9
HO
HO
OH
COOMe
7.10
TBSO
TBSO
OTBS
COOMe

140

(1.2 mg, 9.9 mmol) in 50 mL of anyhrous DCM was added TBS-Cl (34 g, 148
mmol) dropwise at 0°C. The triol (4.7 g, 24.7 mmol) was cannulated to the flask
and stirred overnight at room temperature. The reaction mixture was quenched
with saturated aqueous NH4Cl (125 mL) and extracted with Et2O (3 x 125 mL).
The organic layer was dried with MgSO4, filtered and the solvent removed in
vacuo. The crude reaction mixture was purified on silica gel using EtOAc-hexanes
(10%) as the eluent to afford the protected triol ester (7.10) (11.8g, 89%) as a
clear colorless oil.
1
H NMR (400 MHz, Chloroform-d) 3.67 (m, 1H), 3.65 (d, J =
1.8 Hz, 3H), 3.58 (m, 1H), 3.52 (m, 2H), 2.33 (dt, J = 14.2, 7.2 Hz, 2H), 1.54 (m,
4H), 0.86 (m, 29H), 0.03 (m, 19H).
Methyl (5S,6S)-5,6-bis((tert-butyldimethylsilyl)oxy)-7-
oxoheptanoate (7.11) To a solution of protected triol (7.10) (525 mg, 0.981
mmol) in 14 mL of a 1:1 mixture of CH2Cl2/MeOH was added camphorsulfonic
acid (200 mg, 0.86 mmol) at 0°C. The reaction was quenched after 50 min with
Et3N (0.15 mL, 1.10 mmol) and the solvent was removed in vacuo. The crude
mixture was purified on silica gel using EtOAc-hexanes (12%) as the eluent to
afford the protected triol ester (289 mg, 70%) as a clear colorless oil. To a
solution of the resulting alcohol in 15 mL of CH 2Cl 2 was added 25 drops of
pyridine and Dess-Martin periodinane (445 mg, 1.05 mmols) the mixture was
stirred at room temperature for 20 minutes. The reaction mixture was quenched
7.11
O
TBSO
OTBS
COOMe

141

with 1:1 mixture of saturated aqueous NaHCO 3 and saturated aqueous Na 2S 2O 3 (7
mL) and extracted with Et2O (3 x 7 mL). The organic layer was dried with
MgSO4, filtered and the solvent removed in vacuo. The crude reaction mixture
was purified on silica gel using EtOAc-hexanes (10%) as the eluent to afford the
aldehyde (7.11) (256mg 89%) as clear colorless oil.
1
H NMR (400 MHz,
Chloroform-d) 9.59 (dd, J = 1.8, 0.7 Hz, 1H), 3.86 (m, 2H), 3.66 (s, 3H), 2.31 (t, J
= 7.2 Hz, 2H), 1.53 (m, 4H), 0.89 (d, J = 17.7 Hz, 18H), 0.06 (m, 12H).
13
C NMR
(400 MHz, cdcl 3) 203.71, 173.83, 128.48, 80.89, 75.17, 51.67, 34.16, 33.11, 25.98,
25.95, 20.78, 18.42, 18.26, -4.26, -4.48, -4.52, -4.70.
Methyl (5S,6R,E)-5,6-bis((tert-butyldimethylsilyl)oxy)-8-
iodooct-7-enoate (7.7) To a solution of CrCl2 (1.23 mg, 14 mmol) dissolved in
5 mL of anhydrous THF at 0°C a mixture of aldehyde (7.11) (580 mg, 1.4 mmol)
and CHI3 (1.5 g, 7 mmol) dissolved in 3 mL of anhydrous THF was added via
cannula. The reaction was stirred at 0°C for 3 h and an additional 1 h at room
temperature. The reaction mixture was quenched with brine (50 mL) extracted
with Et2O (3 x 50 mL) and dried over MgSO4. The organic phase was filtered and
the solvent was removed in vacuo to afford a crude oil which was purified on
silica gel using first pure pentanes and then EtOAc-hexanes (2%) as the eluent to
afford the vinyl iodide (7.7) (420 mg, 55%) as a yellow colored oil.
1
H NMR (400
MHz, Chloroform-d) 6.49 (dd, J = 14.5, 7.0 Hz, 1H), 6.17 (m, 1H), 3.89 (dddd, J =
6.3, 4.1, 3.4, 1.7 Hz, 1H), 3.66 (s, 3H), 3.61 (q, J = 5.2 Hz, 1H), 2.34 (m, 2H), 1.78
7.7
TBSO
OTBS
I
COOMe

142

(m, 1H), 1.33 (m, 1H), 1.25 (t, J = 7.2 Hz, 3H), 0.86 (m, 18H), 0.08 (m, 10H).
13
C
NMR (400 MHz, cdcl 3) 174.03, 147.29, 78.56, 78.06, 75.18, 51.63, 34.44, 33.01,
26.09, 26.02, 20.34, 18.37, -3.91, -4.35, -4.67.
(R)-tert-butyldimethyl(oct-1-yn-3-yloxy)silane (7.12) To a flask
with imidazole (1.2 g, 17.6 mmol) and DMAP (100 mg, 0.8 mmol) in 20 mL of
anyhrous DCM was added TBS-Cl (2.64 g, 17.6 mmol) dropwise at 0°C. The
alcohol (7.4) (2 g, 16 mmol) was added to the flask and stirred overnight at room
temperature. The reaction mixture was quenched with saturated aqueous NH4Cl
(125 mL) and extracted with Et2O (3 x 125 mL). The organic layer was dried with
MgSO4, filtered and the solvent removed in vacuo. The crude reaction mixture
was purified on silica gel using EtOAc-hexanes (1%) as the eluent to afford the
protected alcohol (7.12) (3.54 g, 92%) as clear colorless oil.
1
H NMR (400 MHz,
Chloroform-d) 4.33 (td, J = 6.5, 2.1 Hz, 1H), 1.63 (m, 2H), 1.56 (s, 1H), 1.38 (m,
2H), 1.26 (m, 4H), 0.90 (s, 9H), 0.86 (m, 2H), 0.12 (d, J = 9.4 Hz, 5H).
Methyl (5S,6R,E)-8-(2-bromophenyl)-5,6-bis((tert-
butyldimethylsilyl)oxy)oct-7-enoate (7.38) Vinyl iodine (7.7) (100 mg,
0.18 mmols) was cannulated in 2 mL of anhydrous DMF into a mixture of K 3PO 4
(137 mg, 0.65 mmols), (2-bromophenyl)boronic acid (49 mg, 0.24 mmols) and a
7.12
OTBS
TBSO
OTBS
COOMe
Br
7.38

143

catalytic amount of PdP(Ph 3) 4, the reaction was stirred overnight at 65°C. The
reaction was quenched with NH4Cl (5 mL) and extracted with Et2O (3 x 5 mL).
The solvent was removed in vacuo and the crude mixture was purified on silica
gel using EtOAc-hexanes (1%) as the eluent to afford compound (7.38) (39 mg,
38%) as yellow colored oil.
1
H NMR (400 MHz, Chloroform-d) 7.44 (m, 2H), 7.26
(m, 0H), 7.09 (ddd, J = 8.0, 7.3, 1.7 Hz, 1H), 6.80 (m, 1H), 6.13 (dd, J = 15.9, 6.9
Hz, 1H), 4.17 (ddd, J = 7.0, 4.4, 1.2 Hz, 1H), 3.69 (dt, J = 6.1, 4.6 Hz, 1H), 3.66 (s,
3H), 2.31 (t, J = 7.4 Hz, 2H), 1.67 (m, 2H), 1.51 (m, 4H), 0.90 (d, J = 17.3 Hz,
18H), 0.05 (d, J = 7.0 Hz, 9H).
Methyl (5S,6R,E)-8-(3-bromophenyl)-5,6-bis((tert-
butyldimethylsilyl)oxy)oct-7-enoate (7.39) This compound was prepared
from vinyl iodide (7.7), and (3-bromophenyl)boronic acid similarly to compound
(7.38).
1
H NMR (400 MHz, Chloroform-d) 7.46 (m, 1H), 7.35 (ddd, J = 7.9, 2.0,
1.1 Hz, 1H), 7.26 (m, 1H), 7.18 (t, J = 7.8 Hz, 1H), 6.38 (m, 1H), 6.17 (dd, J = 16.0,
7.0 Hz, 1H), 4.11 (ddd, J = 7.0, 4.9, 1.1 Hz, 1H), 3.66 (d, J = 0.4 Hz, 3H), 3.63 (m,
1H), 2.31 (t, J = 7.4 Hz, 2H), 1.65 (m, 2H), 1.49 (m, 2H), 0.85 (m, 18H), 0.11 (m,
9H).
7.39
TBSO
OTBS
COOMe
Br

144

Methyl (5S,6R,E)-8-(4-bromophenyl)-5,6-bis((tert-
butyldimethylsilyl)oxy)oct-7-enoate (7.40) This compound was prepared
from vinyl iodide (7.7), and (4-bromophenyl)boronic acid similarly to compound
(7.38).
1
H NMR (400 MHz, Chloroform-d) 7.41 (m, 2H), 7.19 (m, 2H), 6.37 (m,
1H), 6.14 (dd, J = 16.0, 7.1 Hz, 1H), 4.09 (ddd, J = 7.2, 5.2, 1.1 Hz, 1H), 3.66 (m,
1H), 3.66 (s, 3H), 2.31 (t, J = 7.5 Hz, 2H), 1.67 (m, 2H), 1.51 (m, 2H), 0.84 (m,
18H), -0.04 (m, 12H).
13
C NMR (101 MHz, cdcl 3) 174.11, 136.11, 131.97, 131.80,
130.16, 128.00, 121.25, 77.07, 75.94, 51.59, 34.50, 33.10, 26.15, 26.12, 26.09,
26.07, 26.05, 20.53, 18.38, 18.27, -3.79, -3.94, -4.35, -4.55.
Methyl (5S,6R,E)-8-(3-bromo-5-fluorophenyl)-5,6-bis((tert-
butyldimethylsilyl)oxy)oct-7-enoate (7.41) This compound was prepared
from vinyl iodide (7.7), and (3-bromo-5-fluorophenyl)boronic acid similarly to
compound (7.38).
1
H NMR (400 MHz, Chloroform-d) 7.11 (dt, J = 8.0, 2.0 Hz,
1H), 6.98 (dt, J = 9.6, 1.9 Hz, 1H), 6.39 (d, J = 16.0 Hz, 1H), 6.19 (dd, J = 16.0, 6.7
Hz, 1H), 4.09 (m, 1H), 3.67 (s, 1H), 3.66 (s, 3H), 2.31 (t, J = 7.4 Hz, 2H), -0.02
(m, 12H).
TBSO
OTBS
COOMe
Br
7.40
7.41
TBSO
OTBS
COOMe
Br
F

145

Methyl (5S,6R,E)-5,6-bis((tert-butyldimethylsilyl)oxy)-8-(2-
((R,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)phenyl)oct-7-
enoate (7.42) Catecholborane (54 mg, 0.45 mmol) and acetylene (7.12) (43
mg, 0.075 mmol) was mixed in a pear- shape flask and stir overnight at 65°C. To
the white muddy substance, a catalytic amount of Pd (PP
3
)
4
and 25 mg K
2
CO
3

(34 mg, 0.25 mmols) was added and aryl bromide (7.38) (43 mg, 0.075 mmol)
was cannulated to the mixture after dissolved in 1 mL of 1,4-dioxane and 2 mL
degassed H 2O was added into the mixture and the reaction was allowed to stir
overnight at 80°C. The reaction was quenched with saturated aqueous NH4Cl (5
mL) and extracted with Et2O (3 x 5 mL). The solvent was removed in vacuo and
the crude mixture was purified on silica gel using EtOAc-hexanes (0.5%) as the
eluent to afford compound (7.42) (55 mg, 95%) as colorless oil.
1
H NMR (400
MHz, Chloroform-d) 7.37 (m, 3H), 7.12 (m, 2H), 6.66 (m, 1H), 5.99 (m, 1H), 4.07
(m, 1H), 3.66 (d, J = 1.2 Hz, 1H), 3.66 (s, 3H), 2.28 (m, 2H), 1.71 (m, 4H), 1.47
(m, 3H), 1.29 (m, 3H), 0.84 (m, 31H), 0.17 (d, J = 8.1 Hz, 3H), 0.01 (m, 18H).

TBSO
OTBS
COOMe
OTBS
7.42

146

methyl (5S,6R,E)-5,6-bis((tert-butyldimethylsilyl)oxy)-8-(3-
((R,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)phenyl)oct-7-
enoate (7.43) This compound was prepared from terminal alkyne (7.12),
catecholborane and aryl bromide (7.39) similarly to compound (7.42).
1
H NMR
(400 MHz, Chloroform-d) 7.47 (m, 1H), 7.29 (m, 3H), 7.18 (t, J = 7.8 Hz, 1H),
6.37 (m, 2H), 6.20 (ddd, J = 28.2, 16.0, 7.0 Hz, 2H), 4.09 (m, 2H), 3.69 (m, 1H),
3.66 (s, 3H), 2.39 (m, 2H), 2.31 (t, J = 7.4 Hz, 1H), 1.84 (m, 4H), 1.68 (m, 4H),
1.59 (m, 1H), 0.97 (d, J = 6.7 Hz, 3H), 0.89 (m, 20H), 0.83 (m, 7H), 0.08 (m,
5H), 0.01 (m, 13H).
Methyl (5S,6R,E)-5,6-bis((tert-butyldimethylsilyl)oxy)-8-(4-
((R,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)phenyl)oct-7-
enoate (7.44) This compound was prepared from terminal alkyne (7.12),
catecholborane and aryl bromide (7.40) similarly to compound (7.42).
1
H NMR
(400 MHz, Chloroform-d) 7.27 (m, 4H), 6.45 (ddd, J = 16.0, 4.6, 1.0 Hz, 2H),
TBSO
OTBS
COOMe
TBSO
7.43
7.44
TBSO
OTBS COOMe
TBSO

147

6.07 (m, 2H), 4.20 (m, 1H), 4.10 (ddd, J = 7.4, 5.2, 1.1 Hz, 1H), 3.67 (s, 1H), 3.66
(s, 5H), 2.31 (t, J = 7.4 Hz, 4H), 2.13 (m, 1H), 1.67 (m, 3H), 1.56 (dt, J = 17.1, 5.7
Hz, 3H), 1.35 (m, 1H), 1.30 (m, 1H), 1.30 (d, J = 2.6 Hz, 1H), 0.84 (m, 31H), 0.01
(m, 23H).
13
C NMR (101 MHz, cdcl 3) 174.16, 137.03, 136.54, 136.21, 133.69,
131.10, 130.81, 128.61, 126.73, 126.68, 126.44, 77.34, 76.02, 73.83, 51.58, 38.62,
34.54, 33.12, 32.00, 29.86, 26.12, 26.10, 26.09, 25.12, 22.79, 20.59, 18.29, 14.21, -
3.73, -3.88, -4.06, -4.37, -4.55, -4.58.
methyl (5S,6R,E)-5,6-bis((tert-butyldimethylsilyl)oxy)-8-(3-
((R,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)-5-
fluorophenyl)oct-7-enoate (7.45) This compound was prepared from
terminal alkyne (7.12), catecholborane and aryl bromide (7.41) similarly to
compound (7.42).
1
H NMR (400 MHz, Chloroform-d) 6.89 (m, 2H), 6.43 (dd, J
= 15.9, 3.4 Hz, 2H), 6.18 (ddd, J = 16.0, 6.6, 5.4 Hz, 2H), 4.25 (ddd, J = 13.4, 6.6,
5.3 Hz, 1H), 4.11 (ddd, J = 7.1, 4.9, 1.1 Hz, 1H), 3.66 (s, 4H), 3.64 (d, J = 4.9 Hz,
1H), 2.31 (t, J = 7.3 Hz, 3H), 2.25 (m, 1H), 1.72 (ttd, J = 13.1, 5.5, 2.0 Hz, 3H),
1.58 (t, J = 2.8 Hz, 7H), 0.86 (m, 37H), -0.00 (m, 23H).
TBSO
OTBS
COOMe
TBSO
F
7.45

148

Methyl (5S,6R,E)-5,6-dihydroxy-8-(2-((R,E)-3-hydroxyoct-1-en-
1-yl)phenyl)oct-7-enoate (7.46) To the late stage intermediate (7.42) (55
mg, 0.071 mmol) dissolved in 1 mL of anhydrous THF was added dropwise 6
equivalents of 1M TBAF (0.42 mL, 0.42 mmol) at 0°C. The reaction was
monitored closely via thin layer chromatography and after 4 h the reaction was
quenched with saturated NH4Cl (15 mL) and extracted with Et2O (5 x 15 mL).
The organic layer was rinsed with brine, dried over MgSO4 and filtered. The
solvent was then concentrated and freshly prepared CH2N2 was added to convert
any acid to the methyl ester. The solvent was completely removed in vacuo and
the compound purified via C-18 reversed Phase HPLC using H2O-MeOH mixture
(32%) to afford compound (7.46) (18 mg 58%) as colorless oil.
1
H NMR (400
MHz, Methanol-d 4) 7.41 (m, 2H), 7.19 (m, 2H), 6.91 (ddd, J = 15.8, 11.6, 1.0 Hz,
2H), 6.19 (dd, J = 15.8, 6.7 Hz, 1H), 5.88 (ddd, J = 15.8, 8.0, 1.6 Hz, 1H), 4.10
(ddd, J = 6.5, 5.0, 1.3 Hz, 1H), 3.77 (ddd, J = 8.4, 7.1, 6.1 Hz, 1H), 3.63 (s, 3H),
3.56 (m, 1H), 2.36 (t, J = 7.3 Hz, 2H), 1.81 (m, 1H), 1.61 (m, 3H), 1.51 (m, 1H),
1.37 (m, 1H), 1.30 (m, 4H), 0.88 (m, 3H).
13
C NMR (400 MHz, cd 3od) 205.81,
166.78, 166.27, 163.68, 162.99, 161.72, 160.44, 158.77, 158.63, 157.78, 157.59,
114.07, 107.11, 105.36, 86.56, 81.96, 66.65, 64.76, 62.98, 56.23, 53.70, 52.53,
44.41.
HO
OH
COOMe
OH
7.46

149

Methyl (5S,6R,E)-5,6-dihydroxy-8-(3-((R,E)-3-hydroxyoct-1-en-
1-yl)phenyl)oct-7-enoate (7.47) This compound was prepared from late stage
intermediate (7.43), similarly to compound (7.46).
1
H NMR (400 MHz,
Chloroform-d) 7.39 (m, 1H), 7.26 (m, 3H), 6.60 (ddd, J = 33.0, 15.9, 1.1 Hz, 2H),
6.27 (ddd, J = 22.5, 15.9, 6.9 Hz, 2H), 4.24 (m, 2H), 3.78 (dt, J = 8.9, 3.9 Hz, 1H),
3.66 (s, 3H), 2.36 (td, J = 7.3, 2.1 Hz, 2H), 1.86 (dddd, J = 11.2, 7.4, 5.9, 3.7 Hz,
1H), 1.69 (m, 1H), 1.58 (m, 2H), 1.47 (m, 1H), 1.30 (m, 3H), 1.25 (m, 2H), 0.86
(m, 3H).
13
C NMR (400 MHz, cdcl 3) 174.13, 137.12, 136.61, 133.08, 132.95, 129.82,
128.83, 127.25, 126.04, 125.85, 124.61, 75.85, 73.80, 73.05, 51.57, 33.70, 31.76,
31.47, 30.90, 25.10, 22.59, 21.07, 14.03.
Methyl (5S,6R,E)-5,6-dihydroxy-8-(4-((R,E)-3-hydroxyoct-1-en-
1-yl)phenyl)oct-7-enoate (7.48) This compound was prepared from late stage
intermediate (7.44), similarly to compound (7.46).
1
H NMR (400 MHz,
Methanol-d 4) 7.47 (q, J = 1.5 Hz, 2H), 7.45 (d, J = 1.1 Hz, 2H), 6.61 (m, 2H), 6.28
7.47
HO
OH
COOMe
HO
7.48
HO
OH COOMe
HO

150

(m, 2H), 4.25 (m, 1H), 4.12 (m, 1H), 3.74 (s, 3H), 3.67 (ddd, J = 9.4, 5.0, 3.2 Hz,
1H), 2.46 (t, J = 7.3 Hz, 2H), 2.21 (m, 1H), 1.99 (m, 1H), 1.90 (m, 1H), 1.61 (m,
5H), 1.50 (m, 1H), 1.52 (s, 1H), 1.00 (dd, J = 4.6, 2.9 Hz, 3H).
Methyl (5S,6R,E)-8-(3-fluoro-5-((R,E)-3-hydroxyoct-1-en-1-
yl)phenyl)-5,6-dihydroxyoct-7-enoate (7.49) This compound was prepared
from late stage intermediate (7.45), similarly to compound (7.46).
1
H NMR
(400 MHz, Methanol-d 4) 7.42 (t, J = 1.4 Hz, 1H), 7.16 (m, 2H), 6.74 (dd, J = 26.8,
16.1 Hz, 2H), 6.51 (ddd, J = 42.9, 16.0, 6.6 Hz, 2H), 4.33 (m, 1H), 4.24 (ddd, J =
6.6, 5.2, 1.2 Hz, 1H), 3.80 (s, 2H), 3.73 (ddd, J = 9.4, 5.2, 3.1 Hz, 1H), 2.53 (t, J =
7.3 Hz, 2H), 2.02 (tdd, J = 11.7, 5.7, 3.2 Hz, 1H), 1.72 (m, 4H), 1.57 (m, 2H), 1.51
(dt, J = 6.8, 2.6 Hz, 3H), 1.05 (m, 4H).
(4-methoxy-4-oxobutyl)triphenylphosphonium bromide (7.16)
To a solution of methyl-bromo-butyrate (7.17) (5.0 g, 27.62 mmols) in 75 mL of
anhydrous toluene was added PPh 3 (8.69 g, 33.14 mmols). The mixture was
refluxed overnight. The mixture was then allowed to cool to rt, 20 mL of Et 2O was
7.49
HO
OH
COOMe
HO
F
7.16
COOMe BrPh
3
P

151

added to the flask and the reaction was triturated. The reaction mixture was
filtered, washing with cold Et 2O (50 mL X 3) to produce a white solid. The white
solid was dried over P 2O 5 for overnight to give pure salt (7.16) (11.5 g, 93%).
1
H
NMR (400 MHz, Chloroform-d) 7.79 (m, 5H), 7.70 (m, 3H), 7.59 (m, 7H), 3.97
(m, 2H), 3.58 (s, 3H), 2.86 (td, J = 6.5, 1.3 Hz, 2H), 1.86 (dddd, J = 9.5, 7.1, 4.8,
1.4 Hz, 2H).
Methyl (Z)-6-((4S,5R)-5-(hydroxymethyl)-2,2-dimethyl-1,3-
dioxolan-4-yl)hex-4-enoate (7.18) The Phosphonium salt (7.16) (16.92 g,
38.4 mmols) was added to a flame dried round bottom flask and stored in a
vacuum oven overnight at 70°C. The reaction flask was allowed to cool to rt, and
40 mL of anhydrous THF was added and the mixture was then cooled to -78°C.
To the reaction mixture was added a 1M KHMDS solution (7.8 g, 38.4 mmols)
dropwise via cannula the reaction was then allowed to stir at 0°C for 30 min. The
reaction mixture was then cooled to -78°C and the protected sugar (5.9) (3.15 g,
18.1 mmols) was added dropwise via cannula, the reaction was allowed to warm
to rt and stir overnight. It was then quenched with saturated aqueous NH 4Cl (125
mL) and extracted with Et2O (3 x 125 mL). The combined extract was dried with
Na 2SO 4 and evaporated to give a crude brown oil which was then
chromatographed on silica gel using EtOAc-hexanes (35%) as the eluent to afford
the alcohol (7.18)
1
H NMR (400 MHz, Chloroform-d) 5.43 (m, 2H), 4.15 (m,
7.18
HO
COOMe
O O

152

2H), 3.67 (s, 2H), 3.64 (m, 2H), 2.42 (ddd, J = 7.8, 3.5, 1.5 Hz, 1H), 2.36 (m, 4H),
2.28 (m, 1H), 1.48 (d, J = 0.8 Hz, 3H), 1.37 (t, J = 0.7 Hz, 3H).
Methyl (Z)-6-((4S,5S)-5-formyl-2,2-dimethyl-1,3-dioxolan-4-yl)hex-4-
enoate (7.19) To a solution of the alcohol (7.18) (1.48 g, 5.7 mmol) in 40 mL of
CH 2Cl 2 was added 5.5 mL of pyridine and Dess-Martin periodinane (3.5 g, 8.3
mmols) the mixture was stirred at room temperature for 20 minutes. The
reaction mixture was quenched with 1:1 mixture of saturated aqueous NaHCO 3
and saturated aqueous Na 2S 2O 3 (125 mL) and extracted with Et2O (3 x 125 mL).
The organic layer was dried with MgSO4, filtered and the solvent removed in
vacuo. The crude reaction mixture was then chromatographed on silica gel using
EtOAc-hexanes (9%) as the eluent to afford the aldehyde (7.19) (1.2 g, 85%) as
yellow colored oil.
1
H NMR (400 MHz, Chloroform-d) 9.66 (dd, J = 3.1, 0.6 Hz, 1H),
5.41 (m, 2H), 4.35 (m, 1H), 4.30 (dd, J = 7.2, 3.1 Hz, 1H), 3.66 (s, 3H), 2.31 (m, 6H),
1.59 (d, J = 0.7 Hz, 3H), 1.40 (d, J = 0.7 Hz, 3H).
Methyl (Z)-6-((4S,5R)-5-((E)-2-iodovinyl)-2,2-dimethyl-1,3-
dioxolan-4-yl)hex-4-enoate (7.20) To a solution of CrCl2 (1.56 g, 12.7 mmol)
dissolved in 10 mL of anhydrous THF was cannulated a mixture of aldehyde
(7.20) (326 mg, 1.27 mmol) and CHI3 (2.5 g, 6.35 mmol) dissolved in 4 mL of
7.19
O
COOMe
O O
7.20
COOMe
O O
I

153

anhydrous THF at 0°C. The reaction was stirred at 0°C for 3 h and an additional 1
h at room temperature. The reaction mixture was quenched with brine (50 mL)
extracted with Et2O (3 x 50 mL) and dried over MgSO4. The organic phase was
filtered and the solvent was removed in vacuo to afford a crude oil which was
purified on silica gel using first pure pentanes and then EtOAc-hexanes (3%) as
the eluent to afford the vinyl iodide (7.20) (270 mg, 56%) as a yellow colored oil.

1
H NMR (400 MHz, Chloroform-d) 6.53 (dd, J = 14.5, 7.1 Hz, 1H), 6.43 (dd, J =
14.5, 0.7 Hz, 1H), 5.36 (m, 2H), 4.49 (ddd, J = 7.0, 6.2, 0.8 Hz, 1H), 4.17 (dt, J =
7.8, 6.1 Hz, 1H), 3.68 (d, J = 0.5 Hz, 3H), 2.38 (d, J = 3.2 Hz, 4H), 2.17 (m, 2H),
1.47 (m, 3H), 1.34 (q, J = 0.7 Hz, 3H).
13
C NMR (101 MHz, cdcl 3) 173.92, 142.52,
130.75, 126.27, 109.15, 80.80, 80.31, 77.98, 52.06, 34.30, 29.01, 28.48, 25.88,
23.56.

Methyl (4Z,6S,7R,8E)-6,7-bis((tert-butyldimethylsilyl)oxy)-9-
iodonona-4,8-dienoate (7.15) To a solution of vinyl iodide (7.20) (34 mg,
0.09 mmol) in 0.25 mL of MeOH at 0°C was added 0.25 mL of 1M HCl. The
reaction was stirred at rt and monitored closely by TLC until complete
consumption of starting material. The reaction was then quenched with 5 mL of
aqueous saturated NaHCO 3, extracted with CH 2Cl2 (3 x 5 mL) and dried over
MgSO4. The organic phase was filtered and the solvent was removed in vacuo to
afford a crude oil which was purified on silica gel using first pure pentanes and
7.15
TBSO
OTBS
COOMe
I

154

then EtOAc-hexanes (40%) as the eluent to afford the deprotected vinyl iodide.
To a solution of deprotected vinyl iodide in 3 mL of anhydrous CH 2Cl 2 at 0°C was
added 2,6-lutidine (0.04 mL, 0.34 mmols) and TBS-OTf (90 mg, 0.34 mmols).
The reaction mixture was allowed to stir at rt overnight. The reaction mixture was
quenched with saturated aqueous NH4Cl (15 mL) and extracted with Et2O (3 x 15
mL). The organic layer was dried with MgSO4, filtered and the solvent removed
in vacuo. The crude reaction mixture was purified on silica gel using EtOAc-
hexanes (3%) to afford the silyl-protected vinyl iodide (7.15) (47 mg, 94%) as
yellow colored oil.
1
H NMR (400 MHz, Chloroform-d) 6.52 (dd, J = 14.5, 7.1 Hz,
1H), 6.22 (dd, J = 14.5, 1.0 Hz, 1H), 5.38 (m, 2H), 3.92 (ddd, J = 7.1, 4.8, 1.0 Hz,
1H), 3.67 (s, 3H), 3.59 (td, J = 5.7, 4.8 Hz, 1H), 2.35 (dd, J = 3.3, 1.1 Hz, 4H), 2.21
(m, 2H), 0.88 (d, J = 4.3 Hz, 18H), 0.04 (dd, J = 8.3, 4.1 Hz, 12H).
13
C NMR (101
MHz, cdcl 3) 173.64, 146.91, 129.40, 127.23, 78.63, 78.04, 75.61, 51.70, 34.07,
31.67, 26.07, 26.04, 23.24, 18.38, 18.24, -4.01, -4.14, -4.21, -4.59.
Methyl (4Z,6S,7R,8E)-9-(2-bromophenyl)-6,7-bis((tert-
butyldimethylsilyl)oxy)nona-4,8-dienoate (7.50) Vinyl iodine (7.15) (213
mg, 0.37 mmol) was cannulated in 3 mL of anhydrous DMF into a mixture of
K 3PO 4 (274 mg, 1.3 mmol), (2-bromophenyl)boronic acid (100 mg, 0.48 mmol)
and a catalytic amount of PdP(Ph 3) 4, the reaction was stirred overnight at 65°C.
7.50
TBSO
OTBS
COOMe
Br

155

The reaction was quenched with NH4Cl (5 mL) and extracted with Et2O (3 x 5
mL). The solvent was removed in vacuo and the crude mixture was purified on
silica gel using EtOAc-hexanes (3%) as the eluent to afford compound (7.50) (84
mg, 39%) as yellow colored oil.
1
H NMR (400 MHz, Chloroform-d) 7.47 (m, 2H),
7.26 (m, 1H), 7.26 (s, 1H), 7.09 (ddd, J = 8.0, 7.2, 1.6 Hz, 1H), 6.81 (m, 1H), 6.16
(dd, J = 15.9, 6.9 Hz, 1H), 5.52 (dt, J = 11.0, 7.0 Hz, 1H), 5.39 (m, 1H), 4.20 (ddd,
J = 6.9, 3.8, 1.2 Hz, 1H), 3.73 (td, J = 6.0, 3.8 Hz, 1H), 3.65 (d, J = 0.7 Hz, 3H),
2.23 (m, 6H), 0.90 (dd, J = 20.2, 0.7 Hz, 15H), 0.03 (m, 10H).
13
C NMR (101
MHz, d 2o) 173.69, 137.16, 133.46, 133.04, 129.97, 129.19, 128.73, 127.80, 127.59,
127.19, 123.80, 77.13, 76.79, 51.66, 34.13, 31.82, 26.18, 26.14, 26.13, 23.27, 18.33.

Methyl (4Z,6S,7R,8E)-9-(4-bromophenyl)-6,7-bis((tert-
butyldimethylsilyl)oxy)nona-4,8-dienoate (7.51) This compound was
prepared from vinyl iodide (7.15), and (4-bromophenyl)boronic acid similarly to
compound (7.50).
1
H NMR (500 MHz, Chloroform-d) 7.21 (m, 2H), 6.70 (m,
2H), 6.41 (d, J = 16.0 Hz, 1H), 6.17 (dd, J = 16.0, 7.1 Hz, 1H), 5.52 (dt, J = 10.6,
7.2 Hz, 1H), 5.43 (dtd, J = 7.6, 5.6, 4.5, 2.4 Hz, 1H), 4.12 (ddd, J = 7.0, 4.5, 1.1 Hz,
1H), 3.68 (m, 1H), 3.66 (s, 3H), 2.22 (m, 6H), 0.88 (d, J = 25.2 Hz, 18H), -0.03
(m, 12H).
13
C NMR (500 MHz, cdcl 3) 173.67, 136.16, 133.28, 132.42, 132.16,
7.51
TBSO
OTBS
COOMe
Br

156

131.79, 130.10, 128.65, 128.02, 127.73, 122.04, 77.19, 76.46, 51.66, 34.10, 31.77,
29.86, 26.13, 26.12, 26.07, 25.77, 23.25, 18.27, -4.02, -4.21, -4.34, -4.46.
Methyl (4Z,6S,7R,8E)-6,7-bis((tert-butyldimethylsilyl)oxy)-9-(2-
((R,E)-3-hydroxyoct-1-en-1-yl)phenyl)nona-4,8-dienoate (7.52)
Catecholborane (72 mg, 0.60 mmol) and acetylene (7.12) (145 mg, 0.60 mmol)
was mixed in a pear- shape flask and stir overnight at 65°C. To the white muddy
substance, a catalytic amount of Pd (PP
3
)
4
and K
2
CO
3
(64 mg, 0.46 mmols) was
added and aryl bromide (7.50) (84 mg, 0.14 mmol) was cannulated to the
mixture and dissolved in 2.5 mL of 1,4-dioxane and 5 mL degassed H 2O was
added into the mixture and the reaction was allowed to stir overnight at 80°C.
The reaction was quenched with saturated aqueous NH4Cl (15 mL) and extracted
with Et2O (3 x 15 mL). The solvent was removed in vacuo and the crude mixture
was purified on silica gel using EtOAc-hexanes (0.5%) as the eluent to afford
compound (7.52) (50 mg, 57%) as colorless oil.
1
H NMR (400 MHz, Chloroform-
d) 7.46 (m, 1H), 7.37 (m, 1H), 7.12 (m, 1H), 6.83 (m, 0H), 6.76 (ddd, J = 15.8, 7.1,
1.1 Hz, 1H), 6.12 (m, 1H), 5.98 (m, 1H), 5.48 (m, 1H), 5.37 (m, 1H), 4.13 (m, 1H),
3.72 (ddd, J = 6.2, 4.4, 2.2 Hz, 1H), 3.65 (d, J = 1.6 Hz, 3H), 2.23 (m, 2H), 1.77
(dtd, J = 9.2, 6.4, 3.4 Hz, 1H), 1.46 (m, 1H), 1.28 (m, 5H), 1.26 (s, 3H), 0.82 (m,
28H), -0.06 (m, 16H).
13
C NMR (400 MHz, cdcl 3) 173.66, 138.41, 136.35, 135.30,
7.52
OTBS
OTBS
OTBS
COOMe

157

133.04, 132.91, 132.54, 129.08, 128.68, 127.96, 127.88, 127.59, 126.69, 77.67,
76.59, 74.05, 51.62, 38.93, 34.13, 32.00, 31.67, 29.85, 26.17, 26.14, 26.12, 26.11,
26.02, 23.24, 22.80, 18.43, 14.22, -3.66, -3.78, -3.92, -3.94, -4.10, -4.23, -4.28, -
4.40, -4.45, -4.54, -4.75.
Methyl (4Z,6S,7R,8E)-6,7-bis((tert-butyldimethylsilyl)oxy)-9-(4-
((R,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)phenyl)nona-4,8-
dienoate (7.53) This compound was prepared from terminal alkyne (7.12),
catecholborane and aryl bromide (7.51) similarly to compound (7.52).
1
H NMR
(500 MHz, Chloroform-d) 7.43 (m, 1H), 7.29 (m, 3H), 6.41 (m, 2H), 6.12 (m, 2H),
5.52 (dt, J = 11.0, 4.4 Hz, 1H), 5.39 (m, 1H), 4.22 (m, 1H), 4.12 (ddd, J = 7.3, 4.5,
1.1 Hz, 1H), 3.67 (m, 1H), 3.64 (m, 3H), 2.32 (m, 4H), 2.27 (ddt, J = 14.0, 10.1, 5.8
Hz, 1H), 1.47 (m, 6H), 1.33 (m, 2H), 1.29 (d, J = 4.4 Hz, 1H), 0.84 (m, 29H), -
0.00 (m, 18H).
13
C NMR (500 MHz, cdcl 3) 173.70, 136.52, 136.27, 133.68, 131.04,
130.50, 129.02, 128.62, 127.90, 127.14, 126.92, 126.71, 126.68, 77.46, 76.56,
73.84, 51.66, 38.62, 34.15, 32.00, 29.86, 26.16, 26.14, 26.12, 26.11, 26.09, 25.13,
23.25, 22.80, 18.42, 14.21, -3.80, -4.22, -4.46, -4.57.
7.53
TBSO
OTBS
COOMe
(R) (R)
OTBS

158

Methyl (4Z,6S,7R,8E)-6,7-dihydroxy-9-(2-((R,E)-3-hydroxyoct-
1-en-1-yl)phenyl)nona-4,8-dienoate (7.54) ) To the late stage intermediate
(7.52) (50 mg, 0.07 mmol) dissolved in 1 mL of anhydrous THF was added
dropwise 6 equivalents of 1M TBAF (0.40 mL, 0.40 mmol) at 0°C. The reaction
was monitored closely via thin layer chromatography and after 4 h the reaction
was quenched with saturated NH4Cl (15 mL) and extracted with Et2O (5 x 15
mL). The organic layer was rinsed with brine, dried over MgSO4 and filtered. The
solvent was then concentrated and freshly prepared CH2N2 was added to convert
any acid to the methyl ester. The product was then suspended in a H2O- MeOH
mixture (1:1, 1 mL) and 10 equivalents of LiOH (17 mg, 0.7 mmol) was added.
After 3 h the reaction mixture was dried and purified via C-18 reversed Phase
HPLC using H2O-MeOH mixture (37%) to afford compound (7.54) (14 mg 52%)
as colorless oil.
1
H NMR (400 MHz, Methanol-d 4) 7.42 (m, 1H), 7.37 (m, 1H), 7.16
(m, 2H), 6.92 (dd, J = 21.0, 15.8 Hz, 2H), 6.22 (dd, J = 15.8, 6.7 Hz, 1H), 6.06
(dd, J = 15.7, 6.7 Hz, 1H), 5.52 (dd, J = 5.4, 4.1 Hz, 1H), 4.22 (q, J = 6.5 Hz, 1H),
4.10 (m, 1H), 3.57 (m, 1H), 2.26 (m, 2H), 2.19 (t, J = 7.6 Hz, 2H), 1.54 (m, 1H),
1.31 (m, 3H), 1.28 (s, 11H).
7.54
OH
OH
OH
COOH

159

Methyl (4Z,6S,7R,8E)-6,7-dihydroxy-9-(4-((R,E)-3-hydroxyoct-
1-en-1-yl)phenyl)nona-4,8-dienoate (7.54) To the late stage intermediate
(7.53) (50 mg, 0.07 mmol) dissolved in 1 mL of anhydrous THF was added
dropwise 6 equivalents of 1M TBAF (0.40 mL, 0.40 mmol) at 0°C. The reaction
was monitored closely via thin layer chromatography and after 4 h the reaction
was quenched with saturated NH4Cl (15 mL) and extracted with Et2O (5 x 15
mL). The organic layer was rinsed with brine, dried over MgSO4 and filtered. The
solvent was then concentrated and freshly prepared CH2N2 was added to convert
any acid to the methyl ester. The product was purified via C-18 reversed Phase
HPLC using H2O-MeOH mixture (32%) to afford compound (7.55) (15 mg 52%)
as colorless oil.
1
H NMR (500 MHz, Methanol-d 4) 7.34 (m, 4H), 6.58 (ddd, J =
38.4, 15.9, 1.1 Hz, 2H), 6.37 (dd, J = 16.0, 6.9 Hz, 1H), 6.23 (dd, J = 15.9, 6.8 Hz,
1H), 5.57 (dt, J = 11.1, 7.3 Hz, 1H), 5.42 (m, 1H), 4.10 (m, 1H), 3.97 (m, 1H), 3.65
(td, J = 4.6, 2.1 Hz, 1H), 3.63 (s, 3H), 2.39 (m, 2H), 2.11 (m, 4H), 1.54 (m, 6H).

7.55
HO
OH
COOMe
(R) (R)
OH

160

Methyl (5S,6R)-5,6-bis((tert-butyldimethylsilyl)oxy)oct-7-
ynoate (7.26) Triphenylphosphine (1.5 g, 5.7 mmol) in 2 mL of anhydrous
CH2Cl2 was cannulated in to a solution of CBr4 (942 mg, 2.8 mmol) at 0°C in 4
mL of anhydrous CH2Cl2 . Aldehyde (7.11) (594 mg, 1.4 mmol) in 6 mL of
anhydrous CH2Cl2 was cannulated in to the reaction mixture. The reaction was
stirred for 1 h at 0°C. Without workup the solvent was evaporated in vacuo and
the crude mixture was purified on silica gel using EtOAc-hexanes (1%) as the
eluent to afford the dibromo ester (780 mg, 97%) as a viscous and yellow colored
oil. To a solution of the dibromo ester (690 mg, 1.2 mmol) at -78°C in 15 mL of
anhydrous THF was added 2.0 M solution of LDA (3.6 mL, 7.2 mmol) drop wise
and stirred for 0.5 h. The reaction was quenched with water (30 mL) and
extracted with Et2O (3 x 30 mL), dried using MgSO4, filtered and concentrated.
The crude was then purified using silica gel with a EtOAc- hexanes eluent (2%) to
afford the alkyne product (439 mg, 88%) as a viscous and yellow colored oil.
1
H
NMR (400 MHz, Chloroform-d) 4.22 (dd, J = 4.9, 2.2 Hz, 1H), 3.69 (m, 1H), 3.66
(s, 3H), 2.35 (d, J = 2.2 Hz, 1H), 2.31 (t, J = 7.3 Hz, 2H), 1.57 (m, 4H), 0.90 (dd, J
= 1.6, 0.9 Hz, 18H), 0.06 (m, 11H).

7.26
TBSO
OTBS
COOMe

161

(R)-1-(3-iodophenyl)oct-1-yn-3-ol (7.27) 1,3-Diiodobenzene (7.25)
(413 mg, 1.25 mmol), alkyne (7.4) (100 mg, 0.4 mmols), Pd(PPh
3
)
4
a catalytic
amount, CuI (20 mg) , and NEt
3
(0.09 mL, 0.63 mmol) were added in 5 mL of
anhydrous benzene and stirred at room temperature overnight. Worked up with
saturated ammonium chloride aqueous solution and ether. The reaction was
quenched with saturated aqueous NH4Cl (30 mL) and extracted with Et2O (3 x
30 mL), dried using MgSO4, filtered and concentrated. The crude was then
purified using silica gel with a EtOAc- hexanes eluent (7.5%) to afford the singly
coupled product (7.27) (88 mg, 67%) as a yellow colored oil.
1
H NMR (400 MHz,
Chloroform-d) 7.78 (t, J = 1.7 Hz, 1H), 7.65 (ddd, J = 8.0, 1.8, 1.1 Hz, 1H), 7.38
(ddd, J = 7.7, 1.5, 1.0 Hz, 1H), 6.99 (m, 1H), 4.58 (td, J = 6.6, 5.6 Hz, 1H), 1.73 (m,
2H), 1.46 (m, 2H), 1.30 (m, 4H), 0.87 (m, 3H).
7.27
OH
I

162

(R)-tert-butyl((1-(3-iodophenyl)oct-1-yn-3-
yl)oxy)dimethylsilane (7.28) To a solution of intermediate (7.27) (150 mg,
0.46 mmols) in 10 mL of anhydrous CH 2Cl 2 at 0°C was added 2,6-lutidine (0.21
mL, 0.1.8 mmols) and TBS-OTf (483 mg, 0.1.8 mmols). The reaction mixture was
allowed to stir at rt overnight. The reaction mixture was quenched with saturated
aqueous NH4Cl (125 mL) and extracted with Et2O (3 x 125 mL). The organic
layer was dried with MgSO4, filtered and the solvent removed in vacuo. The
crude reaction mixture was purified on silica gel using EtOAc-hexanes (3%) to
afford the silyl-protected product (7.28) (189 mg, 93%) as yellow colored oil.
1
H
NMR (500 MHz, Chloroform-d) 7.75 (td, J = 1.7, 0.4 Hz, 1H), 7.63 (ddd, J = 8.0,
1.8, 1.1 Hz, 1H), 7.36 (ddd, J = 7.8, 1.6, 1.1 Hz, 1H), 7.03 (td, J = 7.9, 0.5 Hz, 1H),
4.54 (t, J = 6.5 Hz, 1H), 1.70 (m, 2H), 1.42 (m, 2H), 1.25 (m, 4H), 0.92 (m, 9H),
0.89 (m, 3H), 0.15 (d, J = 11.2 Hz, 6H).

OTBS
I
7.28

163

Methyl (5S,6R,E)-5,6-bis((tert-butyldimethylsilyl)oxy)-8-(3-
((R)-3-((tert-butyldimethylsilyl)oxy)oct-1-yn-1-yl)phenyl)oct-7-enoate
(7.56) Catecholborane (29 mg, 0.24 mmol) and acetylene (7.26) (100 mg, 0.24
mmol) was mixed in a pear- shape flask and stir overnight at 65°C. To the white
muddy substance, a catalytic amount of Pd (PP
3
)
4
and K
2
CO
3
(36 mg, 0.40
mmols) was added and aryl iodide (7.28) (53 mg, 0.12 mmol) was cannulated to
the mixture and dissolved in 1 mL of 1,4-dioxane and 2 mL degassed H 2O was
added into the mixture and the reaction was allowed to stir overnight at 80°C.
The reaction was quenched with saturated aqueous NH4Cl (15 mL) and extracted
with Et2O (3 x 15 mL). The solvent was removed in vacuo and the crude mixture
TBSO
OTBS
COOMe
TBSO
7.56

164

was purified on silica gel using EtOAc-hexanes (0.5%) as the eluent to afford
compound (7.56) (83 mg, 95%) as colorless oil.
1
H NMR (400 MHz, Chloroform-
d) 7.28 (m, 4H), 6.43 (d, J = 16.0 Hz, 1H), 6.15 (dd, J = 16.0, 7.2 Hz, 1H), 4.56
(td, J = 6.5, 4.6 Hz, 1H), 4.11 (ddd, J = 7.2, 5.0, 1.1 Hz, 1H), 3.65 (m, 1H), 3.63 (m,
3H), 2.31 (td, J = 7.3, 2.7 Hz, 2H), 1.44 (m, 8H), 1.30 (m, 4H), 0.86 (m, 25H), -
0.00 (m, 17H).
13
C NMR (400 MHz, cdcl 3) 174.14, 137.26, 133.97, 133.78, 128.84,
128.65, 128.59, 126.15, 123.60, 91.44, 84.03, 76.03, 73.57, 63.63, 51.58, 38.81,
34.51, 31.62, 26.11, 26.10, 26.02, 26.00, 25.15, 22.75, 20.62, 18.46, 14.18, -3.78, -
3.91, -4.21, -4.37, -4.54, -4.74.
methyl (5S,6R,E)-5,6-dihydroxy-8-(3-((R)-3-hydroxyoct-1-yn-1-
yl)phenyl)oct-7-enoate (7.57) To the late stage intermediate (7.56) (50 mg,
0.07 mmol) dissolved in 3.5 mL of anhydrous THF was added dropwise 6
equivalents of 1M TBAF (0.40 mL, 0.40 mmol) at 0°C. The reaction was
monitored closely via thin layer chromatography and after 4 h the reaction was
quenched with saturated NH4Cl (15 mL) and extracted with Et2O (5 x 15 mL).
The organic layer was rinsed with brine, dried over MgSO4 and filtered. The
HO
OH
COOMe
HO
7.57

165

solvent was then concentrated and freshly prepared CH2N2 was added to convert
any acid to the methyl ester. The product was purified via C-18 reversed Phase
HPLC using H2O-MeOH mixture (32%) to afford compound (7.57) (17 mg 65%)
as colorless oil.
1
H NMR (500 MHz, Methanol-d 4) 7.48 (dt, J = 6.0, 1.3 Hz, 1H),
7.36 (m, 1H), 7.26 (m, 2H), 6.61 (dd, J = 16.0, 1.2 Hz, 1H), 6.37 (dd, J = 16.0, 6.7
Hz, 1H), 4.51 (td, J = 6.7, 2.4 Hz, 1H), 4.06 (m, 1H), 3.65 (d, J = 6.7 Hz, 3H), 3.57
(ddd, J = 9.2, 5.1, 3.1 Hz, 1H), 2.34 (m, 2H), 1.84 (m, 1H), 1.71 (m, 2H), 1.62 (m,
1H), 1.51 (m, 2H), 1.36 (m, 4H), 1.28 (m, 2H), 0.92 (m, 3H).
13
C NMR (500 MHz,
cd 3od) 175.78, 138.71, 135.29, 131.72, 131.31, 130.36, 129.58, 127.27, 124.58,
91.60, 84.72, 76.79, 75.25, 63.13, 51.86, 38.93, 34.63, 33.10, 32.60, 32.58, 26.42,
26.04, 26.02, 23.57, 22.41, 14.26.
Methyl (5S,6R,E)-5,6-dihydroxy-8-(2-((R)-3-hydroxyoct-1-yn-1-
yl)phenyl)oct-7-enoate (7.58) This compound was produced as a side
product during the suzuki coupling reaction that produced intermediate (7.42),
and was isolated by HPLC during the purification of benzo-lipoxin A 4 analog
(7.46) to produce analog (7.58) (1.09 mg) as a colorless oil.
1
H NMR (400 MHz,
Methanol-d 4) 7.56 (m, 1H), 7.37 (dd, J = 7.7, 1.4 Hz, 1H), 7.25 (m, 1H), 7.19 (dd, J
= 7.5, 1.3 Hz, 1H), 7.05 (m, 1H), 6.42 (dd, J = 16.0, 6.9 Hz, 1H), 4.55 (t, J = 6.7
Hz, 1H), 4.09 (ddd, J = 6.7, 5.1, 1.3 Hz, 1H), 3.63 (s, 2H), 3.58 (ddd, J = 9.2, 5.1,
7.58
HO
OH
COOMe
OH

166

3.1 Hz, 1H), 2.36 (t, J = 7.0 Hz, 2H), 1.74 (m, 4H), 1.62 (m, 1H), 1.51 (m, 2H), 1.40
(m, 1H), 1.35 (m, 4H), 0.90 (m, 3H).
13
C NMR (500 MHz, cd 3od) 175.87, 139.74,
133.51, 132.28, 130.66, 129.51, 128.27, 126.14, 122.90, 96.58, 83.39, 77.22, 75.39,
63.40, 51.97, 39.09, 34.77, 33.24, 32.76, 26.25, 23.71, 22.54, 14.42.

methyl (5S,6R,E)-5,6-bis((tert-butyldimethylsilyl)oxy)-9-
oxonon-7-enoate (7.33) To a flask with
(Triphenylphosphoranylidene)acetaldehyde (255 mg, 0.84 mmols) was
cannulated aldehyde (7.11) (319 mg, 0.76 mmols) in 6 mL of anhydrous THF.
The mixture was refluxed at 65°C overnight. The reaction mixture with no
workup was condensed in vacuo followed by purification of the crude mixture on
silica gel using EtOAc-hexanes (7%) as the eluent to afford the extended aldehyde
(3.21) (253 mg, 75%) as dark red colored oil.
1
H NMR (400 MHz, Chloroform-d)
9.57 (d, J = 8.0 Hz, 1H), 6.85 (dd, J = 15.7, 5.3 Hz, 1H), 6.25 (ddd, J = 15.7, 8.0,
1.4 Hz, 1H), 4.27 (ddd, J = 5.5, 4.4, 1.4 Hz, 1H), 3.68 (m, 1H), 3.67 (s, 3H), 2.28
(m, 2H), 1.61 (m, 2H), 1.56 (s, 2H), 0.89 (d, J = 14.0 Hz, 18H), 0.05 (dd, J = 13.6,
7.3 Hz, 12H).
13
C NMR (400 MHz, cdcl 3) 193.58, 173.86, 157.86, 132.52, 76.16,
75.61, 51.68, 34.24, 33.27, 26.05, 26.01, 20.66, 18.37, 18.29, -3.89, -4.23, -4.39, -
4.61.
7.33
TBSO
OTBS
O
COOMe

167

Methyl (5S,6R,E)-5,6-bis((tert-butyldimethylsilyl)oxy)dec-7-en-
9-ynoate (7.35) ) Triphenylphosphine (530 mg, 2 mmol) in 1.5 mL of
anhydrous CH2Cl2 was cannulated in to a solution of CBr4 (334 mg, 1 mmol) at
0°C in 3 mL of anhydrous CH2Cl2 . Aldehyde (7.33) (224 mg, 0.5 mmol) in 2 mL
of anhydrous CH2Cl2 was cannulated in to the reaction mixture. The reaction
was stirred for 1 h at 0°C. Without workup the solvent was evaporated in vacuo
and the crude mixture was purified on silica gel using EtOAc-hexanes (1%) as the
eluent to afford the dibromo ester (291 mg, 97%) as a viscous and yellow colored
oil. To a solution of the dibromo ester (248 mg, 0.4 mmol) at -78°C in 10 mL of
anhydrous THF was added 2.0 M solution of LDA (1.25 mL, 2.5 mmol) drop wise
and stirred for 0.5 h. The reaction was quenched with water (30 mL) and
extracted with Et2O (3 x 30 mL), dried using MgSO4, filtered and concentrated.
The crude was then purified using silica gel with EtOAc- hexanes eluent (2%) to
afford the alkyne product (155 mg, 88%) as viscous and yellow colored oil.
1
H
NMR (400 MHz, Chloroform-d) 6.17 (ddd, J = 16.0, 6.4, 0.6 Hz, 1H), 5.58 (ddd, J
= 16.0, 2.3, 1.4 Hz, 1H), 3.97 (ddd, J = 6.3, 4.8, 1.4 Hz, 1H), 3.63 (s, 3H), 3.54 (dt,
J = 5.9, 4.6 Hz, 1H), 2.85 (dt, J = 2.3, 0.5 Hz, 1H), 2.26 (t, J = 7.4 Hz, 2H), 1.59
(m, 2H), 1.22 (m, 2H), 0.86 (d, J = 3.0 Hz, 18H), 0.80 (m, 3H), -0.01 (m, 12H).

13
C NMR (400 MHz, cdcl 3) 207.07, 177.72, 128.51, 82.28, 77.69, 76.40, 75.82,
51.63, 34.44, 31.08, 29.46, 26.10, 26.05, 22.81, 20.66, 18.36, 14.28, 12.76, -4.07, -
4.41, -4.60.
7.35
TBSO
OTBS
COOMe

168

Methyl (7S,8R,Z)-7,8-bis((tert-butyldimethylsilyl)oxy)dec-4-en-
9-ynoate (7.36) Triphenylphosphine (2.05 g, 7.8 mmol) in 5 mL of anhydrous
CH2Cl2 was cannulated in to a solution of CBr4 (905 mg, 3.9 mmol) at 0°C in 3
mL of anhydrous CH2Cl2 . Aldehyde (7.19) (500 mg, 1.95 mmol) in 6 mL of
anhydrous CH2Cl2 was cannulated in to the reaction mixture. The reaction was
stirred for 1 h at 0°C. Without workup the solvent was evaporated in vacuo and
the crude mixture was purified on silica gel using EtOAc-hexanes (5%) as the
eluent to afford the dibromo ester (780 mg, 97%) as a viscous and yellow colored
oil. To a solution of the dibromo ester (680 mg, 1.65 mmol) at -78°C in 10 mL of
anhydrous THF was added 2.0 M solution of LDA (5 mL, 9.9 mmol) drop wise
and stirred for 0.5 h. The reaction was quenched with water (50 mL) and
extracted with Et2O (3 x 50 mL), dried using MgSO4, filtered and concentrated.
The crude was then purified using silica gel with EtOAc- hexanes eluent (10%) to
afford the alkyne product (366 mg, 88%) as viscous and yellow colored oil. To a
solution of terminal alkyne (56 mg, 0.22 mmol) in 0.6 mL of MeOH at 0°C was
added 0.6 mL of 1M HCl. The reaction was stirred at rt and monitored closely by
TLC until complete consumption of starting material. The reaction was then
quenched with 5 mL of aqueous saturated NaHCO 3, extracted with CH 2Cl2 (3 x 5
mL) and dried over MgSO4. The organic phase was filtered and the solvent was
removed in vacuo to afford a crude oil, the crude product was purified on silica
gel using first pure pentanes and then EtOAc-hexanes (40%) as the eluent to
COOMe
OTBS TBSO
7.36

169

afford the deprotected terminal alkyne. To a solution of deprotected alkyne in 3
mL of anhydrous CH 2Cl 2 at 0°C was added 2,6-lutidine (0.08 mL, 0.92 mmols)
and TBS-OTf (174 mg, 0.66 mmols). The reaction mixture was allowed to stir at rt
overnight. The reaction mixture was quenched with saturated aqueous NH4Cl (15
mL) and extracted with Et2O (3 x 15 mL). The organic layer was dried with
MgSO4, filtered and the solvent removed in vacuo. The crude reaction mixture
was purified on silica gel using EtOAc-hexanes (3%) to afford the silyl-protected
vinyl iodide (7.36) (91 mg, 94%) as yellow colored oil.
1
H NMR (400 MHz,
Chloroform-d) 5.51 (dt, J = 10.9, 7.0 Hz, 1H), 5.39 (m, 1H), 4.24 (dd, J = 4.5, 2.2
Hz, 1H), 3.72 (dt, J = 6.5, 4.8 Hz, 1H), 3.67 (s, 3H), 2.29 (m, 6H), 2.17 (s, 1H),
0.89 (d, J = 6.3 Hz, 17H), 0.04 (m, 12H).
13
C NMR (400 MHz, cdcl 3) 178.62,
134.40, 132.30, 88.85, 80.80, 78.62, 71.85, 56.55, 39.03, 36.01, 30.97, 30.85,
28.06, 23.26, 23.21, 0.86, 0.62, 0.58, -0.00.
1-azido-3-bromobenzene (7.31) A solution of 1-bromo-3-iodobenzene
(7.32) (555 mg, 1.96 mmol), NaN 3 (134 mg, 2.06 mmol), CuI (37 mg, 0.19
mmol), L-proline (45 mg, 0.39 mmol), and NaOH (16 mg, 0.39 mmol) in 3.5 mL
of DMSO was allowed to stir at 65°C for 5 h. The reaction was quenched with
water (50 mL) and extracted with Et2O (3 x 50 mL), dried using MgSO4, filtered
and concentrated. The crude was then purified using silica gel with pentanes-
hexanes eluent (50%) to afford the aryl azide product (7.31) (320 mg, 82%).
7.31
N
3
Br

170

Methyl (5S,6R)-6-(1-(3-bromophenyl)-1H-1,2,3-triazol-4-yl)-5,6-
bis((tert-butyldimethylsilyl)oxy)hexanoate (7.30) A solution of sodium
ascorbate (15 mg, 0.072 mmol), azide (7.31) (40 mg, 0.2 mmol), and terminal
alkyne (7.26) (80 mg, 0.18 mmol) in 3 mL of t-BuOH and 1.5 mL of H 2O was
stirred at rt for 10 min. CuSO 4 (6 mg, 0.036 mmol) was added to the solution and
it was allowed to stir overnight at rt. The reaction was quenched with brine (15
mL) and extracted with Et2O (3 x 15 mL), dried using MgSO4, filtered and
concentrated. The crude was then purified using silica gel with EtOAc- hexanes
eluent (6%) to afford the click coupling product product (7.30) (20 mg, 20%) as
a colorless oil.
1
H NMR (500 MHz, Chloroform-d) 7.88 (m, 2H), 7.65 (d, J = 8.1
Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 8.1 Hz, 1H), 4.94 (d, J = 3.5 Hz, 1H),
4.00 (q, J = 5.0 Hz, 1H), 3.65 (d, J = 1.9 Hz, 3H), 2.28 (q, J = 7.1 Hz, 2H), 1.75
(dt, J = 13.0, 6.3 Hz, 1H), 1.61 (m, 2H), 1.47 (tt, J = 9.9, 5.7 Hz, 1H), 0.88 (d, J =
2.8 Hz, 23H), -0.08 (m, 12H).
13
C NMR (126 MHz, cdcl 3) 178.59, 154.94, 142.88,
136.33, 135.78, 128.34, 128.07, 125.32, 123.55, 80.66, 76.44, 56.20, 38.91, 37.40,
34.44, 30.68, 30.64, 25.54, 22.98, 22.93, 18.86, 0.54, 0.17, 0.02, -0.00.

7.30
N
N
N
Br
TBSO
OTBS
COOMe

171

Methyl (6S,7R,Z)-7-(1-(3-bromophenyl)-1H-1,2,3-triazol-4-yl)-
6,7-bis((tert-butyldimethylsilyl)oxy)hept-4-enoate (7.59) This
compound was prepared from terminal alkyne (7.36), and aryl azide (7.31)
similarly to compound (7.30).
1
H NMR (400 MHz, Chloroform-d) 7.94 (t, J =
2.0 Hz, 1H), 7.91 (d, J = 0.6 Hz, 1H), 7.65 (ddd, J = 8.1, 2.1, 1.0 Hz, 1H), 7.56
(ddd, J = 8.1, 1.9, 1.0 Hz, 1H), 7.40 (t, J = 8.1 Hz, 1H), 5.37 (m, 2H), 4.94 (m, 1H),
4.03 (td, J = 6.3, 3.1 Hz, 1H), 3.65 (s, 3H), 2.34 (d, J = 4.3 Hz, 2H), 2.18 (m, 2H),
0.88 (d, J = 1.4 Hz, 18H), 0.03 (m, 9H).
13
C NMR (101 MHz, cdcl 3) 173.70, 150.13,
131.69, 131.18, 129.62, 127.21, 123.76, 123.47, 120.86, 119.21, 118.96, 76.46, 71.76,
51.66, 34.03, 31.51, 29.85, 26.09, 26.07, 23.13, 18.39, 18.33, -4.14, -4.46, -4.53.
methyl (5S,6R,E)-8-(1-(3-bromophenyl)-1H-1,2,3-triazol-4-yl)-
5,6-bis((tert-butyldimethylsilyl)oxy)oct-7-enoate (7.60) This compound
was prepared from terminal alkyne (7.35), and aryl azide (7.31) similarly to
compound (7.30).
1
H NMR (400 MHz, Chloroform-d) 7.94 (t, J = 2.0 Hz, 1H),
7.59
OTBS
OTBS
COOMe
N
N
N
Br
7.60
TBSO
OTBS
COOMe
N
N
N
Br

172

7.88 (s, 1H), 7.71 (ddd, J = 8.1, 2.1, 1.0 Hz, 1H), 7.56 (ddd, J = 8.1, 1.8, 1.0 Hz,
1H), 7.40 (t, J = 8.1 Hz, 1H), 6.41 (m, 2H), 4.18 (ddd, J = 6.4, 4.5, 1.0 Hz, 1H),
3.67 (m, 1H), 3.66 (s, 3H), 2.31 (t, J = 7.3 Hz, 2H), 1.66 (m, 2H), 1.53 (m, 2H),
0.89 (d, J = 16.4 Hz, 18H), 0.02 (m, 12H).
13
C NMR (101 MHz, cdcl 3) 174.12,
138.08, 134.43, 131.77, 131.19, 126.74, 123.59, 123.44, 119.03, 119.01, 117.85,
77.48, 76.96, 76.06, 51.61, 34.50, 32.90, 26.14, 26.12, 20.82, 18.43, 18.33, -3.82, -
3.91, -4.40, -4.51.
Methyl (5S,6R)-5,6-bis((tert-butyldimethylsilyl)oxy)-6-(1-(3-
((R,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)phenyl)-1H-1,2,3-
triazol-4-yl)hexanoate (7.37) Catecholborane (63 mg, 0.52 mmol) and
acetylene (7.12) (126 mg, 0.52 mmol) was mixed in a pear- shape flask and stir
overnight at 65°C. To the white muddy substance, a catalytic amount of Pd
(PP
3
)
4
and K
2
CO
3
(47 mg, 0.27 mmols) was added and aryl bromide (7.30) (64
mg, 0.10 mmol) was cannulated to the mixture and dissolved in 1.8 mL of 1,4-
dioxane and 3.6 mL of degassed H 2O was added into the mixture and the reaction
was allowed to stir overnight at 80°C. The reaction was quenched with saturated
aqueous NH4Cl (15 mL) and extracted with Et2O (3 x 15 mL). The solvent was
7.37
TBSO
OTBS
COOMe
N
N
N
TBSO

173

removed in vacuo and the crude mixture was purified on silica gel using EtOAc-
hexanes (5%) as the eluent to afford compound (7.37) (62 mg, 79%) as colorless
oil.
1
H NMR (400 MHz, Chloroform-d) 7.92 (d, J = 0.5 Hz, 1H), 7.72 (dd, J = 2.2,
1.5 Hz, 1H), 7.53 (ddd, J = 7.7, 2.2, 1.4 Hz, 1H), 7.39 (m, 2H), 6.55 (dd, J = 16.0,
1.2 Hz, 1H), 6.30 (dd, J = 15.9, 6.0 Hz, 1H), 4.92 (m, 1H), 4.30 (qd, J = 6.1, 1.3
Hz, 1H), 4.02 (ddd, J = 6.2, 5.2, 3.5 Hz, 1H), 3.64 (s, 3H), 2.28 (ddd, J = 7.9, 7.0,
3.8 Hz, 2H), 1.64 (m, 2H), 1.53 (m, 4H), 1.43 (m, 1H), 1.27 (m, 5H), 0.85 (m,
25H), 0.02 (m, 13H).
13
C NMR (101 MHz, cdcl 3) 174.03, 149.95, 139.34, 137.69,
136.01, 129.98, 127.50, 126.61, 120.92, 119.06, 118.33, 76.12, 73.34, 71.90, 51.60,
38.46, 34.36, 32.88, 32.01, 29.86, 26.13, 26.11, 26.08, 26.07, 25.02, 22.78, 20.99,
18.37, 14.21, -4.04, -4.13, -4.41, -4.56, -4.58.
Methyl (6S,7R,Z)-6,7-bis((tert-butyldimethylsilyl)oxy)-7-(1-(3-
((R,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)phenyl)-1H-1,2,3-
triazol-4-yl)hept-4-enoate (7.61) This compound was prepared from
terminal alkyne (7.12), catecholborane and aryl bromide (7.59) similarly to
compound (7.37).
1
H NMR (400 MHz, Chloroform-d) 7.94 (s, 1H), 7.73 (t, J = 1.8
Hz, 1H), 7.53 (dt, J = 7.8, 1.7 Hz, 1H), 7.37 (m, 2H), 6.51 (m, 1H), 6.30 (dd, J =
15.9, 6.0 Hz, 1H), 5.36 (m, 2H), 4.97 (d, J = 3.1 Hz, 1H), 4.30 (qd, J = 6.1, 1.3 Hz,
7.61
OTBS
OTBS
COOMe
N
N
N
TBSO

174

1H), 4.04 (td, J = 6.3, 3.2 Hz, 1H), 3.64 (s, 3H), 2.27 (m, 4H), 2.17 (m, 2H), 1.51
(m, 6H), 0.86 (m, 31H), -0.08 (m, 17H).
13
C NMR (400 MHz, cdcl 3) 173.72,
149.72, 139.32, 137.73, 135.99, 129.97, 129.57, 127.53, 127.30, 126.57, 121.05,
119.06, 118.34, 76.50, 73.34, 71.80, 51.64, 38.46, 34.06, 32.01, 29.85, 26.11,
26.07, 25.01, 23.14, 22.78, 18.40, 18.34, 14.20, -4.15, -4.46, -4.53, -4.58.
methyl (5S,6R,E)-5,6-bis((tert-butyldimethylsilyl)oxy)-8-(1-(3-
((R,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)phenyl)-1H-1,2,3-
triazol-4-yl)oct-7-enoate (7.62) This compound was prepared from terminal
alkyne (7.12), catecholborane and aryl bromide (7.60) similarly to compound
(7.37).
1
H NMR (400 MHz, Chloroform-d) 7.90 (s, 1H), 7.70 (m, 1H), 7.55 (dt, J
= 7.7, 1.9 Hz, 1H), 7.38 (m, 2H), 6.57 (t, J = 16.8 Hz, 2H), 6.45 (dd, J = 16.1, 6.5
Hz, 1H), 6.30 (dd, J = 15.9, 6.0 Hz, 1H), 4.26 (m, 1H), 4.15 (m, 1H), 3.67 (m, 1H),
3.66 (s, 3H), 2.31 (t, J = 7.3 Hz, 2H), 1.72 (dddt, J = 23.0, 12.8, 7.4, 5.5 Hz, 3H),
1.58 (ddd, J = 15.7, 9.7, 4.1 Hz, 3H), 1.27 (m, 6H), 0.86 (m, 29H), 0.04 (m, 18H).

13
C NMR (400 MHz, cdcl 3) 174.13, 146.54, 139.33, 137.48, 136.04, 133.93, 129.94,
127.47, 126.67, 119.37, 119.12, 118.37, 118.10, 77.03, 76.09, 73.34, 51.59, 38.44,
7.62
TBSO
OTBS
COOMe
N
N
N
TBSO

175

34.50, 32.87, 31.99, 29.84, 26.13, 26.07, 25.80, 25.00, 22.77, 20.83, 18.44, 18.42,
18.32, 14.19, -3.43, -3.82, -3.89, -4.13, -4.40, -4.52, -4.59.
Methyl (5S,6R)-5,6-dihydroxy-6-(1-(3-((R,E)-3-hydroxyoct-1-en-
1-yl)phenyl)-1H-1,2,3-triazol-4-yl)hexanoate (7.29) To the late stage
intermediate (7.37) (10 mg, 0.013 mmol) dissolved in 1 mL of anhydrous THF
was added dropwise 6 equivalents of 1M TBAF (0.08 mL, 0.08 mmol) at 0°C. The
reaction was monitored closely via thin layer chromatography and after 4 h the
reaction was quenched with saturated NH4Cl (15 mL) and extracted with Et2O (5
x 15 mL). The organic layer was rinsed with brine, dried over MgSO4 and filtered.
The solvent was then concentrated and freshly prepared CH2N2 was added to
convert any acid to the methyl ester. The product was purified via C-18 reversed
Phase HPLC using H2O-MeOH mixture (16%) to afford compound (7.29) (3.5
mg 62%) as colorless oil.
1
H NMR (400 MHz, Methanol-d 4) 8.35 (s, 1H), 7.76 (m,
1H), 7.62 (dt, J = 6.6, 2.2 Hz, 1H), 7.41 (m, 2H), 6.55 (dd, J = 15.9, 1.1 Hz, 1H),
6.31 (dd, J = 15.9, 6.3 Hz, 1H), 4.69 (d, J = 5.2 Hz, 1H), 4.26 (m, 1H), 3.84 (s, 1H),
3.56 (s, 3H), 2.25 (m, 2H), 1.92 (m, 1H), 1.72 (m, 1H), 1.47 (m, 5H), 1.35 (dddd, J
= 12.4, 10.9, 6.8, 2.6 Hz, 3H).
13
C NMR (400 MHz, cd 3od) 175.84, 140.57, 138.90,
7.29
HO
OH
COOMe
N
N
N
HO

176

136.89, 131.19, 129.02, 127.76, 122.58, 120.28, 119.08, 111.43, 74.73, 71.48, 51.97,
49.85, 39.37, 34.67, 33.00, 26.41, 23.72, 22.43, 14.38, -4.02.

Methyl (6S,7R,Z)-6,7-dihydroxy-7-(1-(3-((R,E)-3-hydroxyoct-1-
en-1-yl)phenyl)-1H-1,2,3-triazol-4-yl)hept-4-enoate (7.63) This
compound was prepared from late stage intermediate (7.61), similarly to
compound (7.29).
1
H NMR (400 MHz, Chloroform-d) 8.14 (s, 1H), 7.78 (s, 1H),
7.58 (dd, J = 7.4, 1.9 Hz, 1H), 7.39 (m, 2H), 6.63 (dd, J = 15.9, 1.2 Hz, 1H), 6.34
(dd, J = 15.9, 6.4 Hz, 1H), 5.43 (m, 2H), 4.96 (s, 1H), 4.32 (td, J = 6.3, 5.2 Hz,
1H), 4.08 (s, 1H), 3.65 (s, 3H), 2.38 (m, 4H), 2.24 (m, 1H), 1.59 (m, 2H), 1.36 (m,
1H), 1.30 (m, 3H), 0.86 (m, 3H).
13
C NMR (400 MHz, cdcl 3) 174.24, 138.97,
135.09, 135.09, 131.04, 130.09, 128.65, 128.65, 127.03, 126.62, 119.53, 118.46,
110.16, 77.65, 77.36, 72.88, 51.88, 37.50, 33.60, 31.91, 29.85, 25.25, 22.75, 14.19.

7.63
OH
OH
COOMe
N
N
N
HO

177

Methyl (5S,6R,E)-5,6-dihydroxy-8-(1-(3-((R,E)-3-hydroxyoct-1-
en-1-yl)phenyl)-1H-1,2,3-triazol-4-yl)oct-7-enoate (7.64) To the late stage
intermediate (7.62) (50 mg, 0.0625 mmol) dissolved in 1 mL of anhydrous THF
was added dropwise 6 equivalents of 1M TBAF (0.375 mL, 0.375 mmol) at 0°C.
The reaction was monitored closely via thin layer chromatography and after 4 h
the reaction was quenched with saturated NH4Cl (15 mL) and extracted with
Et2O (5 x 15 mL). The organic layer was rinsed with brine, dried over MgSO4 and
filtered. The solvent was then concentrated and freshly prepared CH2N2 was
added to convert any acid to the ester-lactone mixture. The solvent was
completely removed in vacuo and the compound was purified on silica gel using
MeOH-CH2Cl2 (10%) as the eluent to afford an ester/lactone mixture. The
product was then suspended in a H2O- MeOH mixture (1:1, 1 mL) and 10
equivalents of LiOH (17 mg, 0.7 mmol) was added. After 3 h the reaction mixture
was dried and purified via C-18 reversed Phase HPLC using H2O-MeOH mixture
(16%) to afford compound (7.64) (6 mg 22%) as colorless oil.
1
H NMR (400
MHz, Methanol-d 4) 8.60 (s, 1H), 7.90 (d, J = 2.6 Hz, 1H), 7.72 (dt, J = 5.4, 2.5 Hz,
1H), 7.51 (m, 2H), 6.59 (m, 3H), 6.43 (dd, J = 15.9, 6.4 Hz, 1H), 4.25 (q, J = 6.3
7.64
HO
OH
COOH
N
N
N
HO

178

Hz, 1H), 4.15 (t, J = 5.3 Hz, 1H), 3.57 (m, 1H), 2.76 (m, 4H), 2.35 (td, J = 7.4, 4.4
Hz, 2H), 1.87 (tdd, J = 12.1, 7.8, 2.4 Hz, 1H), 1.71 (tdd, J = 12.1, 5.6, 2.8 Hz, 2H),
1.57 (m, 2H), 1.48 (dddd, J = 17.5, 12.6, 8.7, 4.5 Hz, 2H), 1.28 (m, 3H), 0.87 (m,
3H).

13
C NMR (126 MHz, cd 3od) ? 176.11, 146.31, 139.22, 137.27, 135.06, 132.71,
129.73, 127.91, 126.43, 119.28, 118.71, 118.69, 117.55, 76.54, 75.10, 71.92, 50.57,
42.58, 36.94, 33.49, 31.77, 31.59, 24.92, 22.30, 21.17, 12.99.
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APPENDIX.
1
H and
13
C Spectra of Substrates

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500 E G C G
1 . 2 5
1 . 2 2
0 . 9 5
1 . 0 0
1 . 0 5
1 . 0 0
1 . 9 1
1 . 9 1
2 . 7 9
2 . 8 0
2 . 8 4
2 . 8 4
2 . 9 2
2 . 9 3
2 . 9 6
2 . 9 7
4 . 9 8
5 . 3 8
5 . 3 8
5 . 3 8
5 . 3 8
5 . 3 9
5 . 3 9
5 . 3 9
5 . 4 0
5 . 9 5
5 . 9 6
5 . 9 8
5 . 9 9
6 . 5 2
6 . 5 2
6 . 8 9
Fig. 2
1
H NMR (500 MHz, 80% CD 3N/ 20% D 2O) of epigallocatechin gallate (EGCG).
O
O
OH
OH
OH
O
OH
OH
OH
HO
OH

196

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
B Z M
5 . 8 5
1 . 3 2
2 . 3 1
1 . 1 9
1 . 2 4
1 . 3 7
1 . 0 0
1 . 1 5
3 . 6 9
0 . 9 6
0 . 9 6
0 . 8 8
0 . 7 8
0 . 7 9
0 . 8 0
0 . 8 1
1 . 2 1
1 . 2 2
1 . 2 2
1 . 2 3
1 . 2 4
1 . 2 4
1 . 2 5
1 . 3 2
1 . 3 3
1 . 3 4
1 . 3 5
1 . 3 5
1 . 3 6
1 . 3 6
1 . 3 7
1 . 3 9
2 . 9 1
2 . 9 2
2 . 9 3
2 . 9 4
3 . 0 5
3 . 0 6
3 . 0 7
3 . 0 9
3 . 1 8
3 . 1 9
3 . 2 0
3 . 2 1
4 . 7 6
4 . 7 7
4 . 7 7
4 . 7 8
7 . 1 8
7 . 1 8
7 . 1 8
7 . 1 9
7 . 1 9
7 . 1 9
7 . 1 9
7 . 2 0
7 . 2 3
7 . 2 4
7 . 2 4
8 . 6 2
8 . 6 2
8 . 6 2
8 . 6 2
8 . 7 4
8 . 7 4
9 . 1 1
9 . 1 1
B
H
N
O
N
H
O
N
N
OH
OH
Fig. 2
1
H NMR (500 MHz, 80% CD 3N/ 20% D 2O) of bortezomib (BZM).
B
H
N
O
N
H
O
N
N
OH
OH
Fig. 2
11
B NMR (500 MHz, 80% CD 3N/ 20% D 2O) of bortezomib (BZM).
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
f1 (ppm)
-20
-15
-10
-5
0
5
10
15
20
25
30
35
40
45
50
55
60 B Z M
2 7 . 9 6
2 8 . 5 5

197

Fig. 2
1
H NMR (500 MHz, 80% CD 3N/ 20% D 2O) of BZM/EGCG complex (1:0.5)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
f1 (ppm)
0
500
1000
1500
2000
2500
B Z M : 1 / E G C G : 0 . 5
0 . 8 0
4 . 5 3
1 . 4 3
2 . 1 6
0 . 1 9
0 . 3 1
1 . 0 6
0 . 2 2
1 . 1 0
1 . 4 6
1 . 0 0
0 . 3 4
0 . 3 0
0 . 4 6
0 . 4 1
0 . 3 9
0 . 7 2
0 . 5 1
0 . 0 9
1 . 1 3
3 . 8 5
1 . 1 1
1 . 2 1
1 . 0 4
0 . 7 3
0 . 7 5
0 . 7 8
0 . 7 9
0 . 8 1
1 . 1 9
1 . 2 1
1 . 2 3
1 . 2 4
1 . 2 5
1 . 2 6
1 . 3 1
1 . 3 2
1 . 3 3
1 . 3 5
1 . 3 6
1 . 3 8
1 . 3 8
1 . 3 9
2 . 8 0
2 . 8 4
2 . 8 4
2 . 9 0
2 . 9 2
2 . 9 3
2 . 9 4
2 . 9 6
3 . 0 4
3 . 0 6
3 . 0 7
3 . 0 9
3 . 1 7
3 . 1 8
3 . 2 0
3 . 2 2
4 . 7 6
4 . 7 7
4 . 7 8
4 . 7 9
4 . 9 8
5 . 3 7
5 . 3 8
5 . 3 8
5 . 3 9
5 . 9 5
5 . 9 6
5 . 9 8
5 . 9 9
6 . 5 2
6 . 8 9
6 . 9 1
7 . 1 8
7 . 1 9
7 . 2 0
7 . 2 3
7 . 2 3
7 . 2 4
7 . 2 4
7 . 2 5
8 . 6 1
8 . 6 2
8 . 6 2
8 . 6 2
8 . 7 4
8 . 7 4
9 . 1 0
9 . 1 1
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
O
HO OH
OH
OH
HO
Fig. 2
11
B NMR (500 MHz, 80% CD 3N/ 20% D 2O) of BZM/EGCG complex (1:0.5)
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
O
HO OH
OH
OH
HO
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
f1 (ppm)
-20
-15
-10
-5
0
5
10
15
20
25
30
35
40
45
B Z M : 1 / E G C G : 0 . 5
0 . 1 2
1 . 0 0
1 9 . 6 7
2 7 . 4 1

198

Fig. 2
1
H NMR (500 MHz, 80% CD 3N/ 20% D 2O) of BZM/EGCG complex (1:1)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
f1 (ppm)
0
500
1000
1500
2000
2500
3000
3500
4000
B Z M : 1 / E G C G : 1
6 . 1 9
1 . 1 5
1 . 0 7
1 . 1 0
0 . 3 2
0 . 6 7
1 . 4 9
0 . 3 4
1 . 1 3
1 . 4 2
1 . 0 0
1 . 0 5
0 . 5 2
1 . 0 8
0 . 7 6
0 . 7 7
1 . 3 8
1 . 1 0
0 . 1 8
2 . 3 2
1 . 5 6
0 . 9 1
1 . 0 8
1 . 0 1
0 . 7 7
0 . 7 9
0 . 8 1
1 . 2 3
1 . 2 4
1 . 2 5
1 . 2 6
1 . 3 1
1 . 3 2
1 . 3 3
1 . 3 5
1 . 3 6
1 . 3 8
1 . 3 8
1 . 3 9
1 . 4 0
2 . 7 9
2 . 8 0
2 . 8 4
2 . 8 4
2 . 8 4
2 . 9 0
2 . 9 2
2 . 9 3
2 . 9 4
2 . 9 6
3 . 0 4
3 . 0 6
3 . 0 7
3 . 0 9
3 . 1 7
3 . 1 8
3 . 2 0
3 . 2 2
4 . 7 6
4 . 7 7
4 . 7 8
4 . 7 9
4 . 9 8
5 . 3 7
5 . 3 8
5 . 3 8
5 . 3 9
5 . 3 9
5 . 3 9
5 . 9 6
5 . 9 8
6 . 5 2
6 . 8 9
6 . 9 1
7 . 2 3
7 . 2 4
7 . 2 5
8 . 6 1
8 . 6 2
8 . 6 2
8 . 6 2
8 . 7 4
8 . 7 4
9 . 1 0
9 . 1 1
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
O
HO OH
OH
OH
HO
Fig. 2
11
B NMR (500 MHz, 80% CD 3N/ 20% D 2O) of BZM/EGCG complex (1:1)
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
O
HO OH
OH
OH
HO
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
f1 (ppm)
-16
-14
-12
-10
-8
-6
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
B Z M : 1 / E G C G : 1
0 . 2 2
1 . 0 0
1 9 . 5 0
2 7 . 7 6

199

Fig. 2
1
H NMR (500 MHz, 80% CD 3N/ 20% D 2O) of BZM/EGCG complex (1:4)
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
O
HO OH
OH
OH
HO
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
4 . 6 4
0 . 9 3
1 . 2 8
2 . 0 6
1 . 1 8
2 . 4 1
2 . 2 8
1 . 2 3
1 . 0 6
0 . 3 3
1 . 4 6
0 . 4 0
0 . 8 9
2 . 9 4
0 . 8 8
2 . 9 1
2 . 9 6
2 . 7 9
5 . 6 8
0 . 4 2
4 . 8 1
1 . 0 0
1 . 0 0
0 . 9 0
0 . 7 4
0 . 7 5
0 . 7 5
0 . 7 9
0 . 8 0
0 . 8 0
0 . 8 4
0 . 8 6
0 . 8 9
0 . 9 1
1 . 2 4
1 . 2 5
1 . 2 6
1 . 2 7
1 . 3 1
1 . 3 5
1 . 3 8
1 . 4 0
2 . 8 0
2 . 8 0
2 . 8 0
2 . 8 4
2 . 8 5
2 . 9 2
2 . 9 3
2 . 9 6
2 . 9 7
3 . 0 6
3 . 0 7
3 . 0 7
3 . 0 8
3 . 0 9
3 . 0 9
3 . 1 0
3 . 1 7
3 . 1 8
3 . 1 9
3 . 1 9
3 . 2 0
3 . 2 1
3 . 2 2
3 . 2 2
3 . 2 3
3 . 2 3
3 . 2 4
4 . 7 6
4 . 7 8
4 . 8 0
4 . 9 8
5 . 0 5
5 . 0 5
5 . 0 7
5 . 3 8
5 . 3 8
5 . 3 8
5 . 3 8
5 . 3 9
5 . 3 9
5 . 3 9
5 . 9 5
5 . 9 6
5 . 9 9
5 . 9 9
6 . 5 2
6 . 5 2
6 . 8 9
6 . 9 1
8 . 6 0
8 . 6 1
8 . 6 1
8 . 6 1
8 . 7 3
8 . 7 3
8 . 7 4
8 . 7 4
8 . 7 5
9 . 1 0
9 . 1 0
9 . 1 1
9 . 1 1
9 . 1 2
Fig. 2
11
B NMR (500 MHz, 80% CD 3N/ 20% D 2O) of BZM/EGCG complex (1:4)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
f1 (ppm)
-6
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
B Z M : 1 / E G C G : 4
4 . 2 4
1 . 0 0
2 0 . 1 5
2 7 . 8 0
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
O
HO OH
OH
OH
HO

200

B
H
N
O
N
H
O
N
N
OH
OH
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
f1 (ppm)
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
B Z M
2 1 . 3 3
2 2 . 9 3
2 5 . 3 1
3 7 . 8 7
3 9 . 5 7
5 4 . 2 1
1 2 7 . 2 0
1 2 7 . 2 2
1 2 8 . 8 3
1 2 8 . 8 5
1 2 9 . 7 2
1 3 7 . 0 3
1 4 3 . 7 5
1 4 4 . 0 8
1 4 4 . 4 2
1 4 8 . 0 6
Fig. 2
13
C NMR (500 MHz, 80% CD 3N/ 20% D 2O) of BZM.
O
O
OH
OH
OH
O
OH
OH
OH
HO
OH
60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180
f1 (ppm)
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
E G C G
6 9 . 2 8
7 7 . 2 3
9 5 . 0 5
9 5 . 8 1
9 8 . 4 4
1 0 6 . 0 7
1 0 9 . 3 7
1 1 8 . 2 6
1 2 0 . 7 7
1 3 0 . 1 2
1 4 5 . 0 3
1 4 5 . 3 0
1 5 6 . 0 2
1 5 6 . 4 9
1 5 6 . 5 7
1 6 6 . 2 9
Fig. 2
13
C NMR (500 MHz, 80% CD 3N/ 20% D 2O) of EGCG.

201

-128.2 -128.0 -127.8 -127.6 -127.4 -127.2 -127.0 -126.8 -126.6 -126.4 -126.2 -126.0 -125.8 -125.6 -125.4 -125.2 -125.0 -124.8 -124.6 -124.4 -124.2 -124.0 -123.8 -123.6 -123.4 -123.2 -123.0 -122.8 -122.6 -122.4 -122.2
f1 (ppm)
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
F C A T
- 1 2 4 . 9 4
- 1 2 4 . 9 2
- 1 2 4 . 9 1
- 1 2 4 . 9 1
- 1 2 4 . 9 0
- 1 2 4 . 8 9
- 1 2 4 . 8 7
Fig. 3
19
F NMR (400 MHz, 80% CD 3N/ 20% D 2O) of fluorobenzene-1,2-diol (FCAT)
OH
OH
F
Fig. 2
13
C NMR (500 MHz, 80% CD 3N/ 20% D 2O) of BZM/EGCG complex (1:1)
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
O
HO OH
OH
OH
HO
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210
f1 (ppm)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
2 1 . 3 1
2 1 . 3 4
2 2 . 0 1
2 2 . 0 2
2 2 . 0 7
2 2 . 1 2
2 2 . 9 1
2 4 . 0 9
2 4 . 1 0
2 4 . 3 5
2 4 . 5 0
2 5 . 3 0
2 5 . 6 4
2 5 . 9 1
3 7 . 0 3
3 7 . 8 1
3 7 . 8 4
3 8 . 2 0
3 9 . 5 5
4 4 . 2 5
4 4 . 3 5
5 4 . 1 8
5 4 . 6 6
5 4 . 9 1
6 9 . 3 3
7 2 . 3 2
7 2 . 4 2
7 7 . 2 8
9 5 . 0 9
9 5 . 8 5
9 8 . 4 8
9 8 . 5 0
1 0 6 . 0 8
1 0 6 . 1 2
1 0 9 . 4 2
1 2 0 . 8 2
1 2 7 . 1 8
1 2 7 . 1 9
1 2 7 . 6 2
1 2 8 . 7 7
1 2 8 . 8 0
1 2 8 . 8 2
1 2 9 . 0 4
1 2 9 . 6 7
1 2 9 . 6 9
1 2 9 . 7 4
1 2 9 . 7 7
1 3 0 . 1 7
1 3 2 . 2 1
1 3 5 . 6 5
1 3 6 . 8 6
1 3 6 . 9 9
1 3 7 . 0 1
1 3 8 . 2 9
1 4 3 . 6 9
1 4 3 . 7 1
1 4 3 . 7 3
1 4 3 . 8 6
1 4 4 . 0 0
1 4 4 . 0 3
1 4 4 . 0 5
1 4 4 . 3 8
1 4 4 . 4 0
1 4 5 . 0 8
1 4 5 . 3 3
1 4 5 . 3 6
1 4 8 . 0 0
1 5 6 . 0 7
1 5 6 . 5 5
1 5 6 . 6 3
1 6 3 . 6 5
1 6 3 . 6 7
1 6 3 . 9 0
1 6 6 . 3 4
1 7 1 . 4 3
1 7 1 . 4 5
1 7 1 . 8 5

202

Fig. 3
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/FCAT complex (1:1)
-50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45 50
f1 (ppm)
-15
-10
-5
0
5
10
15
20
25
30
35
B Z M : 1 / F C A T : 1
- 0 . 0 7
- 0 . 0 2
1 . 0 0
1 6 . 4 8
1 9 . 7 5
2 8 . 9 7
B
H
N
O
N
H
O
N
N
O
O
F
Fig. 3
19
F NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/FCAT complex (1:1)
-128.8 -128.6 -128.4 -128.2 -128.0 -127.8 -127.6 -127.4 -127.2 -127.0 -126.8 -126.6 -126.4 -126.2 -126.0 -125.8 -125.6 -125.4 -125.2 -125.0 -124.8 -124.6 -124.4 -124.2 -124.0 -123.8 -123.6 -123.4 -123.2 -123.0 -122.8 -122.6
f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000 B Z M : 1 / F C A T : 1
0 . 1 0
1 . 0 0
- 1 2 5 . 7 0
- 1 2 4 . 9 3
- 1 2 4 . 9 2
- 1 2 4 . 9 1
- 1 2 4 . 8 9
- 1 2 4 . 8 8
B
H
N
O
N
H
O
N
N
O
O
F

203

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
f1 (ppm)
-20
-15
-10
-5
0
5
10
15
20
25
30
35
40
45
50 B Z M : 1 / I S O : 0 . 5
0 . 0 3
0 . 0 1
1 . 0 0
1 6 . 1 6
1 9 . 6 4
2 7 . 9 3
Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/ISO complex (1:0.5)
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
f1 (ppm)
-12
-10
-8
-6
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
B Z M : 1 / I S O : 1
0 . 2 0
0 . 1 2
1 . 0 0
1 6 . 0 0
1 9 . 7 1
2 7 . 9 1
Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/ISO complex (1:1)
B
H
N
O
N
H
O
N
N
O
O
O
O
OH

204

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52
f1 (ppm)
-5
0
5
10
15
20
B Z M : 1 / I S O : 2
0 . 7 8
0 . 6 9
1 . 0 0
1 6 . 0 6
1 9 . 7 0
2 7 . 6 7
Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/ISO complex (1:2)
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/ISO complex (1:4)
B
H
N
O
N
H
O
N
N
O
O
O
O
OH
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
f1 (ppm)
-10
-5
0
5
10
15
20
25
30
35
B Z M : 1 / I S O : 4
2 . 9 5
4 . 7 4
1 . 0 0
1 6 . 1 1
1 9 . 6 9
2 7 . 9 6

205

0 5 10 15 20 25 30 35 40 45 50
f1 (ppm)
-20
-15
-10
-5
0
5
10
15
20
25
30
35
40
B Z M : 1 / P Y R O : 0 . 5
0 . 0 5
1 . 0 0
1 9 . 7 2
2 7 . 8 8
Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/PYRO complex (1:0.5)
B
H
N
O
N
H
O
N
N
O
O
OH
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
f1 (ppm)
-3
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
B Z M : 1 / P Y R O : 1
0 . 0 8
0 . 9 4
1 . 0 0
Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/PYRO complex (1:1)
B
H
N
O
N
H
O
N
N
O
O
OH

206

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
B Z M : 1 / P Y R O : 2
0 . 3 8
2 . 3 6
1 . 0 0
1 5 . 9 1
1 9 . 8 9
2 8 . 2 1
Fig. 4, 5
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/PYRO complex (1:2)
B
H
N
O
N
H
O
N
N
O
O
OH
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
f1 (ppm)
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
B Z M : 1 / P Y R O : 4
0 . 7 7
4 . 8 5
1 . 0 0
1 6 . 1 0
1 9 . 9 5
2 8 . 0 3
Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/PYRO complex (1:4)
B
H
N
O
N
H
O
N
N
O
O
OH

207

Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/RES complex (1:0.5)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
f1 (ppm)
-25
-20
-15
-10
-5
0
5
10
15
20
25
30
35
40
45
50
B Z M : 1 / R E S : 0 . 5
0 . 1 1
1 . 0 0
2 0 . 4 5
2 8 . 1 7
H
N
O
N
H
O
N
N
B OH
O HO
O
H
Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/RES complex (1:1)
0 5 10 15 20 25 30 35 40 45 50
f1 (ppm)
-15
-10
-5
0
5
10
15
20
25
30
35
B Z M : 1 / R E S : 1
0 . 2 6
1 . 0 0
2 0 . 0 9
2 8 . 1 4
H
N
O
N
H
O
N
N
B OH
O HO
O
H

208

Fig. 4
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/RES complex (1:4)
0 5 10 15 20 25 30 35 40 45 50
f1 (ppm)
-5
0
5
10
15
20
25
30
35
B Z M : 1 / R E S : 4
0 . 6 9
1 . 0 0
1 9 . 7 0
2 7 . 8 1
H
N
O
N
H
O
N
N
B OH
O HO
O
H
Fig. 4, 5
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/RES complex (1:2)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
f1 (ppm)
-10
-5
0
5
10
15
20
25
30
35
B Z M : 1 / R E S : 2
0 . 4 7
1 . 0 0
2 0 . 2 0
2 8 . 2 5
H
N
O
N
H
O
N
N
B OH
O HO
O
H

209

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
f1 (ppm)
-15
-10
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75 B Z M : 1 / C A T : 2
0 . 3 8
0 . 8 8
1 . 0 0
1 6 . 0 9
2 0 . 0 7
2 8 . 1 4
Fig. 5
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/CAT complex (1:2)
B
H
N
O
N
H
O
N
N
O
O
Fig. 5
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/PHE complex (1:2)
B
H
N
O
N
H
O
N
N
OH
O
-5 0 5 10 15 20 25 30 35 40
f1 (ppm)
-12
-10
-8
-6
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
B Z M : 1 : P H E : 2
0 . 1 5
1 . 0 0
1 9 . 5 9
2 8 . 0 7

210

Fig. 5
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/NCAT complex (1:2)
B
H
N
O
N
H
O
N
N
O
O
NO
2
0 5 10 15 20 25 30 35 40 45 50
f1 (ppm)
-5
0
5
10
15
20
25
30
35
40
45
B Z M : 1 / N C A T : 2
0 . 6 0
3 . 2 9
1 . 0 0
1 6 . 3 4
1 9 . 8 9
2 8 . 0 4
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
f1 (ppm)
-6
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
B Z M : 1 / D H B A : 2
0 . 6 5
1 . 0 2
1 . 0 0
1 6 . 4 8
2 0 . 2 7
2 8 . 4 2
Fig. 5
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/DHBA complex (1:2)
B
H
N
O
N
H
O
N
N
O
O
OH
O

211

Fig. 6
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/RSV complex (1:0.5)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
f1 (ppm)
-20
-15
-10
-5
0
5
10
15
20
25
30
35
40
45
B Z M : 1 / R S V : 0 . 5
1 . 0 0
2 8 . 4 0
H
N
O
N
H
O
N
N
B OH
O HO
O
H
OH
Fig. 6
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/RSV complex (1:1)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
f1 (ppm)
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19 B Z M : 1 / R S V : 1
0 . 0 3
1 . 0 0
1 9 . 7 2
2 7 . 5 7
H
N
O
N
H
O
N
N
B OH
O HO
O
H
OH

212

Fig. 6
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/RSV complex (1:2)
0 5 10 15 20 25 30 35 40 45
f1 (ppm)
-8
-6
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
B Z M : 1 / R S V : 2
0 . 1 5
1 . 0 0
1 9 . 6 2
2 7 . 2 1
H
N
O
N
H
O
N
N
B OH
O HO
O
H
OH
Fig. 6
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/RSV complex (1:4)
0 5 10 15 20 25 30 35 40 45
f1 (ppm)
-6
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
B Z M : 1 / R S V : 4
0 . 6 0
1 . 0 0
1 9 . 6 2
2 7 . 5 9
H
N
O
N
H
O
N
N
B OH
O HO
O
H
OH

213

5 10 15 20 25 30 35 40 45 50
f1 (ppm)
-15
-10
-5
0
5
10
15
20
25
30
B Z M : 1 / C A T N : 0 . 5
0 . 0 5
1 . 0 0
2 0 . 4 8
2 7 . 8 7
B
H
N
O
N
H
O
N
N
O
O
O
OH
OH
HO
Fig. 6
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/CATN complex (1:0.5)
-5 0 5 10 15 20 25 30 35 40 45
f1 (ppm)
-15
-10
-5
0
5
10
15
20
25
30
35
B Z M : 1 / C A T N : 1
0 . 2 4
1 . 0 0
2 0 . 2 8
2 7 . 7 2
Fig. 6
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/CATN complex (1:1)
B
H
N
O
N
H
O
N
N
O
O
O
OH
OH
HO

214

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52
f1 (ppm)
-6
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
B Z M : 1 / C A T N : 2
0 . 0 7
1 . 6 9
1 . 0 0
1 6 . 1 5
1 9 . 8 9
2 8 . 0 4
Fig. 6
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/CATN complex (1:2)
B
H
N
O
N
H
O
N
N
O
O
O
OH
OH
HO
Fig. 6
11
B NMR (400 MHz, 80% CD 3N/ 20% D 2O) of BZM/CATN complex (1:4)
B
H
N
O
N
H
O
N
N
O
O
O
OH
OH
HO
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
f1 (ppm)
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
B Z M : 1 / C A T N : 4
3 . 1 9
1 . 0 0
1 9 . 4 4
1 9 . 7 3
2 7 . 9 1

215

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
4.28
2.73
1.04
0.98
0.55
1.00
3.56
3.58
3.61
3.63
3.65
3.66
3.73
4.23
4.23
4.24
4.24
4.25
4.26
6.08
6.08
6.12
6.12
6.18
6.18
6.19
6.19
6.20
6.20
6.20
6.20
7.11
7.12
7.15
7.16
HO
HO
COOMe
OH
3.14
HO
HO
COOMe
OH
3.14
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
50.66
62.92
71.25
74.35
148.69
167.20
174.13

216

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
-400
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
13.69
23.14
1.84
1.45
0.72
1.01
3.00
0.68
0.04
0.05
0.07
0.88
0.89
0.89
0.89
1.77
1.77
1.78
1.79
1.80
1.83
1.85
1.87
1.87
1.87
1.89
2.35
2.35
2.37
2.37
2.39
2.41
2.42
2.45
3.43
3.45
3.46
3.47
3.58
3.58
3.59
3.60
3.66
3.77
3.78
3.79
TBSO
TBSO
COOMe
OTBS
3.12
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
-5.44
-5.38
-4.88
-4.68
-4.37
-4.11
25.64
25.86
25.89
25.93
25.97
25.98
27.19
30.11
51.42
64.84
72.74
77.10
174.38
TBSO
TBSO
COOMe
OTBS
3.12

217

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
-5.12
-4.66
17.81
17.99
25.52
25.54
25.55
28.14
29.27
51.34
73.76
80.59
173.36
203.06

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
11.86
18.68
2.59
2.08
3.00
1.08
1.11
0.99
0.06
0.08
0.08
0.87
0.91
1.84
1.85
1.85
1.86
1.86
1.91
1.91
1.92
1.92
1.92
1.94
2.36
2.37
2.39
3.67
3.88
3.88
3.89
3.95
3.96
3.96
3.96
9.59
9.60
O
TBSO
COOMe
OTBS
3.15
O
TBSO
COOMe
OTBS
3.15

218

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
f1 (ppm)
0
100
200
300
400
500
600
700
800
900
1000
12.69
19.51
2.30
2.33
3.00
1.05
1.03
1.25
1.22
1.06
0.02
0.04
0.07
0.86
0.90
1.81
1.83
1.83
1.84
1.84
1.85
1.86
2.38
2.38
2.39
2.40
2.41
2.41
3.66
3.73
3.74
3.75
3.76
4.26
4.27
4.28
6.23
6.23
6.24
6.24
6.26
6.26
6.27
6.28
6.81
6.82
6.84
6.85
9.56
9.57
TBSO
COOMe
OTBS
O
3.16
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
-4.81
-4.79
-4.48
-4.05
18.09
25.82
25.86
28.35
29.42
51.55
75.07
75.63
132.42
157.17
193.26
TBSO
COOMe
OTBS
O
3.16

219

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
11.98
19.06
2.19
2.25
1.02
3.00
0.92
0.93
0.98
1.08
0.97
0.00
0.03
0.05
0.87
0.87
0.88
0.88
1.79
1.79
1.80
1.81
2.35
2.36
2.37
2.38
2.39
2.40
3.60
3.61
3.63
3.64
3.66
3.93
3.93
3.94
3.94
3.95
3.95
3.96
3.96
5.64
5.66
5.68
5.70
6.03
6.03
6.03
6.06
6.06
6.06
6.07
6.07
6.07
6.10
6.10
6.10
6.28
6.32
6.98
7.01
7.02
7.04
TBSO
COOMe
OTBS
I
3.11
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-400
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
-4.74
-4.72
-4.03
18.13
18.22
25.90
25.93
25.97
28.16
29.53
51.49
74.97
78.97
131.16
135.19
144.63
174.19
TBSO
COOMe
OTBS
I
3.11

220

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
0
50
100
150
200
250
300
350
400
450
500
11.56
19.70
2.30
2.08
1.26
3.00
1.02
1.98
1.10
1.18
1.26
1.28
0.00
0.02
0.03
0.05
0.87
0.88
3.61
3.62
3.63
3.64
3.66
3.97
3.98
3.98
3.99
4.00
4.41
5.57
5.61
5.72
5.72
5.74
5.76
5.78
6.14
6.17
6.17
6.18
6.18
6.21
6.54
6.57
6.58
6.61
TBSO
COOMe
OTBS
HO
3.17
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
-4.76
-4.68
-4.09
-4.02
14.07
18.11
18.21
19.35
20.55
25.90
25.93
26.99
27.02
29.70
127.54
135.87
174.23
TBSO
COOMe
OTBS
HO
3.17

221

TBSO
COOMe
OTBS
Br
3.5
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
12.95
19.33
2.34
2.38
1.25
3.00
1.07
1.81
1.15
1.16
1.26
1.20
-0.01
0.02
0.03
0.04
0.86
0.88
1.80
1.81
1.82
1.83
2.36
2.38
2.40
3.60
3.62
3.63
3.64
3.65
3.97
3.98
3.99
4.00
4.00
4.08
4.09
5.57
5.61
5.75
5.76
5.79
5.80
6.14
6.17
6.17
6.18
6.21
6.57
6.59
6.61
6.63
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200
f1 (ppm)
-5
0
5
10
15
20
25
30
35
40
45
50
55
-4.77
-4.70
-4.15
-4.04
15.55
18.10
18.20
25.88
25.91
25.95
28.23
29.43
51.48
75.02
76.47
86.04
86.49
109.92
130.37
138.10
142.60
174.16
TBSO
COOMe
OTBS
Br
3.5

222

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6.00
9.12
0.96
1.10
0.99
1.02
1.10
0.07
0.07
0.89
2.62
2.63
2.63
2.74
2.75
2.76
2.77
3.06
3.07
3.07
3.08
3.08
3.08
3.63
3.64
3.66
3.67
3.82
3.83
3.85
3.86
O
OTBS
3.9
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
-5.38
-5.34
18.33
25.84
44.42
52.38
63.72
O
OTBS
3.9

223

-10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120
f1 (ppm)
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
-5.43
12.36
14.14
18.26
23.36
25.83
65.62
70.44
75.04
83.91
TBSO
OH
3.22

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
5.77
8.99
2.87
2.00
1.84
0.97
1.07
1.02
1.03
0.06
0.89
1.08
1.10
1.11
2.14
2.15
2.35
2.36
2.36
2.37
2.48
2.49
3.59
3.60
3.67
3.67
3.69
3.69
3.72
3.73
TBSO
OH
3.22

224

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
2.96
8.89
2.12
1.05
1.00
2.10
1.00
6.29
4.24
1.06
1.08
1.10
1.12
2.07
2.08
2.09
2.10
2.10
2.11
2.12
2.13
2.30
2.30
2.31
2.34
2.35
2.35
2.40
2.40
2.41
2.42
2.42
2.43
2.44
2.46
3.66
3.67
3.67
3.67
3.68
3.92
3.93
3.94
7.41
7.41
7.43
7.43
7.44
7.46
7.71
7.71
7.72
7.73
7.73
7.73
7.74
HO
OTBDPS
3.23
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
f1 (ppm)
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
12.31
13.99
19.24
23.81
26.92
65.54
72.52
75.26
127.60
127.73
129.77
129.82
133.50
133.52
135.62
135.77
HO
OTBDPS
3.23

225

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
f1 (ppm)
0
50
100
150
200
250
300
350
400
450
500
2.92
8.53
1.99
1.14
1.25
1.00
1.14
1.07
6.73
4.39
0.92
0.90
0.92
0.94
1.15
1.93
1.93
1.93
1.95
1.95
1.95
1.95
1.97
2.35
2.35
2.37
2.37
2.37
2.38
2.39
2.39
2.39
2.40
2.41
2.43
2.44
2.45
2.45
2.45
2.45
2.46
2.47
2.47
2.47
4.07
4.07
4.08
4.09
4.09
4.09
4.09
4.10
4.10
4.11
5.35
5.36
5.36
5.38
5.38
5.38
5.46
5.47
5.48
5.48
5.48
5.49
5.50
5.50
5.51
7.39
7.40
7.41
7.41
7.41
7.42
7.45
7.45
7.45
7.45
7.46
7.46
7.66
7.68
7.68
7.69
7.70
9.59
9.60
TBDPSO
O
3.24
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75 13.97
19.31
20.56
26.90
30.97
77.77
122.00
127.71
127.77
129.96
130.00
132.94
133.06
134.92
135.77
203.27
TBDPSO
O
3.24

226

TBDPSO
O
3.25
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
f1 (ppm)
0
50
100
150
200
250
300
350
400
450
3.37
8.66
2.02
2.19
1.00
1.05
1.06
1.01
1.02
6.32
4.25
0.88
0.82
0.84
0.86
1.09
1.77
1.79
1.79
1.80
1.81
2.20
2.22
2.23
2.24
2.26
2.27
2.28
2.30
2.32
2.33
2.35
4.44
4.45
4.47
4.48
5.18
5.20
5.22
5.24
5.39
5.41
5.43
5.45
6.19
6.19
6.21
6.21
6.23
6.23
6.25
6.25
6.70
6.71
6.73
6.75
7.35
7.38
7.38
7.40
7.40
7.40
7.42
7.42
7.43
7.45
7.60
7.60
7.60
7.60
7.62
7.62
7.67
7.67
7.67
7.68
7.69
7.69
9.45
9.47
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
14.02
19.30
20.56
26.96
34.97
72.34
122.31
127.65
127.68
129.92
129.94
130.93
133.16
133.49
134.92
135.75
135.83
158.92
193.53
TBDPSO
O
3.25

227

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
4.88
8.85
3.07
2.07
2.03
0.88
1.00
1.21
1.10
1.00
1.00
0.04
0.06
0.90
0.94
0.96
0.98
2.01
2.02
2.03
2.03
2.05
2.23
2.23
2.25
2.25
2.25
2.25
2.26
2.26
2.27
2.27
2.28
2.28
2.86
2.86
4.16
4.17
4.18
4.19
4.21
4.21
5.28
5.30
5.30
5.30
5.33
5.35
5.44
5.46
5.47
5.49
5.51
5.64
5.68
6.22
6.23
6.26
6.27
TBSO
3.7
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550 -5.14
-4.98
13.81
17.89
20.41
25.50
35.41
71.94
81.76
107.30
107.31
123.48
133.76
147.66
TBSO
3.7

228

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
0
10
20
30
40
50
60
70
80
90
13.43
21.56
2.78
2.81
1.96
2.27
2.20
2.08
1.44
0.94
1.95
1.00
1.03
0.63
1.16
1.62
1.28
1.69
1.00
-0.01
0.02
0.03
0.04
0.85
0.86
0.87
0.88
0.89
0.90
0.90
0.93
0.95
0.97
1.25
1.81
1.81
1.83
1.83
2.00
2.02
2.04
2.21
2.22
2.24
2.25
2.36
2.38
2.40
3.44
3.60
3.61
3.62
3.64
3.65
3.95
3.96
3.97
3.98
4.14
4.15
4.16
4.17
4.18
5.32
5.34
5.43
5.44
5.47
5.49
5.49
5.53
5.57
5.62
5.66
5.68
5.70
5.72
5.73
6.09
6.10
6.11
6.13
6.14
6.15
6.18
6.51
6.54
6.55
6.58
5.5 6.0 6.5
f1 (ppm)
TBSO
TBSO
COOMe
OTBS
3.4
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-5
0
5
10
15
20
25
30
35
40
45
-4.83
-4.78
-4.70
-4.61
-4.12
-4.04
11.29
14.14
18.09
18.19
20.72
25.91
28.24
29.41
35.84
72.43
75.01
76.55
79.06
79.85
83.29
85.91
108.44
110.86
124.01
130.72
133.89
136.79
141.09
145.99
174.20
TBSO
TBSO
COOMe
OTBS
3.4

229

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
10.87
3.46
14.60
7.82
3.15
1.83
1.83
1.21
2.17
1.35
1.10
2.54
0.82
0.99
1.14
1.09
1.87
0.93
2.07
1.00
3.79
1.77
3.77
0.81
0.83
0.84
0.86
0.87
0.88
1.06
1.24
1.25
1.25
1.26
1.27
1.74
1.74
1.74
1.75
1.75
1.75
1.76
1.76
1.77
1.77
1.80
1.81
1.81
1.82
1.82
1.83
1.83
1.83
1.84
1.84
2.10
2.12
2.13
2.37
2.38
2.38
2.40
3.43
3.44
3.44
3.61
3.62
3.63
3.66
3.95
3.96
3.96
3.97
3.97
3.97
3.98
3.98
4.18
4.18
4.19
4.20
5.17
5.19
5.34
5.55
5.57
5.57
5.58
5.58
5.60
5.60
5.69
5.71
5.72
5.74
6.07
6.09
6.11
6.12
6.13
6.15
6.16
6.18
6.53
6.55
6.56
6.58
7.35
7.35
7.35
7.36
7.36
7.36
7.37
7.38
7.40
7.40
7.40
7.41
7.41
7.41
7.42
7.43
7.61
7.62
7.63
7.63
7.67
7.67
7.68
7.68
7.69
5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6
f1 (ppm)
TBSO
TBSO
COOMe
OTBS
3.3
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
-4.77
-4.69
-4.10
-4.03
11.32
14.06
18.11
18.21
20.54
25.93
27.01
28.25
29.43
35.43
51.48
73.12
75.01
76.55
79.11
79.87
83.31
85.94
109.01
110.86
123.24
127.54
129.63
134.09
135.86
141.11
145.26
174.25
TBSO
TBSO
COOMe
OTBS
3.3

230

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
14.70
27.44
4.41
1.94
2.51
1.72
1.93
2.28
2.11
1.11
1.08
0.88
4.00
1.88
2.04
1.76
1.64
0.05
0.06
0.07
0.07
0.09
0.09
0.90
0.91
0.93
1.29
1.29
1.29
1.29
1.30
1.31
1.80
1.82
1.83
1.85
2.01
2.03
2.03
2.05
2.07
2.09
2.24
2.25
2.27
2.40
2.42
2.44
2.99
3.01
3.03
3.05
3.08
3.09
3.11
3.13
3.16
3.18
3.20
3.65
3.67
3.68
3.70
3.71
4.04
4.05
4.06
4.07
4.25
4.25
4.27
4.27
5.33
5.35
5.37
5.37
5.38
5.39
5.42
5.44
5.46
5.47
5.47
5.63
5.65
5.66
5.67
5.68
5.69
5.70
5.72
5.97
6.00
6.03
6.06
6.08
6.18
6.21
6.22
6.23
6.24
6.25
6.28
6.50
6.52
6.52
6.55
6.55
6.56
6.56
6.59
5.4 5.6 5.8 6.0 6.2 6.4 6.6
f1 (ppm)
TBSO
TBSO
COOMe
OTBS
3.27
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
-4.42
-4.37
-4.34
-4.04
-3.60
14.45
19.06
19.15
19.17
21.72
23.70
26.21
26.42
26.52
26.54
26.57
27.56
29.44
30.41
32.75
37.31
54.78
74.36
76.33
78.48
125.84
129.11
129.40
129.82
130.85
133.63
134.41
137.98
141.83
175.78
TBSO
TBSO
COOMe
OTBS
3.26

231

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
3.15
0.80
1.45
0.69
1.30
1.92
3.77
0.95
0.90
0.90
0.70
0.86
2.00
1.22
1.79
0.94
2.37
1.40
0.83
1.35
0.68
0.95
0.96
0.98
1.53
1.53
1.54
1.55
1.56
1.63
1.64
1.66
1.67
1.69
1.70
1.74
1.75
1.76
1.83
1.84
1.85
1.87
1.88
1.90
2.03
2.05
2.06
2.08
2.09
2.23
2.25
2.26
2.28
2.29
2.31
2.32
2.33
2.35
2.37
2.38
2.40
2.88
2.90
2.91
2.93
3.09
3.10
3.12
3.51
3.52
3.53
3.53
3.54
3.98
3.99
4.01
4.12
4.13
4.15
4.16
5.34
5.36
5.36
5.38
5.39
5.40
5.45
5.46
5.48
5.50
5.67
5.68
5.70
5.71
5.73
5.74
5.82
5.83
5.85
5.86
5.88
5.90
5.93
5.95
5.96
5.98
6.00
6.02
6.04
6.07
6.08
6.10
6.13
6.23
6.25
6.26
6.28
6.29
6.35
6.37
6.38
6.40
6.54
6.56
6.57
6.59
6.65
6.68
6.68
6.71
5.5 6.0 6.5
f1 (ppm)
HO
HO
COOH
OH
3.2
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
0
50
100
150
200
250
300
350
400
14.43
21.68
23.70
27.48
32.75
36.25
73.18
76.22
76.52
125.49
126.27
128.74
129.57
130.18
130.24
130.46
132.34
133.14
134.18
134.47
134.62
137.52
182.79
HO
HO
COOH
OH
3.2

232

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
0
50
100
150
200
250
300
350
2.14
1.87
1.39
1.37
1.70
3.81
1.17
1.00
0.80
1.05
1.98
1.17
1.72
1.12
2.87
1.09
0.90
1.41
0.95
0.96
0.97
1.29
1.31
1.32
1.65
1.66
1.67
1.67
1.68
1.70
1.71
1.83
1.83
1.84
1.85
1.85
1.86
1.87
1.87
1.87
2.04
2.05
2.06
2.07
2.09
2.24
2.25
2.26
2.28
2.29
2.31
2.31
2.32
2.32
2.33
2.34
2.36
2.37
2.38
2.39
3.09
3.10
3.12
3.51
3.52
3.53
3.53
3.54
3.54
3.98
3.99
4.00
4.12
4.13
4.14
4.15
5.35
5.36
5.37
5.38
5.39
5.40
5.41
5.45
5.46
5.48
5.48
5.49
5.67
5.68
5.70
5.71
5.72
5.74
5.82
5.83
5.85
5.86
5.94
5.96
5.98
6.00
6.02
6.03
6.04
6.06
6.09
6.10
6.13
6.23
6.25
6.26
6.27
6.35
6.37
6.38
6.39
6.55
6.56
6.57
6.57
6.59
5.4 5.6 5.8 6.0 6.2 6.4 6.6
f1 (ppm)
HO
HO
COOH
OH
3.1
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
14.62
21.71
23.70
27.50
32.78
49.51
49.87
73.16
76.21
76.54
125.47
126.27
128.73
129.57
130.18
130.24
130.43
130.56
133.12
134.18
134.44
134.63
137.49
182.81
HO
HO
COOH
OH
3.1

233

-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
4.68
5.92
1.53
4.06
1.00
1.43
2.32
1.39
0.08
0.90
2.36
2.36
2.37
2.37
2.37
2.38
2.38
2.38
2.47
2.48
2.48
2.49
2.49
2.49
2.49
2.50
2.50
2.51
2.51
2.53
3.45
3.47
3.49
3.50
3.57
3.58
3.59
3.61
3.66
3.67
3.68
3.69
3.72
3.72
3.72
3.73
3.73
3.75
TBSO
OH
O
O
4.12
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
f1 (ppm)
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
-5.25
14.93
18.45
23.57
26.02
33.83
51.91
65.74
66.01
70.51
77.48
77.68
80.60
172.65
TBSO
OH
O
O
4.12

234

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
9.83
1.17
5.33
1.95
2.63
1.00
6.63
4.45
1.07
1.07
1.07
1.08
2.24
2.24
2.25
2.26
2.27
2.28
2.29
2.29
2.35
2.36
2.36
2.37
2.37
2.37
2.37
2.38
2.38
2.38
2.39
2.39
2.39
2.40
2.40
2.40
2.40
2.41
2.41
2.42
2.42
2.43
2.43
2.44
2.44
2.45
2.45
3.61
3.62
3.62
3.63
3.66
3.85
3.86
3.86
3.87
3.87
3.88
3.89
3.89
3.89
3.91
7.36
7.37
7.37
7.37
7.38
7.38
7.39
7.39
7.39
7.39
7.40
7.40
7.41
7.41
7.41
7.41
7.42
7.43
7.43
7.44
7.44
7.46
7.65
7.66
7.67
7.67
7.67
7.68
7.68
7.69
7.69
HO
OTBDPS
O
O
4.13
HO
OTBDPS
O
O
4.13
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
f1 (ppm)
0
50
100
150
200
250
14.85
19.45
23.93
27.11
33.70
51.87
65.67
72.56
77.10
80.51
127.84
127.97
130.03
130.07
133.63
133.65
135.82
135.96
172.63

235

-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
10.25
4.25
1.92
2.94
1.00
1.57
7.00
3.69
0.99
1.10
1.10
1.10
1.11
1.12
1.12
1.12
2.23
2.24
2.24
2.24
2.24
2.25
2.25
2.25
2.26
2.26
2.26
2.27
2.27
2.27
2.28
2.28
2.29
2.29
2.30
2.30
2.31
2.32
2.36
2.37
2.37
2.39
2.44
2.44
2.45
2.48
2.48
3.65
4.05
4.05
4.06
4.06
4.07
4.07
4.08
4.08
5.41
5.42
5.43
5.43
5.44
5.44
7.35
7.35
7.36
7.36
7.37
7.37
7.37
7.37
7.38
7.38
7.38
7.38
7.39
7.39
7.39
7.39
7.40
7.40
7.40
7.41
7.42
7.42
7.42
7.43
7.43
7.43
7.44
7.44
7.44
7.44
7.62
7.62
7.64
7.64
7.64
7.64
7.66
7.66
9.56
9.56
O
OTBDPS
COOMe
4.14
O
OTBDPS
COOMe
4.14
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
13.96
19.48
27.07
31.06
31.73
51.63
77.09
127.95
127.97
130.22
133.05
135.88
135.95
135.97
135.97
177.46
203.09

236

O
OTBDPS
COOMe
4.15
-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
8.79
1.86
2.67
2.67
1.00
1.54
1.09
0.93
9.88
4.62
0.88
1.09
2.12
2.13
2.14
2.14
2.16
2.16
2.17
2.23
2.23
2.24
2.25
2.25
2.26
2.27
2.28
3.64
4.46
4.47
4.48
4.48
4.48
4.49
4.49
4.49
4.49
4.50
4.51
5.30
5.31
5.32
5.33
5.33
5.34
5.35
5.36
5.38
5.38
6.17
6.18
6.19
6.20
6.21
6.22
6.23
6.24
6.68
6.69
6.72
6.73
7.33
7.34
7.34
7.34
7.35
7.35
7.35
7.36
7.36
7.36
7.37
7.37
7.37
7.38
7.38
7.39
7.39
7.40
7.40
7.40
7.41
7.41
7.42
7.42
7.43
7.44
7.44
7.44
7.59
7.60
7.60
7.60
7.61
7.61
7.62
7.66
7.67
7.67
7.68
7.68
7.69
9.46
9.47
O
OTBDPS
COOMe
4.15
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
19.45
22.90
27.11
27.13
33.80
35.03
51.67
53.56
72.27
124.85
127.81
127.83
127.86
130.11
130.85
131.22
133.25
133.58
135.91
135.94
135.97
158.72
173.41
193.64

237

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
9.08
2.05
3.50
3.10
1.00
1.08
1.05
0.79
0.94
0.98
7.75
4.58
1.07
2.15
2.15
2.16
2.16
2.17
2.17
2.18
2.18
2.20
2.22
2.23
2.23
2.24
2.24
2.25
2.25
2.25
2.26
2.26
2.27
2.27
3.65
3.65
3.66
4.15
4.15
4.16
4.17
4.17
4.18
4.18
4.18
4.19
4.20
4.20
5.33
5.34
5.62
5.62
5.64
5.64
5.66
5.66
5.67
5.68
5.84
5.84
5.84
5.84
5.87
5.87
5.87
5.87
5.88
5.88
5.88
5.91
5.91
6.15
6.19
6.88
6.88
6.88
6.91
6.91
6.92
6.92
6.94
6.94
7.33
7.34
7.35
7.35
7.36
7.36
7.37
7.37
7.37
7.38
7.38
7.39
7.39
7.39
7.39
7.40
7.41
7.41
7.41
7.42
7.61
7.61
7.62
7.63
7.63
7.63
7.66
7.66
7.67
7.67
7.68
7.68
OTBDPS
COOMe
I
4.5
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
0
50
100
150
200
250
300
14.26
19.47
27.15
31.73
33.98
53.56
73.16
78.91
126.05
127.62
127.66
127.69
129.75
129.79
129.81
129.91
133.92
134.19
136.01
136.04
136.05
136.70
144.77
173.58
OTBDPS
COOMe
I
4.5

238

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
4.66
15.79
4.10
3.75
3.93
3.25
0.83
1.00
1.24
1.43
6.10
1.03
0.83
1.06
12.65
8.77
0.03
0.05
1.08
1.09
2.00
2.00
2.00
2.01
2.01
2.02
2.02
2.02
2.03
2.03
2.04
2.04
2.04
2.05
2.43
2.43
2.43
2.44
2.44
2.44
2.45
2.45
2.46
2.46
2.47
2.48
2.48
2.66
2.67
2.68
2.69
2.69
2.70
2.71
3.67
4.35
4.35
4.49
4.49
5.21
5.23
5.24
5.24
5.24
5.25
5.26
5.26
5.34
5.35
5.35
5.37
5.37
5.42
5.43
5.43
5.44
5.44
5.45
5.45
5.45
5.46
6.26
7.34
7.35
7.35
7.35
7.36
7.36
7.37
7.37
7.37
7.38
7.38
7.39
7.39
7.39
7.40
7.40
7.41
7.41
7.41
7.42
7.42
7.42
7.43
7.43
7.43
7.68
7.69
7.69
7.69
7.70
7.70
7.71
7.71
7.71
7.72
7.74
7.74
7.75
7.75
7.75
7.76
7.76
7.77
OTBDPS
OTBDPS
COOMe
4.3
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
1.77
1.76
3.85
2.58
1.13
1.39
6.23
0.80
0.69
7.46
2.41
1.37
1.80
1.00
0.96
0.97
0.99
2.07
2.07
2.07
2.09
2.09
2.09
2.09
2.11
2.11
2.11
2.28
2.29
2.29
2.29
2.29
2.30
2.30
2.30
2.30
2.31
2.31
2.31
2.31
2.32
2.32
2.32
2.32
2.33
2.33
2.33
2.34
2.37
2.37
2.44
2.46
2.46
2.46
2.83
2.84
2.84
3.65
5.32
5.37
5.43
5.44
5.44
5.44
5.44
5.45
5.45
5.45
5.45
5.46
5.46
5.46
5.47
5.47
5.48
5.48
5.49
5.49
5.49
5.50
5.67
5.68
5.74
5.74
5.75
5.77
5.77
5.78
5.79
5.79
5.79
5.83
6.16
6.16
6.16
6.17
6.19
6.19
6.19
6.19
6.20
6.20
6.20
6.23
6.23
6.43
6.43
6.43
6.44
6.47
6.47
6.47
6.47
6.52
6.55
7.03
7.03
7.05
7.05
7.06
7.06
7.09
7.09
OH
OH
COOMe
4.2

239

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
0
5000
10000
15000
20000
25000
30000
35000
40000
14.64
21.48
23.94
30.75
34.69
35.99
36.97
48.36
49.64
52.08
63.20
72.37
79.32
84.35
93.78
125.53
127.51
130.92
132.00
132.85
133.13
133.79
138.12
142.52
146.25
175.31
OH
OH
COOMe
4.2
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
3.17
2.49
2.74
1.86
1.78
1.04
2.15
2.90
1.13
0.99
7.22
0.92
1.08
2.16
1.00
0.85
0.86
0.86
1.63
1.63
1.63
1.64
1.64
1.64
1.65
1.65
1.66
1.74
1.74
1.74
1.75
1.75
1.75
1.75
1.76
1.76
1.76
1.77
1.77
1.77
1.77
1.87
1.87
1.88
1.89
1.89
1.90
1.91
2.07
2.07
2.07
2.07
2.08
2.09
2.10
2.10
2.10
2.22
2.23
2.24
2.25
2.25
2.79
2.79
2.79
2.80
2.82
2.82
3.65
3.65
4.57
4.57
4.57
4.58
4.58
4.59
4.59
5.31
5.37
5.37
5.38
5.38
5.38
5.39
5.39
5.39
5.40
5.40
5.41
5.41
5.42
5.42
5.42
5.43
5.44
5.44
5.44
5.45
5.45
5.45
5.45
5.46
5.46
5.47
5.73
5.74
5.76
5.77
6.05
6.05
6.08
6.08
6.10
6.10
6.24
6.26
6.27
6.27
6.29
6.29
6.50
6.50
6.52
6.53
OH
OH
COOH
4.1

240

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
3.16
3.00
0.44
0.55
1.05
0.53
0.99
0.45
0.47
0.54
0.45
0.54
0.44
0.51
1.24
1.24
1.25
1.25
1.37
1.37
1.38
1.38
1.50
1.51
1.52
1.52
1.53
1.54
1.54
1.56
1.56
1.56
1.58
1.65
1.66
1.66
1.68
1.69
1.70
1.70
1.71
1.94
1.95
1.95
1.96
1.98
1.99
1.99
2.00
2.01
2.02
2.02
2.03
2.04
2.05
2.06
3.54
3.55
3.58
3.58
3.63
3.64
3.66
3.67
3.67
3.68
3.70
3.71
3.78
3.80
3.82
3.83
4.03
4.04
4.04
4.05
4.05
4.06
4.08
4.09
4.09
4.10
4.10
4.11
4.19
4.21
4.21
4.22
4.23
4.24
4.32
4.33
4.33
4.34
4.34
4.35
4.35
4.35
4.36
4.39
4.40
4.40
4.41
4.42
4.64
4.65
4.65
4.66

OH
OH
COOH
4.1
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
14.43
21.50
23.69
25.80
32.74
39.00
68.48
73.10
126.11
126.30
128.17
128.76
130.59
131.28
131.29
132.61
132.78
134.74
135.04
138.17
182.26
O
O
O
OH
5.9

241

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2.66
2.14
2.00
1.83
2.13
15.88
0.61
0.63
0.65
1.62
1.62
1.62
1.62
1.64
1.64
1.64
1.64
1.65
1.66
1.66
1.68
1.69
1.70
1.70
1.71
2.10
2.11
2.11
2.11
2.11
2.11
2.11
2.12
2.12
2.13
2.13
2.13
2.13
2.14
2.14
2.15
2.15
2.15
3.07
3.09
3.09
3.10
3.10
3.10
3.11
3.12
3.12
3.12
3.14
5.16
5.17
5.18
5.18
5.19
5.20
5.21
5.21
5.21
5.23
7.46
7.46
7.47
7.47
7.47
7.47
7.47
7.47
7.48
7.48
7.48
7.48
7.48
7.49
7.49
7.49
7.49
7.49
7.50
7.50
7.50
7.50
7.50
7.51
7.52
7.52
7.52
7.52
7.53
7.53
7.53
7.53
7.54
7.55
7.55
7.55
7.55
7.55
7.55
7.61
7.61
7.62
7.62
7.63
7.63
7.63
7.63
7.64
7.64
7.65
7.65
IPh
3
P
5.8
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
14.18
21.07
22.66
23.15
118.17
118.65
119.51
131.08
131.20
134.57
134.66
135.98
136.01
IPh
3
P
5.8

242

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
3.00
3.16
2.89
1.82
1.77
1.56
2.04
2.18
1.40
3.04
0.95
0.95
0.97
0.97
0.99
0.99
1.36
1.37
1.37
1.48
1.48
2.04
2.04
2.05
2.05
2.05
2.06
2.06
2.08
2.08
2.08
2.09
2.29
2.31
2.31
2.31
2.31
2.32
2.33
2.33
2.34
2.34
2.38
2.38
2.38
2.39
2.40
2.40
2.41
2.42
2.78
2.78
2.78
2.78
2.79
2.80
2.80
2.80
2.80
2.80
2.81
2.82
2.82
2.82
3.63
3.64
3.66
4.15
4.17
4.17
4.17
4.18
4.19
4.19
4.19
4.20
4.21
4.21
4.21
4.21
4.22
4.23
4.23
4.24
5.28
5.28
5.29
5.29
5.29
5.30
5.30
5.31
5.31
5.32
5.33
5.37
5.37
5.38
5.39
5.39
5.40
5.40
5.41
5.41
5.41
5.42
5.42
5.42
5.43
5.43
5.44
5.47
5.48
5.48
5.49
5.49
5.50
5.51
5.51
OH
O O
5.5
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-4
-2
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
14.38
20.71
25.55
25.92
27.53
28.26
61.82
76.79
77.91
108.35
124.85
126.67
131.04
132.42
OH
O O
5.5

243

-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
3.00
3.46
3.20
2.18
1.75
2.17
0.92
1.01
1.06
2.92
0.81
0.92
0.93
0.95
1.37
1.37
1.56
2.00
2.01
2.02
2.02
2.03
2.04
2.30
2.31
2.31
2.32
2.33
2.33
2.33
2.33
2.70
2.70
2.71
2.71
2.71
2.72
2.72
2.72
2.72
2.72
2.73
2.73
2.74
2.74
2.74
4.26
4.27
4.28
4.29
4.34
4.35
4.35
4.37
4.38
5.22
5.23
5.23
5.24
5.24
5.24
5.25
5.25
5.25
5.26
5.27
5.27
5.27
5.35
5.35
5.35
5.36
5.36
5.36
5.37
5.37
5.38
5.38
5.38
5.39
5.39
5.39
5.40
5.41
5.45
5.46
5.46
5.47
5.47
5.48
5.48
5.49
9.63
9.63
O
O O
5.12
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-40
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
14.27
20.60
25.25
25.80
27.49
27.93
78.36
81.95
110.60
123.93
126.44
126.44
131.59
132.37
201.66
O
O O
5.12

244

-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
0
50
100
150
200
250
300
350
400
450
3.00
2.67
2.82
1.86
0.74
0.71
1.93
0.88
0.81
4.10
1.55
0.61
0.67
0.70
0.91
0.93
0.95
1.36
1.36
1.50
1.50
1.98
1.98
1.99
1.99
2.00
2.00
2.02
2.02
2.04
2.04
2.05
2.14
2.16
2.18
2.20
2.21
2.26
2.27
2.28
2.30
2.31
2.32
2.70
2.72
2.73
2.75
4.22
4.23
4.24
4.25
4.26
4.26
4.28
4.63
4.64
4.65
4.66
4.68
4.68
5.22
5.23
5.23
5.24
5.24
5.25
5.25
5.26
5.26
5.26
5.27
5.28
5.31
5.32
5.32
5.33
5.34
5.34
5.34
5.35
5.35
5.36
5.36
5.37
5.37
5.38
5.38
5.39
5.39
5.40
5.40
5.41
5.43
5.44
5.44
5.45
5.45
5.46
5.46
5.47
5.47
5.48
5.48
5.48
6.13
6.14
6.15
6.15
6.16
6.17
6.18
6.19
6.21
6.50
6.53
6.54
6.57
7.07
7.09
7.10
7.13
9.55
9.57
O O
O
5.3
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-4000
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
36000
14.31
20.66
25.51
25.90
28.10
28.92
78.11
78.31
108.96
124.57
126.63
130.28
131.00
132.33
140.18
150.57
193.61
O O
O
5.3

245

-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
1.84
3.84
2.00
2.60
2.35
2.36
2.36
2.37
2.37
2.38
2.38
2.39
2.40
2.43
2.43
2.43
2.44
2.44
2.45
2.45
2.45
2.46
2.46
2.47
2.47
2.47
2.47
2.47
2.48
2.48
2.49
2.50
2.51
2.51
2.52
3.62
3.64
3.66
3.67
HO
O
O
5.13
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-5000
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
14.88
23.19
33.77
51.90
61.29
77.85
80.46
172.76
HO
O
O
5.13

246

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
2.00
3.40
1.61
2.93
1.90
2.33
2.33
2.34
2.35
2.36
2.36
2.36
2.37
2.38
2.39
2.40
2.40
2.40
2.40
3.63
3.65
3.66
3.66
5.41
5.43
5.43
5.44
5.44
5.45
5.46
5.46
5.47
5.47
5.48
5.48
5.49
5.49
5.49
5.49
5.49
5.50
5.50
5.50
5.50
5.50
5.51
5.51
5.51
5.52
5.52
5.52
5.53
5.53
5.53
O
O HO
5.14
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-10000
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000
22.92
30.94
33.96
51.77
62.29
127.31
130.90
173.82
O
O HO
5.14

247

-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
4.27
2.02
2.00
3.23
1.26
1.14
2.32
2.32
2.33
2.33
2.33
2.33
2.34
2.34
2.34
2.34
2.34
2.34
2.35
2.35
2.35
2.35
2.36
2.38
2.60
2.60
2.62
2.62
2.62
2.62
2.63
2.64
2.64
2.64
2.64
2.64
2.66
2.66
2.66
3.10
3.12
3.14
3.65
3.65
5.32
5.32
5.33
5.34
5.34
5.35
5.35
5.35
5.35
5.36
5.36
5.37
5.37
5.37
5.38
5.39
5.39
5.44
5.45
5.45
5.45
5.46
5.46
5.46
5.46
5.46
5.46
5.47
5.47
5.48
5.48
5.48
5.49
5.49
5.49
5.50
5.51
5.51
5.51
O
O I
5.15
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190 5.31
23.02
31.38
33.86
51.67
51.68
129.48
130.12
173.41 O
O I
5.15

248

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
20.16
22.43
23.17
33.10
51.44
117.20
118.05
127.17
127.33
130.18
130.41
130.53
133.44
133.54
135.09
135.12
173.16

-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
2.00
1.99
2.07
2.73
1.59
1.13
1.02
7.03
9.83
2.05
2.07
2.07
2.07
2.08
2.09
2.09
2.10
2.11
2.11
2.22
2.24
2.26
2.26
2.34
2.35
2.36
2.36
2.37
2.37
2.38
2.39
2.39
2.40
2.41
2.41
2.42
2.43
2.43
2.43
3.54
3.57
3.58
3.59
3.59
3.60
3.62
5.23
5.24
5.25
5.25
5.26
5.26
5.26
5.27
5.27
5.28
5.28
5.28
5.29
5.29
5.29
5.30
5.31
5.31
5.31
5.55
5.56
5.56
5.57
5.58
5.58
5.59
5.59
5.59
5.60
5.60
5.61
5.62
7.64
7.64
7.64
7.65
7.65
7.65
7.66
7.66
7.66
7.66
7.67
7.67
7.67
7.68
7.68
7.68
7.69
7.69
7.69
7.73
7.74
7.74
7.74
7.74
7.75
7.75
7.76
7.76
7.76
7.77
7.77
7.78
7.78
7.79
7.79
O
O IPh
3
P
5.4
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
20.16
22.43
23.17
33.10
51.44
117.20
118.05
127.17
127.33
130.18
130.41
130.53
133.44
133.54
135.09
135.12
173.16
O
O IPh
3
P
5.4

249

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
0
100
200
300
400
500
600
700
800
900
1000
2.93
3.03
3.27
1.90
0.89
0.99
4.14
2.00
2.10
3.09
1.00
0.94
1.80
6.41
1.19
0.99
2.21
0.88
0.94
0.95
0.96
0.97
1.36
1.37
1.50
1.50
2.02
2.03
2.05
2.06
2.08
2.16
2.16
2.17
2.17
2.18
2.20
2.21
2.30
2.31
2.33
2.34
2.36
2.37
2.38
2.39
2.39
2.40
2.41
2.73
2.75
2.76
2.78
2.87
2.88
2.95
2.97
2.98
3.67
3.67
4.15
4.16
4.17
4.17
4.18
4.19
4.20
4.58
4.59
4.60
4.60
4.61
5.27
5.27
5.28
5.28
5.29
5.29
5.30
5.31
5.31
5.36
5.36
5.37
5.38
5.38
5.39
5.40
5.41
5.41
5.42
5.43
5.44
5.45
5.45
5.46
5.46
5.46
5.47
5.47
5.47
5.48
5.66
5.68
5.68
5.69
5.71
6.01
6.03
6.05
6.06
6.20
6.21
6.22
6.23
6.24
6.25
6.27
6.29
6.32
6.34
6.35
6.37
6.50
6.52
6.53
6.55
O
O
O O
5.2
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
14.41
20.71
22.97
25.72
25.95
26.31
28.35
29.01
34.11
51.73
78.54
79.26
108.41
125.23
126.97
128.42
128.78
128.86
128.90
128.99
130.67
130.96
132.09
132.25
133.91
173.67
O
O
O O
5.2

250

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3.00
2.04
2.44
4.02
3.35
2.07
1.00
3.29
8.07
2.41
0.94
0.90
0.89
0.89
0.91
0.91
0.93
0.93
1.95
1.96
1.96
1.97
1.97
1.97
1.98
1.98
1.99
1.99
1.99
1.99
2.01
2.01
2.01
2.10
2.10
2.11
2.12
2.13
2.14
2.22
2.24
2.27
2.28
2.29
2.30
2.30
2.31
2.71
2.71
2.71
2.72
2.72
2.73
2.73
2.74
2.74
2.74
2.75
2.77
2.87
2.88
2.90
3.03
3.04
3.05
3.06
3.34
3.34
5.31
5.31
5.32
5.33
5.33
5.34
5.34
5.35
5.35
5.36
5.36
5.36
5.37
5.37
5.37
5.38
5.38
5.38
5.39
5.39
5.40
5.40
5.41
5.41
5.42
5.42
5.42
5.43
5.43
5.43
5.44
5.45
5.45
5.50
5.50
5.98
5.98
6.01
6.01
6.04
6.04
6.06
6.09
6.10
6.13
6.35
6.38
6.39
6.42
6.50
6.52
6.53
6.56
O
O
O
5.18
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000 14.45
20.93
23.13
26.05
26.52
30.19
33.97
51.07
57.73
60.13
124.02
127.22
128.81
128.85
128.93
129.17
131.09
131.30
131.38
132.32
132.36
134.25
172.73
O
O
O
5.18

251

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
3.91
3.88
2.77
5.61
2.75
0.94
1.63
0.86
0.86
0.73
3.00
2.95
1.00
3.15
3.39
1.01
0.90
1.99
0.70
0.93
0.94
0.95
0.96
0.97
0.97
0.98
0.99
1.29
1.29
2.02
2.04
2.06
2.08
2.10
2.34
2.36
2.37
2.37
2.38
2.39
2.39
2.40
2.41
2.78
2.88
2.99
3.01
3.11
3.11
3.11
3.12
3.13
3.13
3.13
3.13
3.14
3.14
3.14
3.15
3.47
3.48
3.48
3.48
3.48
3.49
3.49
3.49
3.64
3.64
3.65
3.66
3.76
3.82
5.25
5.28
5.30
5.38
5.39
5.40
5.42
5.43
5.44
5.48
5.72
6.02
6.05
6.07
6.26
6.60
5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8
f1 (ppm)
0
200
400
600
800
O
O
OH
S
NH
2
NH
O
HOOC
5.17
O
O
OH
S
NH
O
HOOC
NH
2
NH
O
HOOC
5.16
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
21000
22000
3.14
0.92
4.14
3.27
4.55
1.86
2.79
2.79
1.19
3.05
1.90
1.31
0.89
6.41
1.18
1.03
1.87
1.00
0.77
0.57
0.93
0.93
0.95
0.95
0.97
0.97
1.06
1.07
1.08
1.27
1.29
1.30
1.31
1.31
1.33
1.35
1.35
2.03
2.04
2.04
2.04
2.05
2.05
2.05
2.06
2.06
2.06
2.07
2.07
2.07
2.07
2.08
2.08
2.08
2.31
2.31
2.31
2.31
2.32
2.33
2.34
2.34
2.34
2.35
2.35
2.36
2.37
2.37
2.38
2.38
2.39
2.39
2.40
2.41
2.52
2.54
2.54
2.56
2.56
2.75
2.76
2.77
2.79
2.79
2.93
2.95
2.95
2.97
2.98
2.99
3.01
3.63
3.64
3.65
3.65
3.66
3.66
3.73
3.76
4.51
4.52
4.53
4.53
4.54
5.29
5.31
5.38
5.38
5.39
5.39
5.40
5.42
5.42
5.43
5.44
5.44
5.68
5.70
5.71
6.04
6.07
6.24
6.25
6.25
6.26
6.27
6.28
6.29
6.60
5.5 6.0 6.5 7.0 7.5
f1 (ppm)
0
1000
2000
3000

252

-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
3.75
3.08
2.00
2.46
2.47
2.47
2.47
2.47
3.63
4.14
4.14
4.15
O
O
HO
6.11
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
21000
22000
14.74
33.31
51.21
51.95
79.32
84.17
172.59
O
O
HO
6.11

253

-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
4.00
2.88
2.21
2.46
2.47
2.47
2.48
2.48
2.49
3.62
3.82
3.82
3.83
O
O
Br
6.8
25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145
f1 (ppm)
0
5000
10000
15000
20000
25000
30000
35000
40000
32.86
51.75
76.00
85.72
O
O
Br
6.8

254

-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
6.35
2.00
4.89
2.30
2.31
2.31
2.32
2.33
2.34
2.34
2.35
2.36
2.37
2.38
2.38
2.38
2.39
2.39
2.40
2.40
2.41
2.43
2.44
2.44
2.45
2.46
3.01
3.02
3.02
3.03
3.04
3.04
3.59
3.60
3.60
3.61
3.61
3.61
O
O
HO
6.5
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
f1 (ppm)
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
9.63
14.54
22.93
33.28
51.72
60.95
75.08
78.47
O
O
HO
6.5

255

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
4.33
2.00
2.45
3.48
4.34
2.31
2.33
2.33
2.33
2.34
2.34
2.34
2.34
2.35
2.35
2.35
2.78
2.78
2.79
2.79
2.79
2.79
2.80
2.80
2.80
2.80
2.80
2.81
2.81
2.82
2.82
2.82
2.82
3.60
3.60
3.62
3.62
3.62
3.63
3.63
5.31
5.32
5.33
5.33
5.33
5.33
5.33
5.34
5.34
5.34
5.34
5.35
5.35
5.36
5.36
5.36
5.36
5.37
5.37
5.37
5.38
5.38
5.38
5.39
5.39
5.39
5.40
5.41
5.41
5.41
5.43
5.43
5.45
5.45
5.45
5.47
5.47
5.47
5.47
5.48
O
O
HO
6.12
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-4000
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
22.85
25.76
30.94
34.01
51.63
62.13
125.99
127.93
129.29
130.60
173.74
O
O
HO
6.12

256

O
O
I
6.13
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
0
100
200
300
400
500
600
700
800
900
3.82
1.84
1.76
2.00
2.90
3.93
2.27
2.28
2.28
2.29
2.30
2.30
2.30
2.31
2.31
2.32
2.32
2.56
2.56
2.58
2.58
2.58
2.59
2.60
2.60
2.61
2.61
2.62
2.71
2.72
2.73
2.73
2.74
2.75
2.75
3.05
3.07
3.09
3.59
5.25
5.26
5.27
5.27
5.28
5.28
5.28
5.29
5.29
5.30
5.30
5.30
5.31
5.31
5.31
5.32
5.32
5.33
5.33
5.33
5.33
5.34
5.34
5.35
5.35
5.35
5.36
5.36
5.37
5.38
5.39
5.39
5.39
5.41
5.41
5.41
5.42
5.42
5.42
5.43
5.43
5.44
5.44
5.45
5.45
5.45
5.46
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
5.18
22.72
25.68
31.34
33.83
51.46
128.07
128.28
128.72
130.12
173.20
O
O
I
6.13

257

-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2.14
3.08
2.71
2.20
3.00
1.88
0.52
14.69
2.17
2.17
2.17
2.18
2.18
2.18
2.18
2.19
2.19
2.19
2.19
2.20
2.20
2.20
2.25
2.26
2.27
2.27
2.27
2.27
2.28
2.28
2.29
2.29
2.41
2.42
2.42
2.42
2.43
2.43
2.44
2.44
2.44
2.45
2.45
2.64
2.64
2.64
2.65
2.66
2.67
3.57
5.27
5.27
5.27
5.28
5.28
5.29
5.29
5.44
5.44
5.44
5.45
5.45
5.45
5.45
5.45
7.71
7.71
7.73
7.73
7.74
7.74
7.74
7.74
7.74
7.75
7.75
7.76
7.78
7.79
7.79
7.80
7.81
7.82
7.82
7.84
7.87
7.87
O
O
IPh
3
P
6.3
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
23.78
26.49
34.55
49.85
52.05
54.82
119.35
120.20
127.35
127.52
129.43
129.45
131.37
131.46
131.50
131.51
131.59
131.63
131.63
131.80
131.82
134.38
134.49
134.77
134.82
134.84
134.87
134.92
134.94
136.23
136.26
136.28
136.31
136.34
136.37
175.18
O
O
IPh
3
P
6.3

258

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
3.00
1.77
2.48
5.15
1.23
1.35
0.84
0.96
1.05
2.60
7.53
1.90
2.02
0.96
0.98
1.00
2.00
2.00
2.00
2.00
2.00
2.01
2.02
2.02
2.03
2.04
2.04
2.22
2.22
2.22
2.23
2.23
2.24
2.24
2.25
2.25
2.25
2.26
2.26
2.26
2.27
2.27
2.28
2.28
2.30
2.30
2.30
2.31
2.32
2.32
2.33
2.40
2.40
2.40
2.40
2.41
2.42
2.42
2.42
2.80
2.80
2.80
2.81
2.81
2.81
2.82
2.89
2.89
2.89
2.90
2.90
3.01
3.14
3.15
3.16
3.44
3.44
3.44
3.45
3.45
5.41
5.42
5.42
5.43
5.44
5.44
5.46
5.46
5.46
5.47
5.47
5.47
5.48
5.48
5.48
5.48
5.49
5.49
5.49
5.50
5.50
5.51
5.51
5.52
5.52
5.52
5.53
5.53
5.53
5.53
5.54
5.54
5.55
5.56
5.56
5.56
5.59
6.13
6.13
6.21
6.47
6.51
5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8
f1 (ppm)
0
100
200
300
O
O
O
6.1
O
O
O
6.1
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
21000
22000 14.41
21.01
23.17
25.99
26.62
30.11
34.02
51.08
57.75
60.26
123.22
127.92
128.54
128.83
128.95
129.16
129.33
131.09
131.42
132.38
134.18
134.67
172.76

259

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
1.90
0.75
3.00
1.60
0.93
0.91
0.90
2.41
2.41
2.43
2.43
2.43
2.44
2.45
2.45
2.47
2.47
2.48
2.49
2.49
2.50
2.50
2.51
2.51
2.52
2.52
2.53
2.53
2.54
2.54
2.54
3.62
3.63
3.73
3.78
3.78
3.78
3.79
3.79
3.79
3.80
3.80
3.80
3.80
3.84
3.85
3.86
3.87
3.87
3.87
3.88
3.89
5.93
5.93
5.94
5.97
5.97
5.98
6.97
6.99
7.01
7.01
7.03
7.05
HO
HO
OH
7.9
COOMe
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
18.84
29.10
3.84
2.19
1.68
1.04
3.00
1.05
0.05
0.06
0.06
0.06
0.07
0.07
0.09
0.87
0.88
0.88
0.88
0.89
0.89
0.91
1.57
1.58
1.58
1.59
1.59
1.60
1.60
1.61
1.61
1.62
1.62
1.62
1.62
1.63
1.64
1.64
1.65
1.65
1.65
1.66
1.66
1.66
1.67
1.68
1.68
1.68
1.69
1.69
1.69
1.70
1.70
1.71
1.71
1.71
1.72
1.73
1.74
1.75
1.75
1.76
1.77
2.29
2.31
2.33
2.35
2.37
2.37
3.54
3.54
3.55
3.56
3.56
3.57
3.57
3.57
3.58
3.58
3.58
3.59
3.60
3.60
3.61
3.61
3.62
3.63
3.63
3.65
3.65
3.66
3.70
3.70
3.71
3.71
3.72
7.10
TBSO
TBSO
OTBS
COOMe

260

-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
12.47
18.43
3.78
1.68
3.00
2.02
0.97
0.07
0.07
0.08
0.08
0.08
0.86
0.87
0.91
0.91
0.92
1.54
1.54
1.55
1.57
1.57
1.58
1.58
1.58
1.59
1.59
1.60
1.60
1.60
1.61
1.61
1.61
1.62
1.62
1.62
1.62
1.63
1.63
1.63
1.64
1.64
1.64
1.65
1.65
1.66
1.66
1.67
1.68
1.69
1.69
2.29
2.31
2.33
3.66
3.87
3.88
3.88
3.89
3.89
3.89
3.90
3.90
3.90
3.91
9.59
9.59
9.60
9.60
O
TBSO
OTBS
7.11
COOMe
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
-4.70
-4.52
-4.48
-4.26
18.26
18.42
20.78
25.95
25.98
33.11
34.16
51.67
75.17
80.89
128.48
173.83
203.71
O
TBSO
OTBS
7.11
COOMe

261

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
10.32
18.35
3.33
1.19
1.49
1.57
0.73
3.00
0.95
0.78
0.73
0.02
0.02
0.03
0.04
0.05
0.05
0.05
0.06
0.06
0.06
0.86
0.86
0.87
0.87
0.88
0.88
0.88
0.89
1.23
1.25
1.25
1.25
1.26
1.26
1.26
1.26
1.26
1.27
1.35
1.37
1.38
1.39
1.40
1.48
1.49
1.50
1.51
1.52
1.52
1.53
1.55
1.57
1.58
1.58
1.59
1.59
1.60
1.60
1.60
1.61
1.61
1.62
1.62
1.64
1.65
1.65
1.66
1.66
1.66
1.67
1.67
1.80
1.81
1.82
1.82
1.82
1.82
1.83
1.83
1.84
1.84
1.84
1.85
2.35
2.36
2.37
2.37
2.38
2.38
2.39
3.59
3.60
3.61
3.63
3.66
3.87
3.87
3.88
3.88
3.88
3.89
3.89
3.89
3.89
3.90
3.90
3.90
3.91
3.91
6.22
6.22
6.23
6.23
6.25
6.26
6.46
6.48
6.50
6.52
TBSO
OTBS
I
COOMe
7.7
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-5000
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000 -4.67
-4.35
-3.91
18.37
20.34
26.02
26.09
33.01
34.44
51.63
75.18
78.06
78.56
147.29
174.03
TBSO
OTBS
I
COOMe
7.7

262

-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
5.43
2.46
9.12
4.46
2.03
1.28
2.07
1.00
0.10
0.11
0.12
0.12
0.12
0.13
0.14
0.86
0.87
0.88
0.89
0.90
1.26
1.27
1.27
1.28
1.28
1.29
1.29
1.30
1.31
1.31
1.31
1.32
1.32
1.33
1.38
1.39
1.39
1.40
1.40
1.41
1.41
1.42
1.42
1.43
1.43
1.43
1.43
1.44
1.45
1.45
1.47
1.56
1.63
1.64
1.65
1.66
1.66
1.67
1.67
1.67
1.68
1.69
1.70
4.31
4.32
4.33
4.33
4.35
4.35
OTBS
7.12
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
8.97
17.84
3.94
1.95
2.47
2.82
1.11
1.00
1.01
0.99
1.01
0.50
2.35
0.04
0.06
0.87
0.92
1.53
1.54
1.54
1.55
1.56
1.56
1.57
1.57
1.57
1.58
1.58
1.59
1.60
1.69
1.71
1.71
1.72
1.72
1.73
1.74
1.74
1.76
1.76
2.29
2.31
2.33
3.66
3.67
3.68
3.69
3.70
3.70
3.71
4.16
4.16
4.17
4.17
4.18
4.18
4.19
4.19
6.10
6.12
6.14
6.16
6.82
6.82
6.86
6.86
6.86
7.06
7.07
7.08
7.08
7.09
7.09
7.10
7.11
7.26
7.26
7.27
7.28
7.28
7.28
7.28
7.47
7.47
7.47
7.49
7.49
7.53
7.53
7.55
7.55
7.55
7.56
7.57
7.58
7.62
7.62
7.64
TBSO
OTBS
COOMe
7.38
Br

263

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
9.42
18.26
2.24
2.15
2.04
0.70
2.98
1.00
0.96
1.00
0.94
1.36
0.86
0.96
0.01
0.03
0.04
0.08
0.86
0.86
0.86
0.87
0.87
0.87
0.88
0.88
0.90
0.90
0.91
0.91
1.51
1.51
1.52
1.53
1.53
1.54
1.54
1.55
1.56
1.56
1.57
1.57
1.58
1.60
1.62
1.65
1.67
1.68
1.69
1.69
1.70
1.70
1.71
1.71
1.71
1.72
1.72
1.73
1.73
1.74
1.74
1.75
1.75
1.76
1.77
2.29
2.31
2.32
3.63
3.64
3.64
3.65
3.66
3.66
4.10
4.10
4.11
4.11
4.11
4.12
4.13
4.13
6.14
6.15
6.18
6.19
6.39
6.39
6.39
6.43
6.43
6.43
7.16
7.18
7.20
7.26
7.26
7.26
7.26
7.26
7.26
7.28
7.28
7.28
7.28
7.34
7.34
7.34
7.34
7.36
7.36
7.36
7.36
7.47
7.47
7.48
7.48
7.48
TBSO
OTBS
COOMe
Br
7.39
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
12.13
17.55
1.54
2.21
2.01
2.92
1.23
1.00
0.97
0.95
1.84
2.11
-0.01
0.02
0.04
0.07
0.86
0.86
0.88
0.89
0.90
1.52
1.53
1.55
1.55
1.56
1.56
1.57
1.58
1.59
1.60
1.62
1.67
1.69
1.69
1.70
1.71
1.71
1.72
1.72
1.73
1.73
1.74
1.74
1.74
1.75
1.76
1.76
1.77
2.29
2.31
2.32
2.33
3.66
3.66
3.67
3.68
4.07
4.08
4.09
4.09
4.09
4.10
4.11
4.11
6.11
6.13
6.15
6.17
6.39
6.39
6.39
6.43
7.20
7.20
7.21
7.21
7.22
7.22
7.22
7.22
7.42
7.42
7.43
7.44
7.44
7.44
7.45
TBSO
OTBS
COOMe
Br
7.40

264

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
-4.55
-4.35
-3.94
-3.79
18.27
18.38
20.53
26.05
26.07
26.09
26.12
26.15
33.10
34.50
51.59
75.94
77.07
121.25
128.00
130.16
131.80
131.97
136.11
174.11
TBSO
OTBS
COOMe
Br
7.40
TBSO
OTBS
COOMe
Br
F
7.41
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
11.83
2.00
3.33
0.86
1.30
0.86
0.83
0.88
0.86
0.01
0.03
0.05
0.06
0.08
2.29
2.31
2.33
2.34
3.66
3.67
4.10
4.11
4.12
4.12
4.12
4.13
4.14
6.17
6.18
6.21
6.22
6.37
6.41
6.97
6.97
6.98
6.99
7.00
7.00
7.09
7.10
7.10
7.11
7.12
7.12

265

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
18.00
2.56
31.32
2.72
2.63
3.73
2.00
2.92
1.40
1.25
1.44
1.35
1.57
2.50
0.01
0.03
0.03
0.04
0.05
0.06
0.06
0.07
0.08
0.09
0.10
0.11
0.16
0.18
0.86
0.87
0.89
0.90
0.91
0.92
0.94
1.29
1.29
1.30
1.31
1.31
1.32
1.33
1.33
1.34
1.34
1.35
1.35
1.36
1.47
1.48
1.48
1.49
1.49
1.50
1.50
1.51
1.51
1.52
1.52
1.52
1.53
1.54
1.54
1.55
1.55
1.56
1.56
1.57
1.58
1.58
1.59
1.59
1.60
1.60
1.61
1.62
1.62
1.62
1.63
1.63
1.65
1.71
1.71
1.73
1.73
1.74
1.74
1.75
1.76
1.76
1.77
1.78
1.79
2.29
2.31
2.32
2.33
3.66
3.66
3.66
3.66
6.04
6.93
6.97
7.16
7.17
7.18
7.19
7.20
7.21
7.22
7.22
7.39
7.40
7.41
7.41
7.47
7.50
7.50
7.50
7.52
7.53
7.53
TBSO
OTBS
COOMe
OTBS
7.42
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-400
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
13.33
4.50
6.98
19.59
3.47
1.09
4.43
3.93
1.19
2.40
3.25
1.00
2.40
1.86
1.96
1.40
2.72
1.28
0.02
0.03
0.03
0.05
0.05
0.05
0.06
0.08
0.09
0.10
0.84
0.84
0.84
0.84
0.85
0.86
0.90
0.91
0.91
0.91
0.91
0.92
0.96
0.98
1.60
1.60
1.60
1.60
1.61
1.61
1.62
1.62
1.63
1.70
1.70
1.71
1.71
1.71
1.72
1.72
1.72
1.73
1.73
1.73
1.73
1.74
1.74
1.74
1.74
1.74
1.75
1.85
1.85
1.86
1.86
1.87
1.87
1.88
1.88
1.88
1.88
1.88
1.89
1.89
1.89
1.90
2.29
2.31
2.40
2.42
2.42
2.44
2.44
2.44
3.66
3.72
4.11
4.11
4.12
4.13
4.13
6.14
6.16
6.18
6.20
6.40
6.41
6.41
7.16
7.18
7.20
7.31
7.33
7.34
7.34
7.35
7.36
7.36
7.37
7.38
7.38
7.38
7.40
7.40
7.41
7.42
7.47
7.48
7.48
TBSO
OTBS
COOMe
TBSO
7.43

266

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
23.00
31.36
1.03
0.68
1.01
3.27
3.23
0.52
3.84
5.06
1.00
1.38
1.00
2.21
2.09
4.42
0.01
0.02
0.04
0.05
0.08
0.87
0.90
0.90
0.92
1.29
1.29
1.30
1.32
1.32
1.33
1.33
1.34
1.34
1.37
1.38
1.38
1.39
1.39
1.40
1.40
1.40
1.41
1.41
1.41
1.42
1.53
1.54
1.54
1.56
1.56
1.57
1.57
1.57
1.58
1.59
1.60
1.68
1.70
1.70
1.70
1.71
1.72
1.72
1.73
1.73
1.74
1.74
1.74
1.75
1.75
1.76
2.15
2.15
2.16
2.16
2.16
2.29
2.30
2.30
2.31
2.32
2.33
3.66
3.67
4.08
4.08
4.09
4.10
4.10
4.10
4.11
4.11
4.12
4.23
4.24
4.25
4.26
4.26
4.26
4.27
4.28
6.10
6.10
6.12
6.14
6.14
6.16
6.16
6.18
6.20
6.20
6.42
6.43
6.43
6.44
6.44
6.47
6.47
6.48
6.48
7.30
7.31
7.31
7.32
TBSO
OTBS COOMe
7.44
TBSO
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
-4.58
-4.55
-4.37
-4.06
-3.88
-3.73
14.21
18.29
20.59
22.79
25.12
26.09
26.10
26.12
29.86
32.00
33.12
34.54
38.62
51.58
73.83
76.02
77.34
126.44
126.68
126.73
128.61
130.81
131.10
133.69
136.21
136.54
137.03
174.16
TBSO
OTBS COOMe
7.44
TBSO

267

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
22.59
36.84
6.83
2.93
0.77
2.82
0.80
3.61
1.33
1.22
2.00
2.06
2.07
0.01
0.03
0.04
0.05
0.08
0.87
0.89
0.91
0.92
1.57
1.58
1.58
1.67
1.68
1.68
1.69
1.70
1.70
1.71
1.71
1.72
1.73
1.73
1.74
1.74
1.75
1.76
1.76
2.25
2.25
2.25
2.27
2.28
2.28
2.29
2.29
2.29
2.31
2.33
2.33
3.63
3.64
3.65
3.65
3.66
4.09
4.09
4.10
4.11
4.11
4.11
4.12
4.12
4.22
4.23
4.24
4.25
4.25
4.27
4.27
4.28
4.28
6.14
6.15
6.16
6.17
6.18
6.19
6.20
6.21
6.41
6.42
6.45
6.46
6.90
6.92
6.92
6.93
6.93
6.95
6.95
6.96
TBSO
OTBS
COOMe
TBSO
7.45
F
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
3.44
3.99
1.32
0.89
2.68
0.72
2.00
1.15
2.72
1.02
1.00
0.92
0.95
1.87
1.69
2.21
0.89
0.89
0.89
0.89
0.90
0.90
0.90
0.91
0.92
1.30
1.30
1.31
1.31
1.32
1.32
1.33
1.33
1.33
1.34
1.34
1.34
1.35
1.39
1.40
1.40
1.42
1.43
1.43
1.44
1.45
1.46
1.52
1.54
1.55
1.63
1.63
1.64
1.64
1.65
1.66
1.66
1.67
1.67
1.68
1.68
1.68
1.69
2.34
2.36
2.38
3.57
3.57
3.58
3.58
3.58
3.59
3.59
3.63
3.76
3.76
3.77
3.78
4.09
4.09
4.10
4.10
4.11
4.12
4.12
5.85
5.86
5.87
5.88
5.89
5.90
5.91
5.92
6.16
6.18
6.20
6.22
6.88
6.88
6.90
6.91
6.92
6.92
6.94
6.95
7.20
7.21
7.21
7.21
7.22
7.22
7.22
7.41
7.42
7.42
7.42
7.43
7.43
7.44
7.44
7.45
7.46
7.46
7.47
7.48
HO
OH
COOMe
OH
7.46

268

20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130 44.41
52.53
53.70
56.23
62.98
64.76
66.65
81.96
86.56
105.36
107.11
114.07
157.59
157.78
158.63
158.77
160.44
161.72
162.99
163.68
166.27
166.78
205.81
HO
OH
COOMe
OH
7.46
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
3.12
1.97
3.27
1.16
1.78
1.31
0.89
2.09
2.57
1.00
2.07
1.96
1.95
3.27
0.94
0.87
0.88
0.88
0.89
0.89
0.90
0.90
0.91
0.91
1.25
1.25
1.26
1.31
1.31
1.32
1.32
1.33
1.33
1.34
1.34
1.49
1.50
1.51
1.51
1.52
1.52
1.53
1.54
1.55
1.59
1.59
1.59
1.61
1.61
1.61
1.62
1.63
1.63
1.64
1.64
1.65
1.65
1.66
1.66
1.69
1.71
1.71
1.71
1.72
1.72
1.73
1.74
1.75
1.77
1.83
1.85
1.85
1.86
1.87
1.87
1.87
1.89
2.34
2.35
2.36
2.37
2.38
2.38
3.66
3.76
3.77
3.78
3.78
3.79
3.80
4.25
4.25
4.25
4.26
4.26
4.27
4.27
4.27
4.27
4.28
4.29
4.29
6.21
6.23
6.25
6.27
6.28
6.31
6.32
6.54
6.54
6.58
6.58
6.62
6.62
6.62
6.66
6.66
7.26
7.27
7.28
7.40
7.41
7.41
HO
OH
COOMe
HO
7.47

269

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-30000
-20000
-10000
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000
170000
180000
190000
14.03
21.07
22.59
25.10
30.90
31.47
31.76
33.70
51.57
73.05
73.80
75.85
124.61
125.85
126.04
127.25
128.83
129.82
132.95
133.08
136.61
137.12
174.13
HO
OH
COOMe
HO
7.47
-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
2.91
0.58
1.02
4.91
0.81
0.82
1.20
1.90
1.04
2.95
1.00
0.80
2.33
1.65
1.52
2.32
0.99
1.00
1.00
1.00
1.01
1.01
1.01
1.52
1.53
1.53
1.53
1.54
1.54
1.55
1.65
1.67
1.68
1.68
1.69
1.70
1.71
1.71
1.72
1.72
1.73
1.74
1.74
1.74
1.75
1.75
1.76
1.76
1.76
1.77
1.77
1.77
1.78
1.79
1.80
1.94
1.95
1.96
2.25
2.25
2.25
2.27
2.44
2.46
2.48
3.65
3.66
3.67
3.67
3.68
3.68
3.74
4.15
4.15
4.15
4.16
4.16
4.16
4.17
4.17
4.18
4.18
4.31
4.31
4.31
4.31
4.32
4.32
4.33
4.33
6.30
6.32
6.34
6.34
6.36
6.36
6.38
6.40
6.40
6.41
6.42
6.44
6.45
6.45
6.47
6.62
6.62
6.66
6.66
6.67
6.67
6.68
6.68
6.71
6.71
6.78
6.78
6.82
6.82
7.45
7.45
7.47
7.47
7.48
7.48
HO
OH COOMe
7.48
HO

270

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
3.62
3.48
1.61
4.45
0.91
1.86
0.99
2.31
1.00
1.14
2.00
2.18
2.22
1.06
1.06
1.06
1.08
1.09
1.10
1.49
1.49
1.50
1.51
1.51
1.52
1.52
1.58
1.58
1.59
1.60
1.61
1.62
1.63
1.63
1.64
1.73
1.74
1.74
1.75
1.75
1.76
1.77
1.78
1.78
1.78
1.79
1.80
1.81
1.82
1.82
1.83
1.84
1.85
1.85
1.86
1.87
1.88
1.89
1.89
1.99
2.00
2.01
2.02
2.02
2.03
2.03
2.04
2.05
2.05
2.06
2.51
2.53
2.55
3.71
3.72
3.73
3.73
3.74
3.74
3.75
3.76
3.80
4.22
4.23
4.24
4.24
4.24
4.25
4.26
4.34
4.34
4.35
4.35
4.37
4.37
4.39
6.43
6.44
6.47
6.48
6.53
6.55
6.57
6.59
6.69
6.73
6.73
6.76
6.80
7.17
7.17
7.18
7.19
7.19
7.20
7.20
7.20
7.21
7.22
7.22
7.42
7.42
7.42
HO
OH
COOMe
HO
7.49
F
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
2.21
2.21
3.02
2.00
7.23
3.15
5.30
1.83
1.83
1.84
1.85
1.85
1.86
1.87
1.87
1.88
1.88
1.89
2.84
2.84
2.85
2.86
2.87
2.87
2.87
3.58
3.98
4.00
4.00
4.01
4.01
4.02
4.02
4.03
4.05
7.60
7.60
7.61
7.61
7.61
7.62
7.62
7.62
7.63
7.63
7.63
7.63
7.64
7.64
7.64
7.65
7.65
7.65
7.70
7.70
7.70
7.70
7.71
7.71
7.72
7.72
7.72
7.72
7.74
7.74
7.74
7.74
7.80
7.80
7.80
7.82
7.82
7.82
7.83
7.83
7.83
7.85
7.85
7.85
COOMe BrPh
3
P
7.16

271

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2.76
2.72
1.13
3.99
0.51
1.87
2.19
2.00
1.82
1.36
1.37
1.37
1.37
1.48
1.48
2.28
2.28
2.29
2.29
2.30
2.31
2.32
2.32
2.32
2.33
2.34
2.34
2.37
2.37
2.38
2.38
2.39
2.39
2.40
2.40
2.41
2.41
2.42
2.42
2.43
2.43
2.44
2.44
3.65
3.65
3.66
3.66
3.66
3.67
3.67
3.67
4.16
4.17
4.18
4.18
4.18
4.19
4.19
4.19
4.20
4.20
4.21
4.21
4.23
5.43
5.45
5.46
5.46
5.47
5.47
5.47
5.48
5.48
5.49
5.49
5.49
5.49
5.51
HO
COOMe
O O
7.18
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
f1 (ppm)
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2.75
2.66
5.92
2.80
1.00
1.04
2.41
0.94
1.40
1.40
1.58
1.59
2.31
2.31
2.31
2.31
2.32
2.32
2.33
2.33
2.33
2.34
2.34
2.34
2.34
2.34
2.35
2.35
2.35
2.36
2.36
2.36
2.36
2.37
2.38
2.38
3.66
4.29
4.30
4.31
4.32
4.36
4.36
4.37
4.37
4.38
4.39
4.39
4.39
4.39
4.39
4.41
4.41
5.42
5.42
5.42
5.43
5.44
5.44
5.45
5.45
5.46
5.46
5.46
5.47
5.47
5.47
5.48
5.48
5.48
5.49
5.49
5.49
5.50
5.50
5.50
5.51
5.51
5.52
5.53
9.66
9.66
9.67
9.67
O
COOMe
O O
7.19

272

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
3.12
3.49
2.48
4.05
3.01
1.12
1.00
2.48
0.95
1.04
1.34
1.34
1.34
1.35
1.48
1.48
1.48
2.17
2.18
2.18
2.19
2.20
2.20
2.21
2.22
2.22
2.23
2.23
2.24
2.24
2.25
2.25
2.27
2.28
2.29
2.29
2.30
2.31
2.31
2.33
2.33
2.37
2.37
2.37
2.38
2.39
3.67
3.68
4.14
4.16
4.16
4.17
4.18
4.19
4.47
4.47
4.49
4.49
4.49
4.50
4.51
5.37
5.38
5.39
5.39
5.40
5.40
5.41
5.41
5.42
5.43
5.43
5.44
5.45
5.45
5.45
5.46
5.46
5.46
5.46
5.47
5.47
5.47
5.48
5.48
5.48
5.49
5.49
5.49
5.50
6.41
6.41
6.45
6.45
6.45
6.50
6.52
6.54
6.56
COOMe
O O
I
7.20
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
23.56
25.88
28.48
29.01
34.30
52.06
77.98
80.31
80.80
109.15
126.27
130.75
142.52
173.92
COOMe
O O
I
7.20

273

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
11.98
18.22
1.78
3.53
0.95
2.86
1.00
2.17
0.92
0.93
0.02
0.03
0.04
0.05
0.83
0.85
0.85
0.85
0.86
0.86
0.86
0.86
0.86
0.86
0.87
0.87
0.87
0.88
0.88
0.89
0.89
0.90
0.90
0.90
0.90
0.90
0.91
0.91
0.92
2.22
2.23
2.23
2.24
2.24
2.25
2.25
2.26
2.26
2.26
2.27
2.27
2.28
2.29
2.32
2.33
2.33
2.33
2.34
2.34
2.34
2.35
2.35
3.57
3.59
3.59
3.60
3.60
3.62
3.67
3.91
3.91
3.92
3.92
3.92
3.93
3.94
3.94
5.38
5.38
5.39
5.39
5.40
5.40
5.40
5.40
5.41
5.41
5.42
5.42
5.42
5.43
5.43
5.43
5.44
5.44
5.45
5.45
5.46
5.47
5.47
5.47
5.48
5.50
6.20
6.20
6.23
6.24
6.49
6.51
6.53
6.55
TBSO
OTBS
COOMe
I
7.15
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
-4.59
-4.21
-4.14
-4.01
18.24
18.38
23.24
26.04
26.07
31.67
34.07
51.70
75.61
78.04
78.63
127.23
129.40
146.91
173.64
TBSO
OTBS
COOMe
I
7.15

274

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
0
100
200
300
400
500
600
700
800
900
9.79
14.98
6.33
2.77
0.97
1.00
0.98
1.01
0.69
0.95
0.90
0.89
0.66
1.96
0.05
0.05
0.06
0.10
0.87
0.87
0.92
0.92
2.24
2.25
2.26
2.27
2.28
2.28
2.28
2.30
2.30
2.31
2.32
2.32
2.33
2.34
2.35
2.35
2.36
2.36
2.36
2.37
2.37
2.38
2.38
2.38
3.65
3.65
3.71
3.72
3.73
3.74
3.75
3.75
4.19
4.19
4.20
4.20
4.20
4.21
4.21
4.21
4.22
5.41
5.41
5.41
5.42
5.43
5.43
5.43
5.44
5.44
5.45
5.49
5.51
5.52
5.52
5.53
5.54
5.55
6.13
6.15
6.17
6.19
6.82
6.82
6.83
6.86
6.86
6.87
7.07
7.07
7.08
7.09
7.09
7.09
7.10
7.11
7.26
7.27
7.27
7.27
7.27
7.27
7.28
7.29
7.29
7.48
7.48
7.50
7.50
7.53
7.53
7.53
7.55
7.55
7.55
TBSO
OTBS
COOMe
Br
7.50
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
18.33
23.27
26.13
26.14
26.18
31.82
34.13
51.66
76.79
77.13
123.80
127.19
127.59
127.80
128.73
129.19
129.97
133.04
133.46
137.16
173.69
TBSO
OTBS
COOMe
Br
7.50

275

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
f1 (ppm)
0
50
100
150
200
250
300
350
400
450
500
550
12.18
17.60
6.14
3.08
1.17
1.00
1.25
1.24
0.92
0.99
2.00
1.93
0.01
0.02
0.03
0.07
0.85
0.90
2.28
2.32
2.34
2.34
2.35
2.35
2.35
2.36
2.37
3.66
3.68
3.68
3.68
3.69
3.70
3.70
3.71
4.10
4.11
4.11
4.12
4.12
4.12
4.13
4.13
5.40
5.41
5.41
5.42
5.43
5.43
5.43
5.44
5.45
5.49
5.51
5.52
5.52
5.53
5.53
5.54
6.15
6.16
6.18
6.20
6.40
6.43
6.70
6.71
6.71
6.72
6.72
6.73
7.21
7.22
7.22
7.23
TBSO
OTBS
COOMe
Br
7.51
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
-4.46
-4.34
-4.21
-4.02
18.27
23.25
25.77
26.07
26.12
26.13
29.86
31.77
34.10
51.66
76.46
77.19
122.04
127.73
128.02
128.65
130.10
131.79
132.16
132.42
133.28
136.16
173.67
TBSO
OTBS
COOMe
Br
7.51

276

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
16.36
27.98
3.21
4.80
1.38
0.50
2.03
2.58
0.78
0.82
1.00
0.86
0.62
0.62
0.64
0.46
1.28
0.94
0.75
-0.03
-0.01
0.00
0.01
0.01
0.02
0.03
0.04
0.04
0.05
0.06
0.07
0.07
0.08
0.08
0.09
0.09
0.10
0.11
0.16
0.18
0.85
0.86
0.87
0.87
0.88
0.88
0.89
0.89
0.89
0.90
0.91
0.91
0.92
0.92
0.93
0.93
0.94
1.24
1.26
1.26
1.28
1.29
1.29
1.30
1.30
1.31
1.32
1.33
1.34
1.34
1.35
1.35
1.36
2.29
2.29
2.30
2.31
2.31
2.32
2.33
2.33
2.34
2.35
2.36
2.36
3.65
3.65
3.72
3.73
4.18
5.44
5.52
5.52
5.53
5.55
6.04
6.06
6.11
6.18
6.22
6.74
6.75
6.78
6.79
6.79
6.94
6.98
7.17
7.21
7.21
7.22
7.22
7.22
7.23
7.39
7.40
7.40
7.42
7.42
7.43
7.44
7.51
7.52
7.53
7.53
7.54
7.55
7.55
OTBS
7.52
OTBS
OTBS
COOMe
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
-4.54
-4.45
-4.40
-4.28
-4.23
-4.10
-3.94
-3.92
-3.78
14.22
18.43
22.80
23.24
26.02
26.11
26.12
26.14
26.17
29.85
31.67
32.00
34.13
38.93
51.62
74.05
76.59
77.67
126.69
127.59
127.88
127.96
128.68
129.08
132.54
132.91
133.04
135.30
136.35
138.41
173.66
OTBS
7.52
OTBS
OTBS
COOMe

277

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
0
50
100
150
200
250
300
350
400
18.18
29.44
0.88
2.13
5.88
1.10
4.42
2.74
1.23
0.75
1.08
0.92
0.85
2.00
2.02
2.82
1.20
0.02
0.02
0.03
0.05
0.05
0.07
0.07
0.08
0.86
0.86
0.87
0.89
0.90
0.90
0.91
0.91
0.91
0.92
0.92
1.26
1.29
1.29
1.33
1.34
1.35
1.35
1.35
1.36
1.37
1.37
1.38
1.38
1.39
1.39
1.39
1.40
1.40
1.40
1.41
1.41
1.42
1.43
1.52
1.53
1.54
1.55
1.56
1.57
1.58
1.58
1.59
1.60
2.28
2.33
2.33
2.34
2.34
2.35
2.35
2.36
2.36
2.37
2.37
2.38
3.65
3.66
3.66
3.67
3.67
3.68
3.68
3.69
3.69
3.70
3.70
4.12
4.12
4.12
4.24
4.24
4.25
5.51
6.13
6.14
6.15
6.15
6.16
6.17
6.18
6.19
6.42
6.42
6.44
6.44
6.45
6.46
6.46
6.47
6.47
7.29
7.31
7.31
7.32
7.33
7.42
7.44
7.54
7.55
7.56
TBSO
OTBS
COOMe
OTBS 7.53
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
-4.57
-4.46
-4.22
-3.80
14.21
18.42
22.80
23.25
25.13
26.09
26.11
26.12
26.14
26.16
29.86
32.00
34.15
38.62
51.66
73.84
76.56
77.46
126.68
126.71
126.92
127.14
127.90
128.62
129.02
130.50
131.04
133.68
136.27
136.52
173.70
TBSO
OTBS
COOMe
OTBS 7.53

278

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
10.94
2.60
0.96
1.89
2.00
1.00
0.91
0.96
1.42
0.93
0.90
1.77
1.91
0.86
0.80
1.28
1.32
1.34
1.35
1.35
1.36
1.56
1.57
1.58
1.60
1.61
2.17
2.19
2.21
2.28
2.30
2.32
2.36
2.38
2.40
2.44
3.59
3.61
3.62
3.63
3.64
4.13
4.14
4.15
4.16
4.16
4.19
4.21
4.23
4.24
5.50
5.51
5.52
5.52
5.53
6.03
6.05
6.07
6.08
6.20
6.21
6.24
6.25
6.87
6.91
6.93
6.96
7.18
7.19
7.19
7.20
7.20
7.22
7.40
7.41
7.41
7.42
7.45
7.46
7.47
7.48
OH
7.54
OH
OH
COOH
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
5.71
3.58
2.32
3.00
1.40
0.82
1.41
1.05
1.04
0.97
1.28
1.89
3.86
1.55
1.56
1.56
1.57
1.57
1.58
1.59
1.59
1.59
1.60
1.60
1.61
1.62
1.62
1.62
1.63
1.64
1.64
1.65
1.66
1.66
1.66
1.67
1.67
1.67
1.68
1.68
1.68
2.13
2.15
2.16
2.17
2.18
2.18
2.19
2.20
2.20
2.21
2.22
2.23
2.23
2.24
2.26
2.26
2.39
2.40
2.41
2.41
2.42
2.43
2.43
2.43
2.44
2.46
3.63
3.64
3.65
3.65
3.65
3.66
3.66
3.66
3.67
3.98
3.98
4.00
4.00
4.10
4.11
4.12
4.12
4.13
4.13
4.17
4.17
4.18
4.20
5.45
5.47
5.48
5.54
5.56
5.57
5.58
6.21
6.22
6.24
6.25
6.35
6.36
6.36
6.38
6.40
6.53
6.53
6.56
6.56
6.60
6.61
6.64
6.64
7.35
7.36
7.37
7.37
7.38
7.39
7.40
7.40
HO
OH
COOMe
OH 7.55

279

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
11.50
17.80
3.71
2.00
0.75
2.75
1.12
0.98
0.07
0.07
0.07
0.08
0.09
0.10
0.10
0.11
0.12
0.14
0.14
0.89
0.89
0.90
0.90
1.58
1.58
1.59
1.59
1.59
1.60
1.61
1.61
1.61
1.61
1.62
1.63
1.63
1.63
1.64
1.65
1.66
1.66
1.66
1.67
1.68
1.68
1.68
1.70
1.70
1.70
1.71
1.71
1.71
1.71
1.72
1.72
1.73
1.73
1.73
1.74
1.74
1.75
1.75
1.76
1.77
2.29
2.31
2.33
2.34
2.35
3.66
3.69
3.70
3.70
3.71
3.71
3.71
3.72
3.72
3.73
4.21
4.21
4.22
4.22
4.23
TBSO
OTBS
7.26
COOMe
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
f1 (ppm)
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2.85
3.64
1.94
1.86
1.00
0.93
0.96
0.93
0.90
0.89
0.90
0.90
0.90
0.91
0.91
0.92
0.92
0.92
0.92
0.93
0.93
1.33
1.33
1.33
1.34
1.34
1.34
1.35
1.35
1.36
1.37
1.48
1.48
1.49
1.49
1.50
1.50
1.51
1.51
1.51
1.52
1.53
1.75
1.76
1.77
1.78
1.78
1.78
1.79
1.79
1.79
1.80
1.80
1.81
4.55
4.57
4.57
4.58
4.59
4.60
7.02
7.02
7.04
7.06
7.06
7.37
7.37
7.37
7.38
7.39
7.39
7.39
7.40
7.63
7.64
7.64
7.64
7.65
7.66
7.66
7.66
7.78
7.78
7.79
7.79
OH
7.22
I

280

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
-4.74
-4.54
-4.37
-4.21
-3.91
-3.78
14.18
18.46
20.62
22.75
25.15
26.00
26.02
26.10
26.11
31.62
34.51
38.81
51.58
63.63
73.57
76.03
84.03
91.44
123.60
126.15
128.59
128.65
128.84
133.78
133.97
137.26
174.14

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
17.28
25.06
4.39
7.76
2.00
3.43
1.03
0.65
1.27
0.60
0.59
3.67
0.01
0.03
0.04
0.08
0.12
0.16
0.18
0.86
0.90
0.90
0.91
0.94
1.32
1.33
1.33
1.34
1.34
1.35
1.35
1.35
1.46
1.47
1.47
1.48
1.48
1.49
1.50
1.50
1.51
1.51
1.52
1.52
1.53
1.55
1.55
1.56
1.57
1.59
1.59
1.60
1.60
1.61
1.62
1.64
1.64
1.64
1.65
1.66
1.67
1.67
1.67
1.68
1.69
1.69
1.70
1.71
1.71
1.71
1.72
1.72
1.73
1.73
1.74
1.74
1.75
1.76
1.76
1.77
2.29
2.30
2.31
2.32
2.33
2.33
3.66
3.66
3.66
3.68
3.69
3.69
3.69
3.70
4.11
4.55
4.55
4.57
4.58
6.12
6.14
6.16
6.18
6.41
6.45
7.31
7.31
7.31
7.31
7.32
7.32
7.32
7.33
7.33
7.33
7.33
7.34
7.34
7.34
7.34
7.35
TBSO
OTBS
COOMe
TBSO
7.56
TBSO
OTBS
COOMe
TBSO
7.56

281

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
3.02
2.39
3.91
1.88
0.83
2.34
1.00
1.86
0.68
3.24
1.00
1.02
0.68
0.63
1.75
1.35
1.02
0.93
0.93
0.94
0.94
0.95
0.95
0.96
1.30
1.30
1.36
1.37
1.37
1.37
1.38
1.38
1.39
1.39
1.40
1.40
1.40
1.40
1.41
1.42
1.42
1.43
1.43
1.44
1.52
1.52
1.53
1.53
1.54
1.54
1.54
1.55
1.55
1.55
1.56
1.56
1.57
1.66
1.66
1.67
1.67
1.68
1.72
1.73
1.73
1.73
1.73
1.74
1.74
1.74
1.75
1.75
1.76
1.76
1.77
1.77
1.77
1.78
1.78
2.36
2.37
2.39
2.39
2.40
3.65
3.66
3.66
4.08
4.08
4.09
4.09
4.10
4.11
4.50
4.51
4.52
4.53
6.35
6.37
6.38
6.40
6.59
6.59
6.62
6.62
7.27
7.27
7.28
7.29
7.31
7.33
7.37
7.40
7.40
7.40
7.41
7.41
7.41
7.42
7.47
7.47
7.47
7.47
7.48
7.48
7.49
HO
OH
COOMe
HO
7.57
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
14.26
22.41
23.57
26.02
26.04
26.42
32.58
32.60
33.10
34.63
38.93
51.86
63.13
75.25
76.79
84.72
91.60
124.58
127.27
129.58
130.36
131.31
131.72
135.29
138.71
175.78
HO
OH
COOMe
HO
7.57

282

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
2.91
4.23
0.83
2.06
1.33
4.01
1.82
0.99
2.02
1.00
1.43
1.02
1.05
1.02
0.90
1.15
1.30
0.91
0.92
0.93
0.93
0.94
1.36
1.37
1.38
1.38
1.41
1.42
1.43
1.44
1.45
1.46
1.46
1.47
1.53
1.55
1.57
1.62
1.63
1.64
1.64
1.65
1.66
1.67
1.68
1.69
1.69
1.70
1.72
1.74
1.75
1.76
1.76
1.77
1.77
1.78
1.79
1.80
1.80
1.82
1.84
1.85
1.86
1.87
1.88
2.34
2.36
2.38
2.38
3.56
3.56
3.57
3.58
3.58
3.59
3.59
3.60
3.63
4.08
4.08
4.09
4.09
4.10
4.11
4.11
4.54
4.55
4.56
4.57
6.40
6.41
6.44
6.45
7.08
7.08
7.11
7.12
7.18
7.18
7.19
7.20
7.20
7.21
7.26
7.26
7.26
7.28
7.28
7.29
7.29
7.36
7.36
7.37
7.38
7.38
7.59
7.61
7.62
HO
OH
COOMe
OH
7.58
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
14.42
22.54
23.71
26.25
32.76
33.24
34.77
39.09
51.97
63.40
75.39
77.22
83.39
96.58
122.90
126.14
128.27
129.51
130.66
132.28
133.51
139.74
175.87
HO
OH
COOMe
OH
7.58

283

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
11.61
17.66
2.45
1.95
1.95
2.85
0.89
1.00
0.96
0.96
0.93
0.02
0.04
0.06
0.08
0.86
0.87
0.87
0.88
0.90
0.91
1.56
1.63
1.64
1.64
1.65
1.66
1.66
1.67
1.67
1.68
1.68
1.69
1.69
1.70
1.70
1.71
1.71
1.72
1.72
1.73
1.73
1.74
1.74
1.75
1.76
1.76
2.29
2.31
2.33
2.33
3.67
3.68
3.69
3.69
3.70
3.71
4.26
4.26
4.27
4.27
4.28
4.28
4.29
6.21
6.22
6.23
6.24
6.25
6.26
6.27
6.28
6.82
6.84
6.86
6.88
9.56
9.58
TBSO
OTBS
O
7.33
COOMe
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100 -4.61
-4.39
-4.23
-3.89
18.29
18.37
20.66
26.01
26.05
33.27
34.24
51.68
75.61
76.16
132.52
157.86
173.86
193.58
TBSO
OTBS
O
7.33
COOMe

284

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
12.24
3.15
18.31
2.24
1.98
1.53
1.03
0.96
2.60
1.00
0.88
0.74
0.00
0.01
0.03
0.80
0.82
0.82
0.83
0.84
0.85
0.86
1.22
1.23
1.23
1.24
1.24
1.24
1.24
1.59
1.61
1.62
1.62
1.63
1.63
1.63
1.64
1.65
1.65
1.66
1.66
1.67
1.67
1.67
1.68
1.68
1.69
1.70
2.25
2.26
2.28
2.84
2.84
2.84
2.85
2.85
2.85
3.52
3.53
3.53
3.54
3.55
3.56
3.62
3.62
3.63
3.63
3.63
3.64
3.64
3.65
3.96
3.96
3.97
3.97
3.97
3.98
3.99
5.56
5.56
5.56
5.56
5.60
5.60
5.60
5.61
6.14
6.14
6.16
6.16
6.18
6.18
6.20
6.20
TBSO
OTBS
7.35
COOMe
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
-4.60
-4.41
-4.07
12.76
14.28
18.36
20.66
22.81
26.05
26.10
29.46
31.08
34.44
51.63
75.82
76.40
77.69
82.28
128.51
177.72
207.07
TBSO
OTBS
7.35
COOMe

285

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
12.14
17.50
0.60
6.46
2.97
1.02
1.00
1.06
1.11
0.05
0.06
0.06
0.08
0.09
0.10
0.10
0.10
0.10
0.11
0.12
0.13
0.14
0.15
0.89
0.89
0.89
0.90
2.17
2.33
2.34
2.34
2.35
2.36
2.36
2.36
2.37
2.37
2.37
2.37
2.38
2.38
2.38
2.39
2.39
2.40
3.67
3.70
3.71
3.72
3.72
3.72
3.73
3.74
4.23
4.24
4.24
4.25
5.40
5.40
5.41
5.42
5.42
5.43
5.43
5.44
5.44
5.44
5.46
5.48
5.50
5.50
5.51
5.52
5.52
5.53
5.54
COOMe
OTBS TBSO
7.36
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240
f1 (ppm)
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
-0.00
0.58
0.62
0.86
23.21
23.26
28.06
30.85
30.97
36.01
39.03
56.55
71.85
78.62
80.80
88.85
132.30
134.40
178.62
COOMe
OTBS TBSO
7.36

286

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
12.46
22.53
1.32
2.11
1.01
1.55
2.55
1.00
1.01
0.91
0.96
0.93
1.90
-0.06
-0.05
0.03
0.03
0.08
0.09
0.10
0.11
0.87
0.88
1.24
1.42
1.44
1.45
1.46
1.47
1.48
1.49
1.50
1.52
1.62
1.64
1.65
1.66
1.67
1.68
1.72
1.73
1.75
1.76
1.77
2.26
2.27
2.28
2.30
2.32
3.65
3.65
3.65
3.99
4.00
4.01
4.02
4.94
4.95
7.38
7.40
7.40
7.41
7.56
7.58
7.64
7.66
7.89
7.93
7.94
N
N
N
Br
TBSO
OTBS
COOMe
7.30
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240
f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
-0.00
0.02
0.17
0.54
18.86
22.93
22.98
25.54
30.64
30.68
34.44
37.40
38.91
56.20
76.44
80.66
123.55
125.32
128.07
128.34
135.78
136.33
142.88
154.94
178.59
N
N
N
Br
TBSO
OTBS
COOMe
7.30

287

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
9.11
18.05
2.00
2.35
3.14
1.04
1.00
1.92
0.95
0.96
0.98
0.91
0.92
0.05
0.07
0.08
0.09
0.85
0.86
0.86
0.87
0.88
0.88
0.89
0.89
2.19
2.19
2.19
2.21
2.22
2.22
2.23
2.33
2.34
2.34
3.64
3.65
4.01
4.02
4.03
4.04
4.05
4.05
4.96
4.97
4.97
4.97
5.39
5.39
5.40
5.40
5.41
5.41
5.42
5.42
5.43
5.43
5.45
5.47
5.47
5.47
5.48
5.49
5.50
7.38
7.40
7.42
7.55
7.55
7.56
7.56
7.57
7.57
7.58
7.58
7.64
7.64
7.64
7.65
7.66
7.66
7.66
7.67
7.91
7.91
7.94
7.94
7.95
7.59
OTBS
OTBS
COOMe
N
N
N
Br
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230
f1 (ppm)
-3000
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
21000
22000
23000
24000
-4.53
-4.46
-4.14
18.33
18.39
23.13
26.07
26.09
29.85
31.51
34.03
51.66
71.76
76.46
118.96
119.21
120.86
123.47
123.76
127.21
129.62
131.18
131.69
150.13
173.70
7.59
OTBS
OTBS
COOMe
N
N
N
Br

288

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
f1 (ppm)
0
100
200
300
400
500
600
700
800
900
1000
11.72
18.40
2.32
2.07
2.12
3.01
0.98
1.00
2.02
1.06
1.02
1.01
0.95
1.03
0.03
0.03
0.04
0.05
0.05
0.06
0.07
0.09
0.09
0.10
0.11
0.86
0.87
0.87
0.88
0.91
0.91
1.53
1.54
1.55
1.56
1.56
1.57
1.57
1.58
1.59
1.59
1.60
1.61
1.62
1.62
1.66
1.66
1.67
1.68
1.68
1.69
1.70
1.70
1.71
1.72
1.72
1.73
1.74
1.74
1.75
1.75
1.76
1.76
1.77
1.77
1.79
2.29
2.31
2.33
3.66
3.66
3.67
3.67
3.68
3.69
3.69
3.70
3.70
4.16
4.17
4.17
4.18
4.18
4.18
4.19
4.19
6.44
6.46
6.48
6.50
6.56
6.56
6.56
6.60
6.60
7.38
7.40
7.42
7.55
7.55
7.55
7.56
7.57
7.57
7.58
7.69
7.69
7.70
7.70
7.71
7.72
7.72
7.88
7.93
7.94
7.94
TBSO
OTBS
COOMe
N
N
N
Br
7.60
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
-3000
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
21000
22000
23000
-4.51
-4.40
-3.91
-3.82
18.33
18.43
20.82
26.12
26.14
32.90
34.50
51.61
76.06
76.96
77.48
117.85
119.01
119.03
123.44
123.59
126.74
131.19
131.77
134.43
138.08
174.12
TBSO
OTBS
COOMe
N
N
N
Br
7.60

289

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
13.43
24.90
5.23
1.14
4.10
1.82
1.94
2.80
1.00
0.89
0.89
0.84
0.90
1.75
0.94
0.93
0.84
0.04
0.06
0.09
0.09
0.10
0.88
0.88
0.89
0.93
1.28
1.28
1.29
1.29
1.30
1.30
1.31
1.31
1.32
1.33
1.33
1.44
1.45
1.45
1.46
1.47
1.48
1.48
1.49
1.51
1.54
1.54
1.55
1.56
1.57
1.58
1.59
1.59
1.60
1.61
1.61
1.62
1.65
1.66
1.67
1.68
1.69
1.70
1.72
1.73
1.74
1.75
1.75
2.26
2.27
2.27
2.28
2.28
2.29
2.29
2.30
3.64
3.64
4.00
4.01
4.01
4.01
4.02
4.02
4.03
4.04
4.29
4.29
4.30
4.31
4.94
4.95
4.95
4.95
6.27
6.28
6.31
6.32
6.53
6.53
6.57
6.57
7.40
7.41
7.42
7.42
7.43
7.43
7.45
7.45
7.47
7.52
7.52
7.52
7.52
7.53
7.54
7.54
7.54
7.72
7.72
7.73
7.73
7.92
7.92
TBSO
OTBS
COOMe
N
N
N
TBSO
7.37
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
-4.58
-4.56
-4.41
-4.13
-4.04
14.21
18.37
20.99
22.78
25.02
26.07
26.08
26.11
26.13
29.86
32.01
32.88
34.36
38.46
51.60
71.90
73.34
76.12
118.33
119.06
120.92
126.61
127.50
129.98
136.01
137.69
139.34
149.95
174.03
TBSO
OTBS
COOMe
N
N
N
TBSO
7.37

290

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
16.87
30.91
6.18
2.09
4.24
3.10
1.13
1.00
1.08
2.38
0.96
1.01
2.17
1.01
1.00
1.01
-0.06
-0.05
-0.04
0.05
0.06
0.06
0.07
0.08
0.08
0.09
0.10
0.10
0.11
0.87
0.88
0.89
0.89
0.93
1.54
1.55
1.55
1.56
1.57
1.57
1.58
1.58
1.59
1.59
1.60
1.61
1.61
1.62
1.63
2.19
2.20
2.21
2.23
2.23
2.30
2.31
2.32
2.33
2.33
2.34
2.34
2.34
2.35
2.35
2.36
3.64
4.02
4.03
4.04
4.05
4.05
4.06
4.27
4.28
4.29
4.29
4.30
4.31
4.97
4.98
5.40
5.40
5.41
5.41
5.41
5.42
5.42
5.43
5.44
5.44
5.47
5.48
5.48
5.49
5.50
5.51
6.27
6.28
6.31
6.32
6.53
6.53
6.53
6.57
6.57
6.57
7.39
7.40
7.40
7.41
7.42
7.42
7.43
7.45
7.47
7.52
7.52
7.53
7.54
7.54
7.55
7.73
7.73
7.74
7.94
OTBS
OTBS
COOMe
N
N
N
TBSO
7.61
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200
f1 (ppm)
-20000
-10000
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000
170000
180000
190000
200000
210000
220000
230000
240000
-4.58
-4.53
-4.46
-4.15
14.20
18.34
18.40
22.78
23.14
25.01
26.07
26.11
29.85
32.01
34.06
38.46
51.64
71.80
73.34
76.50
118.34
119.06
121.05
126.57
127.30
127.53
129.57
129.97
135.99
137.73
139.32
149.72
173.72
OTBS
OTBS
COOMe
N
N
N
TBSO
7.61

291

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
18.41
29.40
5.95
3.06
3.03
2.06
3.04
0.99
1.00
0.89
0.93
0.98
1.93
2.06
0.90
0.92
0.96
0.05
0.05
0.06
0.06
0.09
0.09
0.10
0.88
0.91
0.92
0.92
1.26
1.28
1.29
1.30
1.31
1.32
1.32
1.33
1.34
1.36
1.37
1.37
1.39
1.40
1.40
1.42
1.53
1.55
1.56
1.57
1.58
1.59
1.60
1.61
1.61
1.65
1.66
1.66
1.67
1.68
1.69
1.69
1.70
1.70
1.71
1.72
1.72
1.73
1.74
1.74
1.75
1.76
1.76
1.77
1.78
1.79
2.29
2.31
2.33
3.66
3.67
3.68
3.68
3.69
3.70
3.70
4.16
4.18
4.18
4.18
4.18
4.19
4.27
4.29
4.29
4.30
4.30
6.27
6.29
6.31
6.33
6.42
6.44
6.46
6.48
6.53
6.57
6.57
6.61
7.40
7.40
7.41
7.42
7.42
7.43
7.45
7.47
7.54
7.54
7.55
7.56
7.56
7.57
7.73
7.74
7.74
7.90
TBSO
OTBS
COOMe
N
N
N
TBSO
7.62
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
-4.59
-4.52
-4.40
-4.13
-3.89
-3.82
-3.43
14.19
18.32
18.42
18.44
20.83
22.77
25.00
25.80
26.07
26.13
29.84
31.99
32.87
34.50
38.44
51.59
73.34
76.09
77.03
118.10
118.37
119.12
119.37
126.67
127.47
129.94
133.93
136.04
137.48
139.33
146.54
174.13
TBSO
OTBS
COOMe
N
N
N
TBSO
7.62

292

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
f1 (ppm)
-50
0
50
100
150
200
250
300
350
400
450
500
550
600
650
2.55
5.25
0.96
1.20
1.86
2.59
0.70
0.97
1.00
0.87
1.01
1.70
1.06
1.00
0.82
1.31
1.31
1.32
1.33
1.34
1.34
1.34
1.35
1.35
1.36
1.36
1.36
1.37
1.37
1.38
1.39
1.39
1.41
1.48
1.49
1.50
1.51
1.52
1.52
1.52
1.53
1.53
1.54
1.54
1.55
1.56
1.56
1.57
1.57
1.58
1.59
1.59
1.60
1.61
1.62
1.62
1.63
1.64
1.64
1.66
1.66
1.75
1.77
1.77
1.78
1.78
1.79
1.80
1.80
1.94
1.95
1.96
2.07
2.09
2.11
2.13
2.26
2.27
2.28
2.29
2.29
2.30
2.31
3.56
3.56
3.84
4.27
4.27
4.29
4.29
4.30
4.30
4.31
4.32
4.32
4.68
4.69
4.69
6.28
6.30
6.32
6.34
6.53
6.53
6.57
6.57
7.43
7.43
7.43
7.43
7.43
7.45
7.61
7.61
7.62
7.63
7.63
7.64
7.64
7.78
7.78
7.78
7.79
8.35
HO
OH
COOMe
N
N
N
HO
7.29
-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
-4.02
14.38
22.43
23.72
26.41
33.00
34.67
39.37
49.85
51.97
71.48
74.73
111.43
119.08
120.28
122.58
127.76
129.02
131.19
136.89
138.90
140.57
175.84
HO
OH
COOMe
N
N
N
HO
7.29

293

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
f1 (ppm)
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
3.17
2.74
1.02
2.00
1.43
3.72
2.66
0.97
1.00
0.94
1.80
0.93
0.94
1.86
0.83
0.88
0.90
0.88
0.88
0.89
0.89
0.90
0.90
0.91
1.30
1.31
1.32
1.32
1.33
1.33
1.34
1.34
1.37
1.37
1.37
1.38
1.39
1.40
1.41
1.41
1.41
1.42
1.42
1.43
1.43
1.44
1.44
1.44
1.46
1.46
1.47
1.48
1.59
1.60
1.61
1.61
1.62
1.63
1.64
1.64
1.65
1.66
1.66
1.67
1.68
2.25
2.27
2.28
2.30
2.32
2.33
2.33
2.34
2.38
2.39
2.39
2.40
2.41
2.41
2.42
2.44
2.46
2.46
3.65
4.08
4.09
4.29
4.29
4.30
4.31
4.31
4.33
4.33
4.34
4.34
4.96
5.45
5.47
5.50
5.51
5.54
5.56
5.58
6.32
6.33
6.35
6.37
6.61
6.61
6.65
6.65
7.41
7.42
7.42
7.43
7.44
7.44
7.46
7.48
7.57
7.58
7.58
7.59
7.60
7.78
8.14
OH
OH
COOMe
N
N
N
HO
7.63
OH
OH
COOMe
N
N
N
HO
7.63
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
14.19
22.75
25.25
29.85
31.91
33.60
37.50
51.88
72.88
77.36
77.65
110.16
118.46
119.53
126.62
127.03
128.65
128.65
130.09
131.04
135.09
135.09
138.97
174.24

294

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
3.17
3.44
2.33
2.17
1.96
1.02
2.14
3.87
1.10
1.00
1.18
0.99
3.18
1.94
1.05
1.16
0.79
0.91
0.93
0.93
0.94
1.35
1.36
1.37
1.42
1.42
1.43
1.44
1.45
1.46
1.47
1.48
1.49
1.50
1.51
1.52
1.53
1.54
1.58
1.60
1.60
1.61
1.62
1.63
1.63
1.64
1.64
1.66
1.66
1.67
1.68
1.69
1.69
1.70
1.71
1.72
1.72
1.73
1.73
1.74
1.84
1.85
1.86
1.86
1.87
1.87
1.88
1.89
1.89
1.90
2.33
2.34
2.35
2.36
2.36
2.37
2.38
2.77
2.81
2.89
2.93
3.58
3.59
3.59
3.60
3.60
3.61
3.61
3.62
3.63
4.13
4.15
4.16
4.23
4.24
4.26
4.27
6.40
6.41
6.44
6.45
6.61
6.63
6.65
6.66
6.67
6.68
6.69
6.70
6.71
6.72
6.75
7.52
7.53
7.53
7.53
7.54
7.70
7.71
7.71
7.72
7.73
7.90
7.90
7.91
8.60
HO
OH
COOH
N
N
N
HO
7.64
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
f1 (ppm)
0
5
10
15
20
25
30
35
40
45
50 12.99
21.17
22.30
24.92
31.59
31.77
33.49
36.94
42.58
50.57
71.92
75.10
76.54
117.55
118.69
118.71
119.28
126.43
127.91
129.73
132.71
135.06
137.27
139.22
146.31
176.11
HO
OH
COOH
N
N
N
HO
7.64

Abstract (if available)

Abstract This body of work seeks to use synthetic and analytical chemistry techniques to improve the understanding of biologically interesting compounds. This has been completed in multiple projects investigating the anti‐cancer therapeutic Bortezomib and natural metabolites of essentially fatty acids with high potency promoting the resolution of inflammation and tissue regeneration. ❧ Chapter 1 provides a detailed analytical investigation towards the characterization and evaluation of the interactions of FDA approved anti‐cancer therapeutic Bortezomib with dietary polyphenols. The green tea polyphenol epigallocatechin‐3‐gallate was reported to effectively antagonize the ability of Bortezomib to induce apoptosis in cancer cells. This interaction was attributed to the formation of a covalent adduct between a phenolic moiety of epigallocatechin‐3‐gallate with the boronic acid group of Bortezomib. However, the structural details of this boron adduct and the molecular factors that contribute to its formation and its ability to inhibit Bortezomib’s activity remained unclear. This work describes the use of NMR spectroscopy to characterize the structures and properties of the boron adducts of epigallocatechin‐3‐gallate and related polyphenols. ❧ The remaining chapters are dedicated to the design, synthesis and activity of compounds enzymatically produced from essential omega‐3 and omega‐6 fatty acids during a host’s inflammatory immune response, and their synthetic analogs. This includes a brief review of the class of specialized pro‐resolving lipid mediators (SPMs) including their isolation, identification, and biological activity. It has long been postulated that fatty acids such as docosahexaenoic acid present a variety of health benefits including the production of SPMs containing anti‐inflammatory, pro‐resolving, and tissue regenerative properties. This work helps support these claims by producing the design and synthesis of several SPMs derived from docosahexaenoic acid providing a molecular basis for some of theses health effects. Including the design and synthesis of resolvin D4, aspirin‐triggered resolvin D4, maresin 1, maresin 2, maresin conjugate in tissue regeneration 1 and 2, 16S‐17S‐epoxy‐neuroprotectin D1, and a library of benzo SPM based analogs.

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Asset Metadata

Creator Glynn, Stephen J. (author)

Core Title Analytical investigation of the proteasome inhibitor Bortezomib and the total synthesis of specialized pro-resolving lipid mediators

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 11/08/2016

Defense Date 10/06/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag boron NMR,bortezomib,dietary polyphenols,epigallocatechin-3-gallate,green tea extract,Inflammation,lipid mediators,maresin,OAI-PMH Harvest,proteasome inhibitor,protectin,resolvin,total synthesis

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Petasis, Nicos A. (committee chair), Louie, Stan G. (committee member), Prakash, Surya G. K. (committee member)

Creator Email sglynn@usc.edu,stephen.glynn.sg@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-196661

Unique identifier UC11278327

Identifier etd-GlynnSteph-4022.pdf (filename),usctheses-c40-196661 (legacy record id)

Legacy Identifier etd-GlynnSteph-4022.pdf

Dmrecord 196661

Document Type Dissertation

Format application/pdf (imt)

Rights Glynn, Stephen J.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA