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Total synthesis of specialized pro-resolving lipid mediators and their analogs

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Total synthesis of specialized pro-resolving lipid mediators and their analogs

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Total Synthesis of Specialized Pro-resolving Lipid Mediators
and Their Analogs

by

Min Zhu

A Dissertation 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 2013

Copyright 2013 Min Zhu

i

Dedication

To My Dear

Mentor Nicos A. Petasis

Father Huangzhong Zhu and Mother Jianping Hu

Wife Nina Zhao and Daughter Lindsay Zhu

ii

Acknowledgements

First of all, I would like to thank my research advisor Professor Nicos Petasis for his mentoring
over the past several years, which will have profound influence on my future life as a chemist.
As a knowledgeable and accomplished scientist, Professor Petasis guided me into the realm of
organic chemistry and biochemistry, where I learned the art of organic synthesis and elegance of
nature. As a dedicated and visionary teacher, Professor Petasis helped me to find out solutions to
overcome challenging problems inside and outside the lab, where I developed the way of
thinking as a scholar, as well as the skills of a productive chemist. It is hard, to express my
gratitude thoroughly to my mentor of life, in such a short paragraph.
I would like to thank Professor Surya Prakash, Professor Travis Williams, Professor Barry
Thompson, and Professor Stan Louie for their valuable help and patient discussion with me to
solve problems in my classes, qualification exam and research. I would also like to thank
Professor Charles Serhan and Professor Nicolas Bazan, collaborators of the Petasis research
group, for their help and efforts for my research in this thesis.
I would like to thank all the Petasis group members, who created an atmosphere of inspiring
discussion and diligent working environment, from which I have benefitted during my Ph.D.
studies. I would like to thank the senior group members Dr. Kalyan Nagulapalli, Dr. Kevin
Gaffney, Dr. Jeremy Winkler, Dr. Jamie Jarusiewicz, Dr. Alexey Butkevich, Dr. Rong Yang, Dr.
Jasim Uddin, Charles Arden and Anne-Marie Finaldi, who helped me develop skills with their
hands-on experiences. I would like to thank the group members Nikita Vlasenko, Marcos Sainz
and Steve Glynn, who joined our laboratory after me but always gave me tremendous support
and shared with me their knowledge and ideas. I would also like to thank Carole Phillips for her
continuous dedication and support over the past years.
I would like to thank my parents, who always teach me to be strong and independent, and never
give up in front of seemingly unsolvable problems. I would like to thank my wife Nina, who
always supports me to work hard and sacrifices a lot to take care of the family. I would like to
thank my daughter Lindsay, who is the most precious gift God has given me but to whom I feel
genuinely sorry not to have spent more time with her when she needs love from her father. It is
the unconditional love and sacrifice of all my loved ones that made the completion of this thesis
possible.
Last but not the least, I would like to thank all my friends and colleagues working in Locker
Hydrocarbon Institute. I will always remember the happy and difficult times I had with them,
and it is them who make me feel LHI and USC as my home. I wish everyone nothing but the best.

iii

Table of Contents

Dedication ...................................................................................................................................................... i
Acknowledgements ....................................................................................................................................... ii
List of Figures ............................................................................................................................................... v
List of Schemes ............................................................................................................................................ vi
Abstract ........................................................................................................................................................ vi
Chapter 1. Specialized Pro-resolving Mediators Derived from ω-3 Polyunsaturated Fatty Acids: from
Identification to New Pharmaceuticals ......................................................................................................... 1
1.1. Introduction ................................................................................................................................... 1
1.2. Identification of DHA and EPA-derived SPMs ............................................................................ 3
1.3. Biosynthesis of DHA and EPA-derived SPMs ............................................................................. 6
1.4. Anti-inflammtory / pro-resolving activities and mechanisms of DHA and EPA-derived SPMs 10
1.5. Development of DHA and EPA-derived SPMs to pharmaceuticals ........................................... 12
1.6. References ................................................................................................................................... 13
Chapter 2. Total Synthesis of Maresin 1 (MaR1) and its Stereoisomers .............................................. 21
2.1. Introduction ................................................................................................................................. 21
2.2. Results ......................................................................................................................................... 21
2.2.1. Retrosynthetic analysis of MaR1 stereoisomers ................................................................. 21
2.2.2. Synthesis of building blocks of MaR1 stereoisomers ......................................................... 24
2.2.3. Assembly of MaR1 stereoisomers and spectrum study ...................................................... 26
2.2.4. Structure elucidation of MaR1 and bioactivity exploration of MaR1 stereoisomers .......... 29
2.3. Conclusion .................................................................................................................................. 30
2.4. Experimental Procedures ............................................................................................................ 30
2.5. References ................................................................................................................................... 45
Chapter 3. Total Synthesis of 14S-epoxy-maresin: Biosynthetic precursor of Maresin ....................... 47
3.1. Introduction ................................................................................................................................. 47
3.2. Results ......................................................................................................................................... 48
3.2.1. Retrosynthetic analysis of 13S, 14S-epoxy-maresin ........................................................... 48
3.2.2. Synthesis of building blocks of 13S, 14S-epoxy-maresin ................................................... 49
3.2.3. Assembly of 13S, 14S-epoxy-maresin ................................................................................ 51
3.3. Conclusion .................................................................................................................................. 53

iv

3.4. Experimental Procedures ............................................................................................................ 53
3.5. References ................................................................................................................................... 61
Chapter 4. Total Synthesis of Deuterium Labeled DHA and Hydroxy-DHAs ..................................... 64
4.1. Introduction ................................................................................................................................. 64
4.2. Results ......................................................................................................................................... 65
4.2.1. Total Synthesis of 22d
3
, 20R-HDHA (1) and 22d
3
, 20S-HDHA (2) ................................... 65
4.2.2. Total Synthesis of 22d
3
, 14S-HDHA (3) ............................................................................. 68
4.2.3. Total Synthesis of 22d
3
, 7S-HDHA (4) ............................................................................... 70
4.2.4. Total Synthesis of 22d
3
, 4S-HDHA (5) ............................................................................... 71
4.2.5. Total Synthesis of 22d
3
-DHA (6) ........................................................................................ 73
4.3. Conclusion .................................................................................................................................. 74
4.4. Experimental Procedures ............................................................................................................ 75
4.5. References ................................................................................................................................... 90
Chapter 5. Total Synthesis of Aspirin-Triggered Neuroprotectin D1/Protection D1 (AT-NPD1/PD1)
and its Stereoisomers and Analogs ............................................................................................................. 93
5.1. Introduction ................................................................................................................................. 93
5.2. Results ......................................................................................................................................... 94
5.2.1. Retrosynthetic analysis of AT-NPD1/PD1 stereoisomers and analogs .............................. 94
5.2.2. Synthesis of building blocks of AT-NPD1/PD1 stereoisomers .......................................... 95
5.2.3. Assembly and spectroscopic study of AT-NPD1/PD1 stereoisomers and analogs............. 97
5.2.4. Structure elucidation/biosynthetic pathway study of AT-NPD1/PD1 and bioactivity
exploration of AT-NPD1/PD1 stereoisomers ................................................................................... 100
5.3. Conclusion ................................................................................................................................ 102
5.4. Experimental Procedures .......................................................................................................... 102
Chapter 6. Total Synthesis of Benzo-lipoxin A
4
Analogs .................................................................. 112
6.1. Introduction ............................................................................................................................... 112
6.2. Results ....................................................................................................................................... 113
6.2.1. Retrosynthetic analysis of benzo-lipoxin A
4
analogs ........................................................ 114
6.2.2. Synthesis of building blocks of benzo-lipoxin A
4
methyl ester analogs ........................... 114
6.2.3. Assembly of benzo-lipoxin A
4
methyl ester analogs ........................................................ 116
6.3. Conclusion ................................................................................................................................ 117
6.4. Experimental Procedures .......................................................................................................... 118
6.5. References ................................................................................................................................. 124
Bibliography ............................................................................................................................................. 128

v

Appendix: Selected
1
H and
13
C NMR Spectra .......................................................................................... 143

List of Figures

Figure 1. Dual anti-inflammatory and pro-resolution actions of specialized pro-resolving mediators......... 2
Figure 2. Specialized pro-resolving mediators derived from DHA and EPA ............................................... 3
Figure 3. Basic structures of PD1 (A) and RvD2 (B) deduced based on LC-UV-MS/MS analysis ............. 4
Figure 4. LC-MS/MS matching of PD1 (II in synthetic mixture) and biogenic PD 1 .................................. 5
Figure 5. Development of more stable lipoxin A
4
analogs ......................................................................... 12
Figure 6. Retrosynthetic analysis of MaR1 stereoisomers .......................................................................... 23
Figure 7. Stereoisomers of MaR1 prepared by total synthesis .................................................................... 24
Figure 8.
1
H NMR of olefinic region of MaR1 methyl ester (1) ................................................................. 28
Figure 9.
1
H NMR comparison of olefinic region of MaR1 stereoisomers ................................................ 28
Figure 10. LC-MS/MS profiles of MaR1 and stereoisomers ...................................................................... 29
Figure 11. Retrosynthetic analysis of 13S, 14S-epoxy-maresin methyl ester ............................................. 49
Figure 12.
1
H NMR of olefinic region of 13S, 14S-epoxy-maresin methyl ester (1) .................................. 53
Figure 13. Biosynthesis of 7S-HDHA......................................................................................................... 65
Figure 14. Deuterium labeled DHA and hydroxy-DHAs prepared by total synthesis ................................ 65
Figure 15. Retrosynthetic analysis of 22d
3
, 20R-HDHA (1) and 22d
3
, 20S-HDHA (2) ............................. 66
Figure 16. Retrosynthetic analysis of 22d
3
, 14S-HDHA (3) ....................................................................... 68
Figure 17. Retrosynthetic analysis of 22d
3
, 7S-HDHA (4) ......................................................................... 70
Figure 18. Retrosynthetic analysis of 22d
3
, 4S-HDHA (5) ......................................................................... 72
Figure 19. Retrosynthetic analysis of 22d
3
-DHA(6) ................................................................................... 74
Figure 20. Stereoisomers of NPD1/PD1 Prepared by Total Synthesis ....................................................... 94
Figure 21. Retrosynthetic analysis of NPD1/PD1 stereoisomers and analogs ............................................ 95
Figure 22.
1
H NMR of olefinic region of NPD1/PD1 methyl ester (1) ...................................................... 99
Figure 23.
1
H NMR comparison of olefinic region of NPD1/PD1 stereoisomers ...................................... 99
Figure 24. LC-MS/MS matching: comparisons for biologic and synthetic AT-(NPD1/PD1) .................. 100
Figure 25. Lipoxin A
4
and epi-benzo-lipoxin A
4
analogs prepared via total synthesis ............................ 113
Figure 26. Retrosynthetic analysis of epi-benzo-lipoxin A
4
methyl ester analogs ................................... 114

vi

List of Schemes

Scheme 1. Oxidation of PUFA catalyzed by lipoxygenases ......................................................................... 7
Scheme 2. Stereospecific ring-opening of S, S-epoxy-PUFA catalyzed by hydrolases (A) and alcohol
trapping experiment (B) ................................................................................................................................ 8
Scheme 3. Oxidation of PUFA catalyzed by aspirin/COX-2 or P450 .......................................................... 9
Scheme 4. Proposed biosynthesis of aspirin-triggered RvD2 ....................................................................... 9
Scheme 5. Synthesis of aldehyde 14-19 for total synthesis of MaR1 stereoisomers .................................. 25
Scheme 6. Synthesis of building blocks 20-27 for total synthesis of MaR1 stereoisomers ........................ 26
Scheme 7. Assembly of MaR1 stereoisomer 1-6 ........................................................................................ 27
Scheme 8. Biosynthesis of LTB
4
and hypothesized biosynthesis of MaR1 ................................................ 48
Scheme 9. Synthesis of epoxy aldehyde 8 for total synthesis of 13S, 14S-epoxy-maresin......................... 50
Scheme 10. Synthesis of Wittig salt 13 for total synthesis of 13S, 14S-epoxy-maresin ............................. 51
Scheme 11. Assembly of 13S, 14S-epoxy-maresin ..................................................................................... 52
Scheme 12. Total synthesis of 22d
3
, 20R-HDHA (1) and 22d
3
, 20S-HDHA (2) ........................................ 67
Scheme 13. Total Synthesis of 22d
3
, 14S-HDHA (3) ................................................................................. 69
Scheme 14. Total Synthesis of 22d
3
, 7S-HDHA (4) ................................................................................... 71
Scheme 15. Total Synthesis of 22d
3
, 4S-HDHA (5) ................................................................................... 73
Scheme 16. Total Synthesis of 22d
3
-DHA (6) ............................................................................................ 74
Scheme 17. Synthesis of aldehyde 13-16 for total synthesis of NPD1/PD1 stereoisomers and analogs .... 96
Scheme 18. Synthesis of building block 17-24 for total synthesis of NPD1/PD1 stereoisomers and analogs
.................................................................................................................................................................... 97
Scheme 19. Assembly of NPD1/PD1 Stereoisomer 1-6 ............................................................................. 98
Scheme 20. Biosynthesis of NPD1/PD1 and AT-(NPD1/PD1) ................................................................ 101
Scheme 21. Synthesis of building blocks of epi-benzo-lipoxin A
4
methyl ester analogs ......................... 115
Scheme 22. Assembly of epi-benzo-lipoxin A
4
methyl ester analog 2-6 .................................................. 117

Abstract

vii

Abstract
This dissertation reports research on the total synthesis of a series of DHA-derived specialized pro-
resolving mediators (SPMs), which can be divided into four projects corresponding to four classes of
DHA-derived SPMs, and the design and synthesis of synthetic lipoxin A
4
analogs.
Chapter 1 briefly explains the term “specialized pro-resolving mediator” (SPM) and reviews the
background of research on ω-3 polyunsaturated fatty acids, as well as the identification, biosynthesis,
total synthesis, anti-inflammatory/pro-resolving activities and actions of ω-3 polyunsaturated fatty acid-
derived SPMs. It also briefly reviews some related research on the development of pharmaceuticals based
on SPMs. In Chapter 2, the total synthesis of maresin 1 (MaR1), a most recently discovered DHA-derived
SPM, as well as a series of its stereoisomers, are described. The synthesis helped the structure elucidation
of MaR1 and the discovery of new bioactivities, as well as the structure-activity-relationships of MaR1
and its stereoisomers. In Chapter 3, a previously hypothesized but never isolated biosynthetic
intermediate was prepared by total synthesis. The 13S, 14S-epoxy-maresin has helped prove the proposed
biosynthesis of MaR1 and uncovered more unexplored biological processes this biosynthetic intermediate
was involved in. In Chapter 4, a series of deuterium-labeled monohydroxy-DHAs and deuterium-labeled
DHA were prepared by total synthesis. Work in this chapter not only produced a series of isotope-labeled
DHA derivatives to assist the exploration of DHA metabolic pathways, but also provided facile and
feasible routes to synthesize labeled DHA and its monohydroxy-E, Z-diene derivatives. In Chapter 5, a
newly elucidated stereoisomer of NPD1/PD1, namely the aspirin-trigger NPD1/PD1, was prepared by
total synthesis to help confirm its stereochemical assignments and explore its bioactivities. A series of
other stereoisomers as well as structrual analogs were prepared to study the structure-activity-
relationships of NPD1/PD1 and its isomers. In Chapter 6, a series of novel stable benzo-analogs of
lipoxin A
4
were prepared by total synthesis. These benzo-LXA
4
analogs are expected to either exhibit
higher in vivo stability or help to uncover an optimal structure for future analogs.
Chapter 1. Specialized Pro-resolving Mediators Derived from ω-3 Polyunsaturated
Fatty Acids: from Identification to New Pharmaceuticals

1.1. Introduction
Over decades it has been found that diet supplementation with ω-3 polyunsaturated fatty acids (PUFA),
such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have a beneficial effect on
inflammatory symptoms in diseases such as atherosclerosis, rheumatoid arthritis and inflammatory
bowel
1-4
. Fish oil supplementation, which is rich in DHA and EPA, has been reported to decrease ex vivo
production of pro-inflammatory cytokines tumor necrosis factor α (TNFα), interleukins IL1β and IL-6 in
humans and in many animal models
5-9
. Also, the anti-inflammatory and anti-angiogenic effect of DHA
and EPA have been reported
10
, and DHA, which is the major component of phospholipids in the outer
segments of photoreceptors, plays a key protective role for photoreceptor function and vision
11
. In
addition, DHA-supplemented infant formula enhances maturation of retinal function
12
, visual acuity
13-14
,
and mental performance
13-14
in preterm and term infants. It has been found that DHA acts as a competitive
substrate for the ω-6 PUFA metabolism that converts arachidonic acid into pro-inflammatory mediators
such as prostaglandins and leukotrienes
15
, and more detailed mechanisms by which ω-3 PUFA exert
beneficial effects in disease states are of wide interests and are being extensively investigated.
It has been postulated for a long time that inflammation related diseases are related to lipid mediators
16
,
which collectively include those derived from fatty acids, phospholipids, lysophospholipids and others,
among which the fatty acid derived lipid mediators include prostaglandins, thromboxane, leukotrienes,
lipoxins, epoxyeicosatrienoic acids and other fatty acid epoxides
15
. It has been known that the ω-6 PUFA
arachidonic acid (AA)-derived lipid mediators, such as prostaglandins and leukotrienes, possess pro-
inflammatory activities
17-18
and in contrast, recent results indicate that as inflammation proceeds, AA
begins to be converted into lipoxins, which are protective lipid mediators to actively terminate

2

inflammation and promote resolution
19-21
. In addition to AA-derived lipoxins, a series of novel lipid
mediators derived from ω-3 PUFAs, such as protectins and resolvins, were recently discovered and found
to have anti-inflammatory and pro-resolving properties
22-25, 57
. A key conceptual advance of Serhan and
colleagues is that resolution as well as propagation of inflammation can be an active process
25-26
, and
increases in lipoxins, protectins and resolvins as well as shifts in patterns of cytokines, chemokines and
other regulatory molecules can lead to active reduction in inflammation. These families of endogenous
pro-resolution molecules, termed as specialized pro-resolving mediators (SPM), stimulate and accelerate
resolution via multi-level mechanisms and they selectively stop neutrophil and eosinophil infiltration,
stimulate nonphlogistic recruitment of monocytes, activate macrophage phagocytosis of microorganisms
and apoptotic cells, increase lymphatic removal of phagocytes, and stimulate expression of antimicrobial
defense mechanisms
27, 24
(Figure 1).

Figure 1. Dual anti-inflammatory and pro-resolution actions of specialized pro-resolving mediators
26

3

The SPM protectins, resolvins and the most recently discovered maresins are derived from EPA and/or
DHA, and molecular structures of several thus far identified members are shown in Figure 2. Notably,
EPA-derived SPM include resolvin E1 (RvE1) and resolvin E2 (RvE2), and DHA-derived SPM include
resolvin D1 (RvD1), resolvin D2 (RvD2), protectin D1 (PD1), maresin 1 (MaR1) and others. This chapter
details the identification, biosynthetic pathways, bioactivities of several recently uncovered SPMs derived
from EPA and DHA, where related research methods are emphasized. The current work and outlook for
development of SPMs intopharmaceuticals are also mentioned.
COOH
OH
OH
COOH
OH
OH
COOH
HO
OH
HO
COOH
OH
OH
HO
RvE1
RvD1
COOH
OH
OH
RvE2
PD1
MaR1
COOH
OH
OH
OH
RvD2

Figure 2. Specialized pro-resolving mediators derived from DHA and EPA

1.2. Identification of DHA and EPA-derived SPMs
Recently Serhan and coworkers devised a new lipid mediator lipidomics and informatics approach to
profile the appearance and/or loss of mediators in resolving inflammatory exudates, and by using this
approach, a series of novel bioactive SPMs were discovered and identified after structural elucidation and
bioactivity confirmation
21, 24, 30-33
.

4

By using liquid chromatography-ultraviolet-tandem mass spectrometry (LC-UV-MS/MS), accurate and
prompt analysis of lipid mediators can be achieved. The foundation of the lipidomics approach lies in that
MS/MS spectra have been used extensively to identify and elucidate the structures of lipid mediators
derived from PUFAs
34-37
, and many lipid mediators derived from PUFAs possess conjugated double bond
systems that are critical components for their bioactions with characteristic UV spectra
34-36
. Based on this
method, known lipid mediators are identified by comparing the spectra and chromatographic behaviors
acquired from sample tissues with authentic standards. When an unknown lipid mediator is encountered,
basic chemical structures can be deduced on the basis of the relationship between structures, features of
their spectra and chromatographic behaviors combined with knowledge of established biosynthesis
32
. As
examples, the basic structure of resolvin D2 was deduced as 7S,16,17S-trihydroxy DHA, and protectin D1
was deduced as 10, 17S-docosatriene, based on the above mentioned lipidomics method

(Figure 3). The
established (S) or (R) configurations in basic structures are based on the established lipoxygenase
biosynthetic pathways
31
.

Figure 3. Basic structures of PD1 (A) and RvD2 (B) deduced based on LC-UV-MS/MS analysis
30

However, the basic structures obtained from the above lipidomics method still contained undecided
alcohol chirality and double bond geometry of the conjugated polyene system. As the lipid mediators are

5

commonly generated in only nanogram quantities, which limits the use of most characterization methods,
it was necessary to synthesize a series of stereo isomers likely to be biosynthesized prepared by
stereocontrolled total synthesis, and the complete stereochemical assignment can be achieved if a certain
isomer matches side-by-side all the spectra, chromatographic behaviors and bioactions of the endogenous
compounds
31, 33
. As an example, PD1, whose basic structure was deduced as 10, 17S-docosatriene, was
shown to have complete stereochemical assignment as 10R, 17S-dihydroxy-docosa-4Z, 7Z, 11E, 13E, 15Z,
19Z-hexaenoic acid by direct composition with synthetic isomers
31
(Figure 4). The stereoisomers prepared
via total synthesis in this matching approachusually have structures based on several possible established
biosynthetic pathways, typically the products with the involvement of epoxide-containing intermediates,
double dioxygenation intermediates, and non-enzymatic hydrolysis of epoxide-containing intermediates
31

(detailed in 1.3).

Figure 4. LC-MS/MS matching of PD1 (II in synthetic mixture) and biogenic PD 1
30

6

Novel anti-inflammatory and pro-resolving lipid mediators have been encountered in the study of
inflammatory exudates, and authentic standard compounds are needed following the same rationale to
achieve their structural elucidation. It has been detailed in Chapter 2, Chapter 4 and Chapter 5 the total
syntheses of those molecules prepared via stereocontrolled strategies.
1.3. Biosynthesis of DHA and EPA-derived SPMs
Several enzymatic conversions of PUFA are commonly observed and the biosynthetic pathways are
established, which form the basis to hypothesize the biosynthesis and to deduce the basic structure of
novel lipid mediators (as discussed in 1.2). These common biosynthetic pathways, as well as the
commonly used methods to prove their occurrence, are discussed in this section.
Oxidation catalyzed by lipoxygenases (LO) occurs to DHA, EPA and other PUFAs (shown in Scheme
1(A)). The three major lipoxygenases (5-LO, 12-LO, and 15-LO) can each convert DHA and EPA to S-
hydroperoxy containing products, which are subsequently enzymatically reduced to S-hydroxy containing
products, or converted by enzymes such as human PMN 5-lipoxgenase to an S, S-epoxide containing
intermediate
38-41, 52
.
The occurrence of lipoxygenation can be confirmed by the
18
O labeling experiment
31
, where the hydroxyl
group generated contains an
18
O which is from oxygen and can be detected by LC-MS/MS. As an
example,
18
O
2
labeling showed that 10S, 17S-diHDHA carried
18
O in the carbon-10 position alcohol and
this indicates that 10S, 17S-diHDHA was biosynthesized via sequential lipoxygenation
42-43
(shown in
Scheme 1(B)).
In the brain, 12-LO of pineal body converts DHA to 14S-HDHA and 15-LO to 17S-HDHA
44
and retina
converts DHA to both mono- and di-hydroxy products via LOs
45
. The resulting S-monohydroxy DHA or
EPA could function as key signaling molecules or biosynthetic intermediates for other lipid mediators.
For example, 17S-HDHA, a product of 15-LO, inhibits human neutrophil 5-LO production of
leukotrienes from endogenous substrate
46
or it is further converted to resolvin D2.

7

COOH
LO
18
O
2
(S)
OOH
5-/12-/15-LO
(S)
OH
(S)
(S)
O
enzymatic reduction
enzymatic epoxidation
O
2
PUFA
S-hydroperxoy-PUFA
S-hydroxy-PUFA
S, S-epoxy-PUFA
(A)
(B)
(S)
COOH
OH
(S)
OH
18
O labled 10, 17-diHDHA
COOH
(S)
HOO
O
2
5-LO
17S-H(p)DHA

Scheme 1. Oxidation of PUFA catalyzed by lipoxygenases
The S, S-epoxide-containing intermediate formed as in Scheme 1 (A) can undergo aqueous enzymatic
hydrolysis to produce R, S-triene in a highly stereospecific way (shown in Scheme 2 (A)), similar to that
established for related enzymes such as leukotriene A
4
hydrolase
47-48
, or to a single 16, 17S-vicinol
alcohol by the actions of an appropriate epoxide hydrolase in a reaction similar to that demonstrated
earlier in the biosynthesis of LXA
4
49-51
. Once produced, these dihydroxy-containing docosatrienes exert
their actions. The epoxide-containing intermediate could also be opened via nonenzymatic hydrolysis to a
diastereomeric mixture, namely, 16R/S, 17S-diHDHA or 10R/S, 17S-diHDHA. The occurrence of the
epoxide-containing intermediate can be confirmed by the methanol trapping experiment
31
. As an example,
the results of methanol trapping experiments provide evidence for a 16(17)-epoxide intermediate
generated from the 17S-hydroperoxy DHA precursor (shown in Scheme 2 (B)), which was subsequently
hydrolyzed to docosatrienes and 17S series resolvins
30
.

8

S,S-epoxy-PUFA
(S)
(S)
O hydrolase
H
2
O
(S)
OH
(R)
HO
R, S-dihydroxy-E, E, Z-PUFA
(B)
(A)
COOH
O
CH
3
OH
COOH
detected by LC-MS/MS
H
3
CO
OH

Scheme 2. Stereospecific ring-opening of S, S-epoxy-PUFA catalyzed by hydrolases (A) and alcohol
trapping experiment (B)
Another important enzymatic oxidation pathway of PUFA involves the use of aspirin in the presence of
cyclooxygenase-2 (COX-2). As the catalytic region of COX-2 is larger than that of COX-1, the acetylated
COX-2 remains active producing hydroperoxy products but with the oxygen insertion in the R
configuration rather than S as is the case with lipoxygenases
53-54
(Scheme 3). This mechanism accounts for
the biosynthesis of a series of aspirin-triggered lipid mediators, and a well-known example is the aspirin
triggered-15R-lipoxin A
4
, which is produced in vivo in humans
55-56
. An alternative uncovered route to
generate an R-hydroxyl group in DHA or EPA is via NADPH-dependent oxidation catalyzed by
cytochrome P450 monooxygenase
57-58
. The produced R-HDHA is also of great interest and as an example,
it was found that 17R-HDHA was further converted to 17R-hydroxy-containing resolvins
30
.

9

(R)
(R)
(R)
(R)
enzymatic reduction
enzymatic cyclization
aspirin/COX-2
or P450
OOH
OH
O
O
2
PUFA
R-hydroperxoy-PUFA
R-hydroxy-PUFA
R, R-epoxy-PUFA

Scheme 3. Oxidation of PUFA catalyzed by aspirin/COX-2 or P450
Based on these established biosynthetic routes, the biosynthesis of novel lipid mediators can be proposed.
As an example, the biosynthesis of aspirin-triggered RvD2 derived from DHA, is shown as in Scheme 4,
where all the above mentioned enzymatic conversions are involved
59
. The hypothesized biosyntheses of
several other SPMs were recently reviewed
59
.
COOH
O
2
COX-2/aspirin
or P450
COOH
(R)
HOO
COOH
(R)
HO
reduction
5-LO
O
2
COOH
(S)
(S)
(R)
HO
O
(S)
COOH
(R)
HO
HOO
5-LO hydrolase
(R)
(R)
(S)
COOH
OH
OH
AT-RvD2
OH
H
2
O

Scheme 4. Proposed biosynthesis of aspirin-triggered RvD2
As is detailed in Chapter 4 and Chapter 5 the stereocontrolled total synthesis of DHA-derived lipid
mediators is of great importance for elucidating these novel metabolic pathways. In Chapter 3, an S, S-

10

epoxide containing intermediate is prepared via total synthesis as a key intermediate in the proposed
pathway.
1.4. Anti-inflammatory / pro-resolving activities and mechanisms of DHA and EPA-derived SPMs
It is now well recognized that inflammation plays a key role in the pathogenesis of many diseases that
were not previously considered classic inflammatory diseases. As examples, (1) many cancers arise from
sites of infection, chronic irritation and inflammation, and tumour cells have co-opted some of the
signaling molecules of the innate immune system, such as selectins, chemokines and their receptors for
invasion, migration and metastasis
58
; (2) it has been found that inflammatory mechanisms couple
dyslipidaemia to atheroma formation, and inflammatory pathways promote thrombosis, a late and
dreaded complication of atherosclerosis responsible for myocardial infarctions and most strokes
59
; (3)
evidence now suggests that syndromes such as Alzheimer’s disease and stroke have important
inflammatory and immune components
60
; and many other examples on pathology-inflammation
relationship aside from (1), (2) and (3) have been reviewed
61-62
. Thus, these most challenging human
diseases may be amenable to treatment by anti-inflammatory and immunotherapeutic approaches.
Therefore, inflammation, which usually initiates from an acute phase, must be resolved to prevent the
inflammation from spreading, becoming chronic inflammation or other diseases. As mentioned in 1.1,
resolution of inflammation was previously considered to be passive
63
but now recognized as an active
biochemical and metabolic process, mediated by locally biosynthesized specialized dual-acting anti-
inflammatory and pro-resolution lipid mediators, such as lipoxins, resolvins and protectins.
Specialized proresolving mediators derived from ω-3 PUFAs, such as resolvins and protectins, have
potent multi-level mechanisms of action in disease models and promote resolution in animal models. As
examples, (1) resolvin E1 prevents periodontitis tissue destruction, and both soft tissue and bone that were
lost during disease were regenerated
64
; (2) protectin D1 promotes brain cell survival by reducing the
expression of pro-inflammatory genes and upregulating the expression of anti-apoptotic genes, and

11

reduces leukocyte infiltration and pro-inflammatory gene expression in brain ischemia-reperfusion
injury
65-67
; (3) protectin D1 protects retinal pigment epithelium from oxidative-stress-induced apoptosis
by limiting pro-inflammatory gene expression
65-67
; (4) when treated with resolvins before bilateral renal
ischemia, mouse kidneys were protected from injury, and creatinine serum levels were lower in treated
mice compared to control mice
68
. Aside from above examples, many other researches have been reported.
Some related progresses are also to be discussed in Chapter 2 and Chapter 5 in this thesis.
Specialized proresolving mediators derived from ω-3 PUFAs are believed to function in signaling
pathways by activating their specific receptors, similar as is established for lipoxin A
4
, which is derived
from the ω-6 PUFA arachidonic acid and activates the lipoxin A
4
receptor (ALX) which is a G-protein-
coupled receptor
69-70
. Two GPCRs have been identified to be involved in the actions of resolvin E1, and
they are CMKLR1 (also known as ChemR23, on mononuclear and dendritic cells) which attenuates
TNFα-stimulated NF-κB activation in response to resolvin E1 binding
31
, and BLT1 (leukotriene B
4

receptor, on neutrophils) which attenuates leukotriene B
4
-dependent pro-inflammatory signals when
resolvin E1 binds with it as an antagonist
71
. While more targets for the other SPM derived from ω-3
PUFAs need to be identified, signaling functions of ω-3 PUFA derived SPMs were reported, such as (1)
resolvins block TNF-induced transcripts for pro-inflammatory cytokine interleukin-1β, which is
expressed rapidly in response to neuronal injury
28, 72
; (2) protectin D1 blocks T-cell migration in vivo,
reduces TNF and interferon-γ secretion and promotes T-cell apoptosis
73
; (3) Resolvin E1 and protectin D1
upregulate the expression of CCR5on dying neutrophils, which acts as a “terminator” of chemokine
signaling
74
. Generally, pro-resolution signals mediated by resolvins and protectins reduce neutrophil
influx and stimulating macrophage ingestion of apoptotic neutrophils, and enhance the number of
phagocytes present in lymph nodes and spleen
75
.
In Chapter 2, Chapter 3 and Chapter 5, more anti-inflammtory and proresolving activities of DHA- and
EPA-derived SPMs are discussed.

12

1.5. Development of DHA and EPA-derived SPMs to pharmaceuticals
Since many current and widely used non-steroidal anti-inflammatory drugs (NSAIDs), such as selective
COX2 inhibitors and certain lipoxygenase inhibitors, have proven to be toxic to the tissue programs of
resolution by delaying the return to homeostasis
27, 75-76
, the PUFA-derived SPMs help open new avenues
for treatment of a series of inflammation-related diseases. However, formulations and routes of delivery
of PUFA-derived SPMs confront considerable challenges. To be specific, lipid mediators are generally
susceptible to various enzymatic degradation mechanisms such as β- and ω-oxidation, and oxidations
mediated by 15-hydroxyprostaglandin dehydrogenase
54, 77, 78
or lipoxygenases (see 1.3), which typically
clear them from the body in minutes after they are intravenously injected. Synthetic analogs of PUFA-
derived SPMs have been synthesized and evaluated, and several most studied analogs of lipoxin A
4
, the
SPM derived from arachidonic acid, are shown in Figure 5. Degradation-resistant modifications, such as
replacement of the ω-oxidation-sensitive ω-6 tail with a fluorophenoxy group, replacement of the light
and acid sensitive tetraene unit with a trienyne group, effectively decrease enzymatic inactivation of
lipoxin A
4
79, 80
. Apart from developing enzymatically stable analogs, it is also important to uncover
structure-activity relationships in PUFA-derived SPMs, which may provide information for structures
which are more synthetically accessible. Our continuous studies on developing more stable analogs are
detailed in Chapter 2, Chapter 5 and Chapter 6.
O
OH
HO OH
HO
O
O
OH
HO OH
HO
F
O O
O
ONa
HO OH
HO
F
O
O
ONa
HO OH
O
OH
F
ZK-192
ZK-142
ATLa
LXA
4
Figure 5. Development of more stable lipoxin A
4
analogs

13

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21

Chapter 2. Total Synthesis of Maresin 1 (MaR1) and its Stereoisomers

2.1. Introduction
Maresin 1 (MaR1) is a recently discovered specialized pro-resolving lipid mediator, which is
biosynthesized by macrophages from docosahexaenoic acid. By analyzing self-resolving inflammatory
exudates, it was found that both DHA and 14S-hydroperoxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic
acid (the 14-lipoxygenated DHA product ) can be converted by macrophages to MaR1, which has the
basic structure of 7,14S-dihydroxydocosa-4Z,8,10,12,16Z,19Z-hexaenoic acid. Preliminary bioactivity
tests showed that MaR1 possessed potent anti-inflammatory and pro-resolving activity with a potency
similar to RvE1 and NPD1/PD1.
1
However, the stereochemical configurations of the two hydroxyl-
attached stereogenic centers, and that of the 8, 10, 12-triene still remained unclear. It was also desirable to
investigate other activities related to inflammation pro-resolution of MaR1, such as wound healing and
pain control. For both purposes, a scalable total synthesis of MaR1 has to be performed and up to
milligram scale of a series of MaR1 isomers has to be prepared in enatiomerically pure form. Related
synthesis and bioactivity exploration of MaR1 and its stereoisomers have been achieved and are discussed
in this chapter.
2.2. Results
2.2.1. Retrosynthetic analysis of MaR1 stereoisomers
Multiple possible combinations of chirality at C
7
and C
14
, as well as double bond configuration at C
8
, C
10

and C
12
, require a highly convergent synthesis strategy in which late-stage synthesis produces isomers
with varied stereochemistry in a timely efficient manner. Based on the above considerations, a
retrosynthetic analysis of MaR1 stereoisomers was designed featuring a highly convergent and stereo-
controlled strategy (shown in Figure 6). MaR1 isomers with varied stereochemistry (7R, 8E, 10E, 12Z,
14S) (1), (7S, 8E, 10E, 12Z, 14S) (2), (7S, 8E, 10E, 12Z, 14R) (3), (7R, 8E, 10Z, 12E, 14S) (4), (7S, 8E,

22

10Z, 12E, 14S) (5), (7R, 8E, 10E, 12E, 14S) (6) have been synthesized via the designed route and more
possible combinations can be achieved similarly (shown in Figure 7).
To control the accurate E/Z configurations in MaR1 stereoisomers, reactions featuring high E/Z product
selectivity were employed. The 4Z, 16Z and 19Z double bonds were produced from the hydrogenation of
triple bonds catalyzed by palladium poisoned with lead (Lindlar catalyst)
2
and the triple bonds are all
from commercially available alkynes. For isomers 1-6, the two trans double bonds in the 8, 10, 12-triene
moiety were generated from Takai olefination and Wittig homologation, and both reactions give
predominant E-products
3, 4
. For isomers 1-3, the 12Z is the last generated double bond and the 10Z is for 3
and 4, all of which were produced from a mild hydrogenation protocol which avoids over-hydrogenation
of alkenes.
5, 6
For isomer 6, the 12E is the last generated double bond from Negishi coupling which gives
high E-selectivity.
7
The accurate stereo-control at C
7
and C
14
was achieved starting from enantiomerically
pure compounds which are readily available and the chirality is retained in the final synthetic compounds
1-6, as no reactions which can potentially cause chirality inversion or loss is involved.
A series of C-C bond formation reactions were used to construct the backbone of isomers 1-6, including
epoxide ring-opening by alkynyl lithium in the presence of a Lewis acid
8
, cross-coupling between
terminal alkyne and propargyl halide
9
in the presence of Cu(I), Takai olefination, Wittig homologation,
Corey-Fuchs reaction
10
and palladium-catalyzed Sonogarshira
7
and Negishi cross-coupling reactions.
With aldehyde 14-19 prepared (Scheme 7), different combinations of reactions mentioned above were
carried out to quickly prepare the MaR1 stereoisomers.

23

O OR
2
R
1
O
O
R
1
= Si
t
BuMe
2

R
2
= Si
t
BuPh
2
OR
2
Br
OR
2
OR
1
TMS
COOMe
O
O
O
COOMe
OH
OH
COOMe
OH
OH
COOMe
OH
OH
COOMe
OH
OH
COOMe
OH
OH
8E, 10E, 12Z 8E, 10Z, 12E
8E, 10E, 12E
O
R
1
O
O

Figure 6. Retrosynthetic analysis of MaR1 stereoisomers

24

(S)
(S)
COOMe
OH
OH
(S)
(R)
COOMe
OH
OH
COOMe
(R)
(S)
OH
COOMe
(S)
(S)
OH
OH
OH
(S)
OH
(R)
OH
COOMe
1
2
4 5
6
(R)
(S)
COOMe
OH
3
OH

Figure 7. Stereoisomers of MaR1 prepared by total synthesis
2.2.2. Synthesis of building blocks of MaR1 stereoisomers
The synthesis originates from protection of (S)- or (R)-glycidol (7 or 8) as silyl ethers
11
(9 or 10), and
protection of 4-pentynoic acid as an ortho ester
12
(11) (Scheme 5 (A)). In the presence of the Lewis acid
boron trifluoride etherate, TBS-protected glycidols are opened by alkynyl lithium, which was generated in
situ by treating 11 with n-butyl lithium. The ortho-ester protecting group was subsequently removed by
an acid cleavage in the presence of HCl and a following trans-esterification in the presence of
triethylamine. After protection of the secondary alcohol to form the di-protected dio (12 or 13), the
primary hydroxyl group was selectively deprotected
13
. Hydrogenation with Lindlar catalyst afforded the
Z-olefin and a subsequent Swern oxidation
14
yielded the aldehyde 14 and 15 for later conversions
(Scheme 5 (B)). Similarly, the TBS-protected glycidols were opened by lithium TMS-acetylene, and after
protecting the secondary hydroxyl group as a bulky tert-butyldiphenylsilyl ether, the trimethylsilyl group
was cleaved in the presence of sodium carbonate in methanol. The unmasked alkyne was coupled to the
propargyl bromide in a key coupling reaction mediated by Cu (I) and sodium iodide to form the 1,4-diyne
(16 or 17). The primary alcohol was selectively deprotected and the two triple bonds were reduced to the
two double bonds simultaneously. The alcohol was then oxidized to aldehyde 18 and 19 for subsequent
reactions (Scheme 5 (C)).

25

4) TBDPS-Cl, Imidazole, DMAP, rt,
CH
2
Cl
2
,97%
2) HCl, H
2
O/THF, rt.,75%
3) MeOH, Et
3
N, 79%
1) CSA, MeOH/CH
2
Cl
2
, rt.,86%
2) H
2
/Lindlar catalyst, quinoline, rt,
EtOAc, 95%
3) DMSO, (COCl)
2
, Et
3
N, -78
o
C,
CH
2
Cl
2
,98%
14: (7R)
15: (7S)
TMS
,n-BuLi, BF
3
.
Et
2
O,
2) TBDPS-Cl, Imidazole, DMAP, rt.,89%
3) MeOH, Na
2
CO
3
,rt.,98%
4)
Br
,CuI, NaI, K
2
CO
3
, 88%
1) CSA, rt.,88%
OTBS
2) H
2
/Lindlar catalyst, quinoline, rt,
EtOAc, 85%
3) DMSO, (COCl)
2
, Et
3
N, -78
o
C,
CH
2
Cl
2
,95%
COOMe
OTBDPS
O
TBSO
OTBDPS
O
OTBDPS
O
OTBS
O
O
O
TBDPSO
COOMe
1)
-78
o
C, THF,84%
11, n-BuLi, BF
3
.
Et
2
O, -78
o
C, THF,85% 1)
O
OTBS
18: (7R)
19: (7S)
2) , DCC, CH
2
Cl
2
, 98%
2) BF
3
.
Et
2
O, rt, CH
2
Cl
2
,85%
COOH
O
OTBS
9: (2R)
10: (2S)
O
OH
7: (S)-glycidol
8: (R)-glycidol
11
9: (2R)
10: (2S)
9: (2R)
10: (2S)
1) TBS-Cl, Imidazole,
DMAP,rt, CH
2
Cl
2
,97%
12: (7R)
13: (7S)
16: (7R)
17: (7S)
(A)
(B)
(C)
O
HO

Scheme 5. Synthesis of aldehyde 14-19 for total synthesis of MaR1 stereoisomers

Aldehydes 14-15 were converted to the corresponding vinyl iodides 20-23, while aldehydes 18-19 were
converted to the corresponding terminal alkyenes 24-27, which are direct building blocks for the final
coupling step. Aldehyde 14 and 15 were either converted to E, E- dienyl iodide 22 or 23 via a Wittig
homologation followed by a Takai olefination (Scheme 6 (A)). Aldehyde 18 or 19 were either converted
to ene-yne 26 and 27 via a Wittig homologation and a two-step Corey-Fuchs reaction (Scheme 6 (B)).

26

COOMe
OTBDPS
I
OTBDPS
20: (7R)
21: (7S)
COOMe
OTBDPS
I
OTBDPS
22: (7R)
23: (7S)
CHI
3
, CrCl
2
, 0
o
C, THF, 56%
2) CHI
3
, CrCl
2
, 0
o
C, THF, 58%
, toluene, 90
o
C, 2h, 80%
PPh
3
O
1)
, toluene, 90
o
C, 2h, 78%
PPh
3
O
1)
2) CBr
4
, PPh
3
, 0
o
C, 1h, 89%
3) LDA, THF, -78
o
C, 80%
1) CBr
4
, PPh
3
, 0
o
C, 1h, 88%
2) LDA, THF, -78
o
C, 83%
14: (7R)
15: (7S)
COOMe
OTBDPS
O
OTBDPS
O
18: (7R)
19: (7S)
26: (7R)
27: (7S)
24: (7R)
25: (7S)
(A)
(B)

Scheme 6. Synthesis of building blocks 20-27 for total synthesis of MaR1 stereoisomers

2.2.3. Assembly of MaR1 stereoisomers and spectroscopic studies
With the building blocks 20-27 prepared, stereo isomers of MaR1 were assembled in a timely-efficient
way. Variations at C
7
and C
14
chirality were achieved using building blocks with the corresponding
chirality, and variations of triene E/Z arrangment at C
8
-C
12
were achieved by carrying out corresponding
coupling reactions. To be specific, isomers 1, 2 and 3 were prepared from the hydrogenation of the
corresponding 12-yne (28-30), which were produced from the coupling of the dienyl iodide (22 or 23) and
the terminal alkyne (24 or 25) (Scheme 7 (A)). Isomers 4 and 5 were synthesized from the hydrogenation
of the 10-yne precursors (31 or 32), which was prepared from the coupling of the vinyl iodide (20 or 21)
and ene-yne 27 (Scheme 7 (B)). In contrast, isomer 6 was directly generated from the coupling of dienyl
iodide 23 and the terminal alkyne 25 followed by silylether protecting group removal, without the need of
a hydrogenation step (Scheme 7 (C)).

27

COOMe
OTBDPS
I
COOMe
COOMe
OH
(S)
OH
(R)
OH
COOMe
OH
1: (7R,8E,10E,12Z,14S)
2: (7S,8E,10E,12Z,14S)
3: (7S,8E,10E,12Z,14R)
COOMe
OTBDPS
I
OH
4: (7R,8E,10Z,12E,14S)
5: (7S,8E,10Z,12E,14S)
COOMe
(R)
I
22: (7R)
6: (7R, 8E,10E,12E,14S)
OTBDPS
1) 24 or 25 Pd(PPh
3
)
4,
CuI, rt, C
6
H
6.
2) TBAF, THF, rt.
1) 27, Pd(PPh
3
)
4,
CuI, rt, C
6
H
6.
2) TBAF, THF, rt.
Zn(Cu/Ag), rt, MeOH/H
2
O
Zn(Cu/Ag), rt, MeOH/H
2
O
1) 25, Cp
2
ZrHCl, ZnCl
2
,Pd(PPh
3
)
4
,rt.
2) TBAF, THF, rt.
(A)
(B)
(C)
COOMe
OH
COOMe
OH
OH
22: (7R)
23: (7S)
20: (7R)
21: (7S)
28: (7R,8E,10E,14S)
29: (7S,8E,10E,14S)
30: (7S,8E,10E,14R)
31: (7R,8E,12E,14S)
32: (7S,8E,12E,14S)
OH OH

Scheme 7. Assembly of MaR1 stereoisomer 1-6

The proton NMR spectra are effective to prove the stereochemistry of the triene. For example, in the
olefinic region of isomer 1, five of the six triene protons were resolved and the coupling constants
unambiguously proved the 8E, 10E, 12Z geometry (Figure 8). The
1
H NMR olefinic regions of MaR1
isomer 2, 4 and 6 were also compared and their difference is significant (Figure 9).

28

H11
H10 H9 H12 H8
H4,5,13,16,17,19,20

Figure 8.
1
H NMR of olefinic region of MaR1 methyl ester (1)

Figure 9.
1
H NMR comparison of olefinic region of MaR1 stereoisomers

29

2.2.4. Structure elucidation of MaR1 and bioactivity exploration of MaR1 stereoisomers
The LC-MS/MS profiles and potencies to stop PMN infiltration of MaR1 stereoisomers prepared by
above synthesis were compared with endogenous MaR1 in inflammation exudates, and the (7R, 8E, 10E,
12Z, 14S) (isomer 1) was confirmed to match the endogenous compound (shown in Figure 10). Hence the
structural elucidation of MaR1 was successfully achieved
15
.
With MaR1 synthesized in milligram scale, our collaborators were able to explore more biological
profiles of this signaling molecule. Results show that MaR1 is highly potent to promote tissue
regeneration in a planaria model and to control pain in mice
15
. MaR1 and its synthetic isomers are also
studied for their wound healing potency in a human dermal fibroblasts model and MaR1 itself shows the
highest potency
16
.

Figure 10. LC-MS/MS profiles of MaR1 and stereoisomers
15

30

2.3. Conclusion
The total synthesis of MaR1, the first discovered member of the maresins, and a series of its
stereoisomers has been achieved successfully. Based on the work of synthesis which produced
enantiomerically pure authentic compounds, the complete stereochemistry assignment has been achieved.
MaR1 was showed to be a potent anti-inflammatory and pro-resolving SPM which may help the
development of theraputics for the treatment of inflammation, wound healing, pain and more.
2.4. Experimental Procedures
All reactions, unless otherwise noted, were carried in flame dried flasks under argon atmosphere. “Dried
and concentrated” refers to removal of residual water with anhydrous MgSO
4
, followed by evaporation of
the solvent on the rotary evaporator. THF was freshly distilled from sodium-benzophenone, benzene and
dichloromethane from CaH
2
and anhydrous DMF, EtOH, and MeOH were purchased from commercial
sources.
1
H and
13
C NMR spectra were recorded on a Varian Mercury 400 or 600 MHz using residual
1
H
or
13
C signals of deuterated solvents as internal standards. UV spectra were recorded on a Hewlett-
Packard 8350 instrument. HPLC analyses were performed on a Rainin dual pump HPLC system
equipped with a Phenomenex ODS column and an UV-VIS detector.
O
O
O
11

1-(but-3-ynyl)-4-methyl-2,6,7-trioxabicyclo[2.2.2]octane (11): To a solution of DCC (1.0 g, 4.40 mmol)
in dry CH
2
Cl
2
(15 mL) was added the mixture of pent-4-ynoic acid (0.45g, 4.58mmol), 3-hydroxymethyl-
3-methyloxetane (1.0 g, 10.09 mmol) and DMAP (30 mg, 0.02 mmol) in dry CH
2
Cl
2
(5 mL) at 0
°
C. The
solution was then stirred at room temperature under argon for overnight. The precipitated was filtered off,
and washed with CH
2
Cl
2
. The solvent was removed and the crude product was purified on a silica
column using 30% EtOAc/hexanes as mobile phase to give pure (3-methyloxetan-3-yl)methyl pent-4-

31

ynoate (0.8 g, 98%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
4.43 (d, J = 6.0 Hz, 2H), 4.30 (d, J = 6.0 Hz, 2H),
4.12 (s, 2H), 2.53 (m, 2H), 2.44 (m, 2H), 1.93 (t, J = 2.8 Hz, 1H), 1.26 (s, 3H);
13
C-NMR (400 MHz,
CDCl
3
) δ
C
171.6, 82.1, 79.3, 79.3, 69.0, 68.7, 38.8, 33.1, 21.0, 14.2. To a stirred solution of half-ester 8
(0.80 g, 4.39 mmol) in dry CH
2
Cl
2
was added BF
3
·OEt
2
(0.14 mL, 1.09 mmol) drop-wise at room
temperature. The reaction mixture was then stirred at room temperature for 1 h. After an hour Et
3
N (1.0
mL) was added. After stirring for 10 min, diethyl ether (20 mL) was added to dilute the solution. The
precipitate was filtered off and the solvent was removed using a rotatory evaporator. The product was
purified by column chromatography on a silica gel pre-treated with 1% Et
3
N in hexanes using 30%
EtOAc/hexanes as the solvent system to give 9 (0.57 g, 72%).
1
H NMR (400 MHz, CDCl
3
) δ
H
3.84 (s,
6H), 2.28 (m, 2H), 1.88 (m, 2H), 0.76 (s, 3H);
13
C NMR (400 MHz, CDCl
3
) δ
C
108.0, 84.0, 72.0, 72.0,
72.0, 67.8, 35.7, 30.2, 14.4, 12.8.
O
OTBDMS
9
O
OTBDMS
10

TBDMS-protected S-glycidol (9) and TBDMS-protected R-glycidol (10). To a mixture of imidazole
(5.5 g, 81.0 mmol), TBDMS-Cl (12.2 g, 81.0 mmol), and DMAP (0.4 g, 3.3 mmol) in dry CH
2
Cl
2
(100
mL) at 0
°
C was added corresponding glycidol (5.0 g, 67.5 mmol). The reaction mixture was warmed to
room temperature, and stirred for overnight. The reaction mixture was quenched with a saturated aqueous
solution of NH
4
Cl, extracted with ether, washed with brine, dried over MgSO
4
, and concentrated under
reduced pressure to give a crude product. The crude product was purified on silica column using 3%
EtOAc/hexane as the eluent to give the TBDMS-protected glycidol 9 or 10 (12.5 g, 98%) as a colorless
oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
3.84 (1H, dd, J = 11.5, 3.1 Hz), 3.65 (1H, dd, J = 11.5, 4.8 Hz), 3.06
(1H, m), 2.76 (1H, dd, J = 5.3, 4.5 Hz), 2.62 (1H, dd, J = 5.3, 2.6 Hz).
13
C-NMR (400 MHz, CDCl
3
) δ
C

64.1, 52.0, 44.2, 25.9, 18.0, -5.0.

32

OTBDPS
TBDMSO
O
OMe
12

Methyl (7R)-8-(t-butyldimethylsilyloxy)-7-(t-butyldiphenylsilyloxy)oct-4-ynoate (12). To a solution
of 9 (0.45 g, 2.483 mmol) in dry THF (10 mL) at -78
o
C was slowly added n-BuLi (1.25 mL, 1.6 M
solution in hexane, 2.00 mmol). The mixture was stirred for 15 min, and then added BF
3
·OEt
2
(0.25 mL,
2.00 mmol) solution at -78
o
C and stirred for additional 15 min. A solution of TBS-protected S-glycidol
10a (0.36 g, 1.91 mmol) in THF (3.0 mL) was slowly added to the reaction mixture at -78
o
C by using a
cannula. The reaction mixture was stirred for 3 h at -78
o
C and then brought to room temperature and
stirred for an additional 40 min. The reaction was quenched with a saturated solution of NH
4
Cl, extracted
with ether, washed with brine. The organic layers were combined, dried over MgSO
4
, and concentrated
under reduced pressure. The crude product was then dissolved in THF-H
2
O (1:1) (3.0 mL), and then was
added 1M HCl (0.4 mL, 0.4 mmol) at 0
°
C and stirred for 1 h. The reaction was quenched with saturated
aqueous solution of NaHCO
3
, extracted with ether, dried over MgSO
4
, and then concentrated in vacuo to
give the crude triol. The crude product was purified on a silica column using 50% EtOAc/hexanes as the
solvent system to give the pure triol 7R-8-(t-butyldimethylsilyloxy)-3'-hydroxy-2'-(hydroxymethyl)-2'-
methylpropyl-7-hydroxy-oct-4-ynoate (0.60 g, 81% in two steps).
1
H –NMR (400 MHz, CDCl
3
) δ
H
4.20
(d, J = 6.4 Hz, 2H), 3.61 (m, 2H), 3.55 (m, 4H), 3.38 (m, 2H), 3.72 (m, 1H), 2.95 (t, J = 4.0 Hz, 1H), 2.75
(t, J = 4.0 Hz, 1H, -OH), 2.70 (d, J = 4.0 Hz, 1H, -OH), 2.58 – 2.48 (m, 4H), 1.60 (m, 2H), 0.87 (s, 9H),
0.82 (s, 3H), 0.04 (s, 6H). The triol (0.30g, 0.77mmol) was dissolved in 20ml anhydrous MeOH and
triethylamine was added (0.02ml, 0.13mmol). The mixture was stirred overnight at room temperature.
Remove the solvent in vacuo and purify the mixture on a silica column using 12% EtOAc/hexanes to give
the pure product methyl (7R)-8-(t-butyldimethylsilyloxy)-7-hydroxyoct-4-ynoate (0.18 g, 79%).
1
H-
NMR (400 MHz, CDCl
3
) δ
H
3.68 (m, 1H), 3.62 (s, 3H), 3.60 (dd, J = 10.0, 3.6 Hz, 1H), 3.53 (dd, J = 10.0,
6.4 Hz, 1H), 2.57 (d, J = 4.8 Hz, 1H), 2.30 (d, J = 4.8 Hz, 1H), 2.43 (m, 4H), 0.82 (s, 9H), 0.01 (s, 6H);

33

13
C-NMR (400 MHz, CDCl
3
) δ
C
172.7, 104.1, 80.3, 70.3, 65.5, 51.7, 33.5, 25.7, 25.6, 23.3, 14.6, -5.5. To
a solution of TBDPS-Cl (0.13 mL, 0.50 mmol), imidazole (34 mg, 0.50 mmol) and DMAP (2 mg, 0.01
mmol) in dry CH
2
Cl
2
(10 mL) at 0
°
C was added the secondary alcohol (0.1 g, 0.34 mmol) in CH
2
Cl
2
(2.0
mL) through a cannula. The mixture was warmed to room temperature and stirred for overnight. The
reaction was quenched with a saturated aqueous solution of NH
4
Cl, extracted with ether, washed with
brine, dried over MgSO
4
, and concentrated under reduced pressure. The crude was purified on a silica gel
column using 2% EtOAc/hexane as the eluent to give pure 12 (0.17 g, 93%) as a colorless oil.
1
H-NMR
(400 MHz, CDCl
3
) δ
H
7.73 (m, 4H), 7.39 (m, 6H), 3.83 (m, 1H), 3.67 (s, 3H), 3.55 (m, 2H), 2.46-2.30 (m,
6H), 1.07 (s, 9H), 0.86 (s, 9H), -0.02 (s, 3H), -0.05 (s, 3H);
13
C-NMR (400 MHz, CDCl
3
) δ
C
172.5, 135.7,
135.8, 135.5, 134.7, 134.1, 134.0, 129.6, 127.6, 127.4, 79.5, 77.8, 72.3, 65.6, 51.7, 33.6, 26.8, 25.8, 23.8,
19.3, 18.2, 14.7, -5.6.
O
OTBDPS
COOMe
14

Methyl (7R, 4Z)-7-(t-butyldiphenylsilyloxy)-8-oxooct-4-enoate (14). To a solution of protected diol 13
(0.25 g, 0.46 mmol) in a 1:1 mixture of CH
2
Cl
2
/MeOH (15 mL) was added camphorsulfonic acid (86.3
mg, 0.37 mmol) at room temperature. The progress of the reaction was monitored by TLC. The reaction
was over by an hour, it was then quenched with Et
3
N (0.30 mL, 2.30 mmol). The solvent was evaporated
to dryness to give a crude mixture, which was then purified on a silica gel column using 15%
EtOAc/hexane to afford the primary alcohol (0.18 g, 90%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.67 (m, 4H),
7.39 (m, 6H), 3.87 (m, 1H), 3.64 (s, 3H), 3.61 (m, 2H), 2.41-2.23 (m, 6H), 1.06 (s, 9H);
13
C-NMR (400
MHz, CDCl
3
) δ
C
172.7, 135.8, 135.6, 135.5, 133.4, 129.8, 127.7, 127.6, 80.3, 77.3, 72.3, 65.4, 51.6, 33.5,
26.9, 26.8, 23.7, 19.2, 14.6. To a solution of the primary alcohol (0.24 g, 0.56 mmol) in EtOAc (50 mL)
was added 20 mg of Lindlar catalyst and 2 drops quinoline. The reaction mixture was stirred at room
temperature under the static atmosphere of hydrogen gas, and its progress was monitored by TLC. The

34

reaction was over by 2 h at room temperature. The reaction mixture was then filtered through celite and
solvent was evaporated under reduced pressure. The crude product was purified on a silica column using
13% EtOAc/hexane as the eluent to give the pure 4Z alkene (0.235 g, 96%) as colorless oil.
1
H-NMR
(400 MHz, CDCl
3
) δ
H
7.68 (m, 4H), 7.37 (m, 6H), 5.32 (m, 2H), 3.80 (quintet, J = 4.8 Hz, 1H), 3.63 (s,
3H), 3.49 (m, 2H), 2.34-2.18 (m, 6H), 1.07 (s, 9H);
13
C-NMR (400 MHz, CDCl
3
) δ
C
173.5, 135.8, 135.6,
133.7, 133.6, 129.8, 129.7, 127.6, 127.5, 126.0, 73.3, 65.2, 51.4, 33.6, 31.3, 26.9, 22.5, 19.2. To a solution
of DMSO (0.13 mL, 1.65 mmol) in dry CH
2
Cl
2
(10 mL) at -78
o
C was slowly added oxalyl chloride (0.10
mL, 1.10 mmol) and stirred for 15 min, and then added the 4Z-alcohol (0.23 g, 0.55 mmol) in CH
2
Cl
2
(3
mL) through a cannula, and stirred for 50 min. Et
3
N (0.38 mL, 2.75 mmol) was then added to the
reaction mixture, and stirred for 3 h at -78
°
C. The reaction mixture was quenched with a saturated
aqueous solution of NH
4
Cl, extracted with ether, washed with brine, dried over anhydrous MgSO
4
, and
concentrated under reduced pressure. The crude product was purified on a silica column using 10%
EtOAc/hexane as the eluent to give the desired aldehyde 14 (0.22 g, 95%) as a colorless oil.
1
H-NMR
(400 MHz, CDCl
3
) δ
H
9.53 (d, J = 1.6 Hz, 1H), 7.62 (m, 4H), 7.37 (m, 6H), 5.40 (m, 2H), 4.04 (td, J = 6.4,
1.6 Hz, 1H), 3.62 (s, 3H), 2.46-2.20 (m, 6H), 1.09 (s, 9H);
13
C-NMR (400 MHz, CDCl
3
) δ
C
203.3, 173.3,
135.7, 132.9, 132.8, 130.7, 130.0, 127.8, 124.4, 77.5, 51.5, 33.6, 30.9, 26.9, 22.7, 19.3.
OTBDPS
COOMe I
22

(7R,4Z,8E,10E)-methyl-7-(tert-butyldiphenylsilyloxy)-11-iodoundeca-4,8,10-trienoate (22). To 0.28g
(0.66mmol) aldehyde 14 and 240mg (0.73mmol) triphenyl phosphanylidene acetaldehyde, 5 ml toluene
was added. The mixture was stirred in 90
°
C oil bath for 2hrs. Monitor the reaction by TLC. Added more
50% more of the Wittig reagent, and heated the reaction at 90
o
C for 1.5hr. Evaporate the solvent, and the
crude product was purified on a silica column using 8% EtOAc/hexane as the eluent to give the product
(0.25 g, 86%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
9.47 (d, J = 8.2Hz, 1H), 7.65 (m, 4H),
7.38 (m, 6H), 6.73 (dd, J =15.5Hz and 4.8Hz, 1H), 6.63 (ddd, J = 15.5Hz, 8.2Hz and 1.4Hz), 5.32 (m,

35

2H), 4.51 (m, 1H), 3.65 (s, 3H), 2.33 (m, 6H), 1.10 (s, 9H).
13
C-NMR (400 MHz, CDCl
3
) δ
C
193.3, 173.2,
158.5, 135.7, 135.6, 133.3, 132.9, 130.9, 130.7, 129.8, 128.6, 123.5, 72.0, 51.4, 33.7, 26.8, 25.4, 22.6,
19.1. At 0
°
C, to 350mg (2.84mmol) chromium chloride suspended in 3.0 ml THF, 140mg (0.31mmol)
aldehyde and 560mg (1.42mmol) iodoform in 10ml THF was added and stirred for 3hrs at 0
o
C. Workup
with ether and brine. Ran the column (silica, 3% ethyl acetate in hexanes) and 22 was obtained (100mg,
56%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.68 (m, 4H), 7.39 (m, 6H), 6.90 (dd, J = 8.0Hz and 14.0Hz, 1H),
6.18 (d, J = 14.4Hz, 1H), 5.87 (dd, J = 10.8 Hz and 15.2 Hz, 1H), 5.65 (dd, J = 6.4Hz and 15.2Hz, 1H),
5.34 (m, 2H), 4.12 (m, 1H), 3.65 (s, 3H), 2.24-2.16 (m, 6H), 1.09 (s, 9H).
13
C-NMR (400 MHz, CDCl
3
)
δ
C
144.8, 136.7, 136.1, 134.2, 133.9, 129.9, 127.8, 126.1, 78.9, 73.3, 64.6, 53.7, 51.8, 35.8, 34.1, 31.9,
30.9, 27.3, 23.1, 22.9, 21.4, 19.6, 14.5.
OTBDMS
OTBDPS
17

(2S)-2,2,8,8,9,9-hexamethyl-5-(octa-2,5-diynyl)-3,3-diphenyl-4,7-dioxa-3,8-disiladecane (17). To a
solution of TMS-acetylene (11.30 mL, 80.0 mmol) in dry THF (90 mL) at -78
°
C was added n-BuLi (50
mL of 1.6 M solution in hexane, 80.0 mmol) dropwise. The mixture was stirred for 15 min at -78
°
C. A
solution of BF
3
·OEt
2
complex (10.13 mL, 80.0 mmol) was then added at the same temperature and stirred
for an additional 15 min. A solution of protected R-glycidol 10 (10.0 g, 53.0 mmol) in dry THF (10 mL)
was added to the reaction mixture at -78
°
C by a cannula. The reaction mixture was stirred at -78
°
C for 2
hrs, and warmed to room temperature by removing the dry-ice bath, and stirred for additional 30 min.
The reaction was then quenched with a saturated aqueous solution of NH
4
Cl, extracted with ether, washed
with brine, dried over MgSO
4
. The organic extracts were combined and concentrated in vacuo. The
crude product was then purified on a silica column using 10% EtOAc/hexanes as the solvent system to
afford pure product (14.2 g, 95%) as a colorless oil.
1
H-NMR (400 MHz, CDCl
3
) δ
H
3.78 (m, 1H), 3.71
(dd, J = 10.4, 4.4 Hz, 1H), 3.62 (dd, J = 9.6, 5.2 Hz, 1H), 2.53 (d, J = 5.2 Hz, 1H, -OH), 2.45 (t, J = 6.0

36

Hz, 2H), 0.90 (s, 9H), 0.14 (s, 9H), 0.08 (s, 6H);
13
C-NMR (400 MHz, CDCl
3
) δ
C
102.8, 86.9, 70.1, 65.4,
25.8, 24.5, 18.3, -0.03, -5.4, -5.5. To a mixture of imidazole (2.57 g, 37.8 mmol), TBDPS-Cl (10.4 g, 37.8
mmol), and DMAP (0.2 g, 1.6 mmol) in dry CH
2
Cl
2
(60 mL) at 0
°
C was added the secondary alcohol
(9.04 g, 31.5 mmol). The reaction mixture was warmed to room temperature, and stirred for overnight.
The reaction mixture was quenched with a saturated aqueous solution of NH
4
Cl, extracted with ether,
washed with brine, dried over MgSO
4
, and concentrated under reduced pressure to give a crude product.
The crude product was purified on silica column using 3% EtOAc/hexanes as the eluent to furnish the
pure product (16.3 g, 99%) as a colorless oil.
1
H-NMR (600 MHz, CDCl
3
) δ
H
7.71 (m, 4H), 7.38 (m, 6H),
3.85 (m, 1H), 3.56 (m, 2H), 2.40 (m, 1H), 2.34 (m, 1H), 1.13 (s, 9H), 0.90 (s, 9H), 0.03 (s, 3H), 0.08 (s,
3H), 0.00 (s, 3H).
13
C-NMR (600 MHz, CDCl
3
) δ
C
136.1, 135.9, 129.8, 129.7, 128.5, 127.8, 127.7, 69.9,
65.4, 27.1, 26.0, 23.7, 19.5, 18.4, 3.4, 0.2, -0.54. 3.0 g TMS-protected alkyne was dissolved in 30ml
MeOH and one scupula of Na
2
CO
3
powder was added, stirred overnight at room temperature. Filtrated off
the solids and concentrated in vacuo. The crude product was purified on silica column using 3%
EtOAc/hexanes as the eluent to furnish the pure product (2.5 g, 99%) as a colorless oil.
1
H-NMR (600
MHz, CDCl
3
) δ
H
7.71 (m, 4H), 7.38 (m, 6H), 3.85 (m, 1H), 3.56 (m, 2H), 2.40 (m, 1H), 2.34 (m, 1H),
1.92 (t, J = 3.0Hz, 1H), 1.13 (s, 9H), 0.90 (s, 9H), 0.03 (s, 3H), 0.00 (s, 3H).
13
C-NMR (600 MHz, CDCl
3
)
δ
C
136.1, 135.9, 129.8, 129.7, 128.5, 127.8, 127.7, 72.1, 69.9, 65.4, 27.1, 26.0, 23.7, 19.5, 18.4, 0.2, -0.54.
At room temperature, 623mg (4.24 mmol) 1-bromopent-2-yne, 1.1g (2.12 mmol) acetylene, 810mg
(4.24mmol) CuI, 585mg (4.24mmol) potassium carbonate, 636mg (4.24mmol) sodium iodide in 30ml
DMF was stirred overnight. Workup with saturated NH
4
Cl aqueous solution. Keep extracting the aqueous
layer with hexanes until no product was left in the aqueous layer which was confirmed by TLC. The
organic layer was combined and was rinsed with brine twice. Dried over magnesium sulfate, and
evaporate the solvent. The crude product was purified on silica column using 3% EtOAc/hexanes as the
eluent to furnish the pure product 17 (1.1 g, 88%) as a colorless oil.
1
H-NMR (600 MHz, CDCl
3
) δ
H
7.71
(m, 4H), 7.36 (m, 6H), 3.83 (m, 1H), 3.53 (m, 2H), 2.39 (m, 1H), 3.02 (m, 2H), 2.31 (m, 1H), 2.17 (m,
2H), 1.13 (s, 9H), 0.90 (s, 9H), 1.10 (t, J = 7.2Hz , 3H), 0.03 (s, 3H), 0.00 (s, 3H).
13
C-NMR (600 MHz,

37

CDCl
3
) δ
C
136.1, 136.0, 134.4, 134.2, 129.7, 129.6, 128.4, 127.6, 81.9, 76.2, 73.8, 72.6, 65.8, 27.1, 26.0,
24.2, 19.5, 18.4, 14.0, 12.5, 9.9, 0.2, -0.54.
O
OTBDPS
19

(2S,4Z,7Z)-2-(tert-butyldiphenylsilyloxy)deca-4,7-dienal (19). To a solution of protected diol 17 (0.6 g,
1.15 mmol) in a 1:1 mixture of CH
2
Cl
2
:MeOH (20 mL) was added camphorsulfonic acid (0.27 g, 1.15
mmol) at room temperature. The progress of the reaction was monitored by TLC. Quenched with Et
3
N
(1ml). The solvent was evaporated to dryness and the crude product was purified on a silica gel column
using 15% EtOAc/hexanes to afford the primary alcohol (0.41 g, 88%).
1
H-NMR (600 MHz, CDCl
3
) δ
H
7.67 (m, 4H), 7.39 (m, 6H), 3.92 (m, 1H), 3.63 (m, 2H), 3.02 (m, 2H), 2.40 (m,1H), 2.32 (m, 1H), 2.15 (m,
2H), 1.07 (s, 9H), 1.10 (t, J = 7.2 Hz, 3H).
13
C-NMR (600 MHz, CDCl
3
) δ
C
136.0, 135.8, 133.7, 133.6,
130.1, 130.0, 127.9, 127.8, 82.1, 76.5, 73.5, 72.5, 60.8, 27.1, 24.0, 19.4, 14.0, 12.5, 9.8. Under hydrogen
atmosphere, Lindlar catalyst, and 350mg diyne in 30ml EtOAc with 1 drop quinoline were stirred
vigorously for 2-3hrs. The reaction was monitored by TLC. Filtrated through celite. Evaporated the
solvent, and the crude product was purified on a silica gel column using 12% EtOAc/hexanes to afford the
hydrogenated product (0.29 g, 85%).
1
H-NMR (600 MHz, CDCl
3
) δ
H
7.68 (m, 4H), 7.39 (m, 6H), 5.35
(m, 2H), 5.22 (m, 2H), 3.81 (m, 1H), 3.53 (m, 1H), 3.48 (m, 1H), 2.60 (m, 2H), 2.32 (m, 1H), 2.20 (m,
2H), 1.99 (m, 2H), 1.78 (m, 1H), 1.07 (s, 9H), 0.94 (t, J = 7.2 Hz, 3H).
13
C-NMR (600 MHz, CDCl
3
) δ
C
136.0, 135.8, 133.9, 133.8, 132.2, 130.9, 130.0, 129.9, 127.9, 127.8, 126.9, 124.7, 73.8, 65.8, 31.8, 27.2,
25.6, 20.7, 19.5, 14.4. At –78
°
C, to 142mg (0.14ml 1.82mmol) DMSO in 4 ml CH
2
Cl
2,
185mg (0.13ml,
1.46mmol) (COCl)
2
was added. Stirred for 15 min. Then add 300mg (0.73mmol) alcohol in 4 ml DCM
through cannula. Stirred at –78
°
C for 45 min. Then inject 0.51ml (3.65mmol) Et
3
N, stirred at –78
°
C for 3
hr. Warm up slowly to room temperature. After 20 min, check by TLC. Work up with H
2
O, extract with
ether. Evaporated the solvent and the crude product was purified on a silica gel column using 10%

38

EtOAc/hexanes to afford 19 (0.27 g, 95%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
9.56 (d, J = 1.6 Hz , 1H),
7.68 (m, 4H), 7.39 (m, 6H), 5.42 (m, 4H), 4.07 (m, 1H), 2.67 (m, 2H), 2.45 (m, 1H), 2.38 (m, 1H), 2.02
(m, 2H), 1.07 (s, 9H), 0.95 (t, J = 7.2 Hz, 3H).
13
C-NMR (400 MHz, CDCl
3
) δ
C
203.2, 135.9, 133.2, 133.1,
132.3, 131.6, 130.2, 130.1, 127.9, 127.8, 127.7, 126.7, 123.2, 78.2, 77.9, 77.8, 31.2, 27.1, 25.7, 20.7, 19.5,
14.3.

OTBDPS
25

Tert-butyldiphenyl((2S,5Z,8Z)-undeca-5,8-dien-1-yn-3-yloxy)silane (25). Carbon tetrabromide (90mg,
0.27mmol) was dissolved in 0.5ml CH
2
Cl
2
and cooled to 0°C and triphenylphosphine (141mg, 0.54mmol)
was added in via canula in 1ml CH
2
Cl
2
solution. Stirred at 0°C for 1 hr and aldehyde 19 (110mg,
0.27mmol) was added in via canula in 1ml CH
2
Cl
2
solution. Stirred at 0°C for 1 hr. Work up with sodium
bicarbonate solution, extract with ether. Evaporated the solvent and purified the mixture on a silica
column using 1% EtOAc/hexanes to afford the dibromo olefin (110mg, a little impure). At -78°C, the
dibromo olefin was dissolved in 2 ml anhydrous THF and lithium diisopropylamide in THF solution
(2.0M, 0.29ml) was added dropwise. Stirred at -78°C for 1hr. Work up with NH
4
Cl solution, extract with
ether. Evaporated the solvent and purified the mixture on a silica column using 1% EtOAc/hexanes to
afford the product 25 as colorless oil (64mg, 59% for two steps).
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.68 (m,
4H), 7.39 (m, 6H), 5.45 (m, 2H), 5.35 (m, 1H), 5.25 (m, 1H), 4.36 (m, 1H), 2.68 (m, 2H), 2.45 (m, 2H),
2.33 (d, J = 3.0Hz, 1H), 2.02 (m, 2H), 1.09 (s, 9H), 0.96 (t, J = 7.2 Hz, 3H).
13
C-NMR (400 MHz, CDCl
3
)
δ
C
136.2, 136.0, 133.6, 133.5, 132.1, 131.2, 129.9, 129.8, 128.5, 127.7, 127.6, 124.3, 72.8, 63.6, 36.4,
31.7, 27.0, 25.8, 22.8, 20.7, 19.4, 14.4.

39

OTBDPS
27

Tert-butyldiphenyl((S,3E,7Z,10Z)-trideca-3,7,10-trien-1-yn-5-yloxy)silane (27). Vinyl iodide (50mg,
0.094mmol) was dissolved in pyrrolidine (1 ml) and 5mg Pd(PPh
3
)
4
and 0.1 ml TMS acetylene was added.
The reaction was stirred for 30 minutes. After removing pyrrolidine, the mixture was purified on a silica
column with 1% EtOAc/hexanes. The product was dissolved in MeOH and 200 mg Na
2
CO
3
was added
and the reaction was stirred overnight. The slurry solution was filtered and purified on a silica column
with 1% EtOAc/hexanes to afford the product (30mg, 75% for two steps).
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.65 (m, 4H), 7.40 (m, 6H), 6.20 (dd, J = 16.0 Hz and 4.0 Hz, 1H), 5.57 (dd, J = 16.0 Hz and 4.0 Hz, 1H),
5.40-5.20 (m, 4H), 4.20 (m, 1H), 2.85 (d, J = 4.0 Hz, 1H), 2.55 (m, 2H), 2.20 (m, 2H), 1.95 (m, 2H), 1.07
(s, 9H), 0.94 (t, J = 7.2 Hz, 3H).
13
C-NMR (400 MHz, CDCl
3
) δ
C
147.3, 136.0, 135.9, 133.9, 133.8, 132.2,
129.9, 129.8, 128.8, 128.7, 128.6, 127.7, 108.5, 77.4, 73.0, 35.5, 29.8, 27.1, 20.6, 19.5, 14.4.
OTBDPS
COOMe I
20

(R,4Z,8E)-methyl 7-((tert-butyldiphenylsilyl)oxy)-9-iodonona-4,8-dienoate (20). Prepared similarly as
22 from aldehyde 14.
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.65 (m, 4H), 7.40 (m, 6H), 6.48 (dd, J = 12.0 Hz
and 4.0 Hz, 1H), 6.00 (dd, J = 16.0 Hz and 4.0 Hz, 1H), 5.35 (m, 2H), 4.10 (m, 1H), 3.65 (s, 3H), 2.30-
2.10 (m, 6H), 1.07 (s, 9H), 0.88 (t, J = 7.2 Hz, 3H).
13
C-NMR (400 MHz, CDCl
3
) δ
C
173.6, 147.9, 136.1,
136.0, 133.9, 133.6, 130.3, 129.4, 129.9, 127.8, 127.7, 125.6, 75.7, 51.7, 35.3, 34.8, 34.0, 31.8, 27.1, 25.4,
22.9, 22.8, 19.5, 14.3.

40

OH
OH
COOMe
28

(4Z,7R,8E,10E,14S,16Z,19Z)-methyl-7,14-dihydroxydocosa-4,8,10,16,19-pentaen-12-ynoate (28).
50mg (0.087mmol) vinyl iodide 22, 50mg (0.13mmol) alkyne 25, 6mg (0.005mmol) Pd(PPh
3
)
4
, 1.9mg
(0.01mmol) CuI, and 0.14ml (1mmol) NEt
3
were added in 1.5ml benzene and stirred at room temperature
overnight. Worked up with saturated ammonium chloride aqueous solution and ether. The organic layer
was combined and washed with brine. Evaporated the solvent and purified the mixture on a silica column
using 3.5% EtOAc/hexanes to afford 60 mg impure product. To 60 mg protected product from last step in
4ml THF, added 0.37ml (0.37mmol) 1.0 M TBAF. The mixture was stirred at room temperature
overnight. Workup with NH
4
Cl solution and extracted with ether, then wash the organic layer with brine.
Evaporated the solvent and purified the mixture on a silica column using 35% EtOAc/hexanes to afford
the product 28 as colorless oil (9 mg, 62% for two steps).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.57 (dd,
J=15.7Hz and 10.8Hz, 1H), 6.29 (dd, J=15.8Hz and 11.4Hz, 1H), 5.83 (dd, J=15.4Hz and 5.8Hz, 1H),
5.7-5.3 (m, 7H), 4.53 (m, 1H), 4.24 (m, 1H), 3.67 (s, 3H), 2.84 (m, 2H), 2.50 (m, 2H), 2.38 (m, 6H), 2.09
(m, 2H), 0.98 (t, J=7.5Hz, 3H).
13
C-NMR (600 MHz, CDCl
3
) δ
C
173.8, 141.6, 138.4, 132.5, 132.4, 131.4,
129.3, 126.9, 126.1, 123.8, 110.8, 92.4, 84.2, 71.5, 68.3, 62.6, 41.5, 35.4, 33.7, 31.1, 29.9, 20.8,14.3.

41

(4Z,7R,8E,12E,14S,16Z,19Z)-methyl 7,14-dihydroxydocosa-4,8,12,16,19-pentaen-10-ynoate (31).
Prepared similarly as 28 from vinyl iodide 20 and acetylene 27.
1
H-NMR (600 MHz, CDCl
3
) δ
H
6.17 (dd,
J=18.0Hz and 6.0Hz, 1H), 6.16 (dd, J=18.0Hz and 6.0Hz, 1H), 5.87 (dd, J=18.0Hz and 12.0Hz, 2H), 5.7-
5.3 (m, 6H), 4.24 (m, 2H), 3.67 (s, 3H), 2.80 (m, 2H), 2.45 – 2.3 (m, 8H), 2.09 (m, 2H), 0.98 (t, J=7.5Hz,
3H).
13
C-NMR (600 MHz, CDCl
3
) δ
C
173.9, 145.0, 144.8, 132.5, 132.4, 131.6, 126.7, 125.9, 124.2, 110.2,
110.0, 88.4, 88.3, 71.7, 71.5, 41.5, 36.2, 33.7, 25.9, 25.4, 20.9, 18.9, 14.3.
(S)
(R)
COOMe
OH
OH
1

(4Z,7R,8E,10E,12Z,14S,16Z,19Z)-methyl-7,14-dihydroxydocosa-4,8,10,12,16,19-hexaenoate (1).
200mg Zn dust was weighed in N
2
glove box and 3mL degassed water was added. Argon was bubbled
through the mixture for 15min. Then 50mg Cu(OAc)
2
was added and stirred for 20min. After that, 50mg
AgNO
3
was added and stirred for another 30min. The precipitate was rinsed by water (2 x 3mL), MeOH
(2 x 3mL), acetone (2 x 3mL), and ether (2 x 3mL) and dried under in vacuo. Weigh 100mg powder
prepared in this way and transferred into a 25ml flask. 0.5ml of degassed water was added in, then added
in 1.0 mg acetylene 28 dissolved in 0.5ml MeOH. The suspension was stirred at room temperature for 4
hrs and the progress of reaction was monitored by HPLC. 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 (0.60mg, 60%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.51 (dd, J = 14.8Hz and 12.0Hz, 1H), 6.32 (ddd,
J = 14.8Hz, 10.4Hz and 0.8Hz, 1H), 6.24 (dd, J = 14.4Hz and 10.8Hz, 1H), 6.09 (dd, J = 10.8 Hz and
10.8Hz, 1H), 5.78 (dd, J = 14.4Hz and 6.0Hz), 5.60-5.25 (m, 7H), 4.62 (m, 1H), 4.24 (m, 1H), 3.67 (s,
3H), 2.81 (m, 2H), 2.39 (m, 8H), 2.07 (m, 2H), 1.99 (d, J = 4.0Hz, 1H), 1.66 (d, J = 3.6Hz, 1H), 0.98 (t,

42

J=7.5Hz, 3H).
13
C-NMR (400 MHz, CDCl
3
) δ
C
173.8, 136.8, 134.0, 133.4, 132.4, 131.9, 131.3, 130.3,
130.2, 127.8, 126.9, 126.3, 124.6, 71.8, 67.8, 53.6, 51.8, 51.1, 35.6, 35.5, 33.8, 25.9, 22.9, 20.7, 14.4.
(S)
(S)
COOMe
OH
OH
2

(4Z,7S,8E,10E,12Z,14S,16Z,19Z)-methyl-7,14-dihydroxydocosa-4,8,10,12,16,19-hexaenoate (2).
Prepared similarly as 1, starting with corresponding glycidols.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.51 (dd, J
= 14.8Hz and 12.0Hz, 1H), 6.32 (ddd, J = 14.8Hz, 10.4Hz and 0.8Hz, 1H), 6.24 (dd, J = 14.4Hz and
10.8Hz, 1H), 6.09 (dd, J = 10.8 Hz and 10.8Hz, 1H), 5.78 (dd, J = 14.4Hz and 6.0Hz), 5.60-5.25 (m, 7H),
4.62 (m, 1H), 4.24 (m, 1H), 3.67 (s, 3H), 2.81 (m, 2H), 2.39 (m, 8H), 2.07 (m, 2H), 1.99 (d, J = 4.0Hz,
1H), 1.66 (d, J = 3.6Hz, 1H), 0.98 (t, J=7.5Hz, 3H).
13
C-NMR (400 MHz, CDCl
3
) δ
C
173.8, 136.8, 134.0,
133.4, 132.4, 131.9, 131.3, 130.3, 130.2, 127.8, 126.9, 126.3, 124.6, 71.8, 67.8, 53.6, 51.8, 51.1, 35.6,
35.5, 33.8, 25.9, 22.9, 20.7, 14.4.
(R)
(S)
COOMe
OH
3
OH

(4Z,7S,8E,10E,12Z,14R,16Z,19Z)-methyl-7,14-dihydroxydocosa-4,8,10,12,16,19-hexaenoate (3).
Prepared similarly as 1, starting with corresponding glycidols.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.51 (dd, J
= 14.8Hz and 12.0Hz, 1H), 6.32 (ddd, J = 14.8Hz, 10.4Hz and 0.8Hz, 1H), 6.24 (dd, J = 14.4Hz and

43

10.8Hz, 1H), 6.09 (dd, J = 10.8 Hz and 10.8Hz, 1H), 5.78 (dd, J = 14.4Hz and 6.0Hz), 5.60-5.25 (m, 7H),
4.62 (m, 1H), 4.24 (m, 1H), 3.67 (s, 3H), 2.81 (m, 2H), 2.39 (m, 8H), 2.07 (m, 2H), 1.99 (d, J = 4.0Hz,
1H), 1.66 (d, J = 3.6Hz, 1H), 0.98 (t, J=7.5Hz, 3H).
13
C-NMR (400 MHz, CDCl
3
) δ
C
173.8, 136.8, 134.0,
133.4, 132.4, 131.9, 131.3, 130.3, 130.2, 127.8, 126.9, 126.3, 124.6, 71.8, 67.8, 53.6, 51.8, 51.1, 35.6,
35.5, 33.8, 25.9, 22.9, 20.7, 14.4.
COOMe
OH
OH
4

(4Z,7R,8E,10Z,12E,14S,16Z,19Z)-methyl 7,14-dihydroxydocosa-4,8,10,12,16,19-hexaenoate (4).
Prepared similarly as 1 from 31.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.74 (dd, J = 8.8 and 15.2 Hz , 2H), 5.99
(m, 2H), 5.77 (ddd, J = 15.2, 6.0 and 1.6Hz, 2H), 5.25-5.60 (m, 6H), 4.27 (m, 2H), 3.65 (s, 3H), 2.81 (m,
2H), 2.38 (m, 8H), 2.07 (m, 2H), 0.97 (t, J = 7.5Hz, 3H).
13
C-NMR (600 MHz, CDCl
3
) δ
C
171.3, 136.9,
136.7, 132.4, 132.0, 131.3, 129.2, 129.1, 126.9, 126.3, 125.7, 125.6, 124.7, 72.1, 71.9, 55.3, 35.5, 33.8,
30.5, 29.9, 25.9, 23.0, 14.4.
COOMe
OH
5
OH

(4Z,7S,8E,10Z,12E,14S,16Z,19Z)-methyl 7,14-dihydroxydocosa-4,8,10,12,16,19-hexaenoate (5).
Prepared similarly as 4.
1
H-NMR (400 MHz, CDCl3) δ
H
6.74 (dd, J = 8.8 and 15.2 Hz , 2H), 5.99 (m, 2H),
5.77 (ddd, J = 15.2, 6.0 and 1.6Hz, 2H), 5.25-5.60 (m, 6H), 4.27 (m, 2H), 3.65 (s, 3H), 2.81 (m, 2H), 2.38

44

(m, 8H), 2.07 (m, 2H), 0.97 (t, J = 7.5Hz, 3H).
13
C-NMR (600 MHz, CDCl3) δ
C
171.3, 136.9, 136.7,
132.4, 132.0, 131.3, 129.2, 129.1, 126.9, 126.3, 125.7, 125.6, 124.7, 72.1, 71.9, 55.3, 35.5, 33.8, 30.5,
29.9, 25.9, 23.0, 14.4.
OH
OH
COOMe
6

(4Z,7S,8E,10E,12E,14S,16Z,19Z)-methyl-7,14-dihydroxydocosa-4,8,10,12,16,19-hexaenoate (6). To a
0.2ml THF suspension of 19mg (0.074) zirconocene chloride hydride (Schwartz's reagent) was added
alkyne 25 (30mg, 0.074mmol) dissolved in 0.3ml THF. The suspension was heated up to 50°C and
maintained for 40 minutes. Cooled to room temperature and 0.05ml (0.096mmol, 1.9M in 2-
methyltetrahydrofuran) ZnCl
2
was added and stirred for 10 minutes. Then vinyl iodide 22 (30mg,
0.052mmol) dissolved in 0.2ml THF was added through cannula, together with 3mg (10%) Pd(PPh
3
)
4

catalyst. Stir at room temperature for 3 hours. Work up the reaction with saturated NH
4
Cl solution and
extract with ether, then wash the organic layer with brine. Remove the solvents in vacuo and purified the
mixture on a silica column using 3.5% EtOAc/hexanes to afford 20mg impure protected 6. To 20 mg
impure protected product from last step in 4ml THF, added 0.12ml (0.12mmol) 1.0 M TBAF. The
mixture was stirred at room temperature for 4 hours. Workup with NH
4
Cl solution and extracted with
ether, then wash the organic layer with brine. Evaporated the solvent and purified the mixture on a silica
column using 35% EtOAc/hexanes to afford the product 6, which was further purified on a HPLC
preparatory column using 28% water/MeOH as eluent (0.50 mg, 34% for two steps).
1
H-NMR (400 MHz,
CDCl
3
) δ
H
6.22 (m, 4H), 5.74 (ddd, J = 14.4, 6.0 and 1.2 Hz, 2H), 5.25-5.60 (m, 6H), 4.22 (m, 2H), 3.65
(s, 3H), 2.80 (m, 2H), 2.38 (m, 8H), 2.07 (m, 2H), 0.97 (t, J = 7.5Hz, 3H).
13
C-NMR (600 MHz, CDCl
3
)
δ
C
173.8, 136.0, 135.9, 135.6, 132.4, 132.3, 131.3, 131.2, 130.5, 130.4, 129.2, 128.2, 125.1, 123.7, 72.1,
72.0, 51.8, 35.5, 35.4, 34.1, 25.9, 22.9, 18.6, 14.3.

45

2.5. References
1. 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.
2. Jin, J.; Weinreb, S. M. Application of a stereospecific intramolecular allenylsilane imino ene
reaction to dnantioselective total synthesis of the 5,11-methanomorphanthridine class of
amaryllidaceae alkaloids. J. Am. Chem. Soc. 1997, 119, 5773-5784.
3. Takai, K.; Nitta, K.; Utimoto, K. Simple and selective method for aldehydes (RCHO) .fwdarw.
(E)-haloalkenes (RCH:CHX) conversion by means of a haloform-chromous chloride system. J.
Am. Chem. Soc. 1986, 108, 7408-7410.
4. For a review see: Petasis, N. A. In Science of Synthesis: Alkenes; Meijere, A. d. Ed.; Thieme-
Verlag: Stuttgart, 2009; pp. 161-246.
5. Boland, W.; Schroer, N.; Sieler, C.; Feigel, M. Sterospecific syntheses and spectroscopic
properties of ssomeric 2,4,6,8-undecatetraenes. New hydrocarbons from the marine brown alga
giffordia mitchellae. Part IV. Helv. Chim. Acta 1987, 70, 1025-1040.
6. Chemin, D.; Linstrumelle, G. A short stereocontrolled synthesis of leukotriene B
4
. Tetrahedron
1992, 48, 1943-1952.
7. Nicolaou, K. C.; Bulger, P. G.; Sarlah, D. Palladium-catalyzed cross-coupling reactions in total
synthesis. Angew. Chem. Int. Ed. 2005, 44, 4442-4489.
8. Mohr, P.; Tamm, C. Stereoselective synthesis of functionalized erythro/1,3-diols. Tetrhedron
Letters 1987, 28, 391.
9. Altundas, R.; Mahadevan, A.; Razdan, R. K. A synthetic route to anandamide analogues carrying
a substituent at the terminal carbon and an acetylene group in the end pentyl chain. Tetrhedron
Letters 2004, 45, 5449-5491.
10. Corey, E. J.; Fuchs, P. L. A synthetic method for formyl→ethynyl conversion (RCHO→RC≡CH
or RC≡CR’) Tetrahedron Letters 1972, 13, 3769-3772.

46

11. Uddin, J. Design and synthesis of novel anti-inflammatory lipid mediators and anticancer small
molecules. Ph.D. dissertation 2008.
12. Corey, E. J.; Raju, N. A new general synthetic route to bridged carboxylic ortho esters.
Tetrahedron Letters 1983, 24, 5571.
13. Nicolaou, K. C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M. R. V.;
Yang, Z. Total syntheses of epothilones A and B via a macrolactonization-based strategy. J. Am.
Chem. Soc. 1997, 119, 7974.
14. Mancuso, A. J.; Huang, S. L.; Swern, D. Oxidation of long-chain and related alcohols to
carbonyls by dimethyl sulfoxide "activated" by oxalyl chloride. J. Org. Chem. 1978, 43, 2480.
15. Serhan, C. N., Dalli, J., Karamnov, S., Choi, A., Park, C., Xu, Z., Ji, R., Zhu, M., Petasis, N. A.
Macrophage pro-resolving mediator maresin 1 stimulates tissue regeneration and controls pain.
The FASEB Journal. 2012, 26, 1755-1765.
16. Zhu, M., Chan, N.; Petasis, N. A., Serhan, C. N. Structure-activity reationship: wound healing
activities of DHA-derided maresin 1 and stereoisomers in a human dermal fibroblasts model.
2013, manuscript in preparation.

47

Chapter 3. Total Synthesis of 14S-epoxy-maresin: Biosynthetic precursor of Maresin 1

3.1. Introduction
Leukotriene B
4
(LTB
4
) is regarded as an important chemical mediator in a variety of acute and chronic
inflammatory diseases, e.g. nephritis, arthritis, dermatitis, and chronic obstructive pulmonary disease
1-3
.
The biosynthesis of LTB
4
starts from the ω-6 unsaturated fatty acid arachidonic acid (AA) and 5-
lipoxygenase, assisted by 5-lipoxygenase-activating protein, converting AA into 5S-hydroperoxy-6-trans-
8,11,14-Z-eicosatetraenoic acid (5-HpETE), which is further transformed to the epoxide (5S, 6S)-E-5,6-
oxido-7,9-E-11,14-Z-eicosatetraenoic acid called leukotriene A
4
(LTA
4
). Enzymatic hydrolysis of LTA
4

by leukotriene A
4
hydrolase (LTA
4
H)
4
leads to LTB
4
(Scheme 8(A)). LTA
4
H is a bifunctional zinc
metalloenzyme that catalyzes the hydrolysis of the LTA
4
into LTB
4
and also exhibits an
amidase/peptidase activity
5-6
.
A series of recently discovered potent anti-inflammatory and pro-resolving lipid mediators biosynthesized
from the ω-3 unsaturated fatty acid DHA were found to possess similar features and their biosynthesis
were proposed to proceed similarly. These include the hypothesized biosynthesis of neuroprotectin D1/
protectin D1 (Scheme 23 in Chapter 5) and maresin 1 (Scheme 8(B)). However a direct proof of the
biosynthetic pathway for these mediators has not been reported, and the hydrolase involved in the
hydrolysis of the hypothesized LTA
4
-like epoxide precursor is not known. On the other hand, it is
difficult to isolate the postulated LTA
4
-like epoxide from biological systems due to its labile nature
7
.
Therefore there is a great need to prepare a series of LTA
4
-like epoxides via total synthesis and they could
be powerful tools to study biosynthetic pathways and elucidate anti-inflammatory signaling mechanisms
of new lipid mediators. This chapter details the total synthesis of the LTA
4
-like epoxide precursor of
maresin 1, named as 13S, 14S-epoxy-maresin.

48

COOH
12/15-LO
(S)
COOH
(S)
(S)
O
COOH
DHA
HOO
14-HpDHA 13S, 14S-epoxy-Maresin
COOH
(S)
COOH
HOO
(S)
COOH
O
5-LO
AA
5-HpETE
LTA
4
hydrolase
(S)
COOH
(R)
OH OH
unknown hydrolase
(S)
(R)
COOH
OH
OH
LTA
4
LTB
4
MaR1
(A)
(B)

Scheme 8. Biosynthesis of LTB
4
and hypothesized biosynthesis of MaR1
3.2. Results
3.2.1. Retrosynthetic analysis of 13S, 14S-epoxy-maresin
The retrosynthetic analysis of 13S, 14S-epoxy-maresin methyl ester (1) is shown in Figure 11. Due to the
particular instability of the triene moiety, the last C-C bond constructed is the 7-Z double bond, which is
generated from a Z-selective Wittig reaction
8
. The epoxide-containing building block has the S, S-epoxide
generated from asymmetric epoxidation
9
(Sharpless epoxidation) and the backbone is constructed via two
Cu (I) mediated coupling reactions
10
. On the other hand, the carboxylester-containing building block has
its cis-double bond generated from reduction of a triple bond, which was introduced to achieve the
backbone construction via alkynylation of alkyl halides.

49

(S)
(S)
O
COOMe
1
Br
TMS
Br
OH
(S)
(S)
O
O
OH
COOMe
I
HO
COOMe
HO
COOMe
Ph
3
P
I

Figure 11. Retrosynthetic analysis of 13S, 14S-epoxy-maresin methyl ester

3.2.2. Synthesis of building blocks of 13S, 14S-epoxy-maresin
Synthesis of the key synthetic intermediates 8 is shown in Scheme 9. TMS propargyl bromide was
coupled to propargyl alcohol mediated by Cu (I) iodide and the propargyl alcohol was selectively reduced
to E-allylic alcohol using lithium aluminium hydride
11
. After removal of the TMS protecting group, the
terminal alkyne was coupled to 1-bromo-2-pentyne under the same Cu (I) mediated coupling conditions
10

50

and a subsequent Lindlar hydrogenation
12
reduced the two triple bonds to Z-double bonds simultaneously.
A subsequent Sharpless asymmetric epoxidation
9
afforded the S, S-epoxide catalyzed by (+)-
diethyltatarate, and the primary alcohol was then oxidized to the aldehyde 8 using the Dess-Martin
periodinane
13
. A one-step Wittig reaction afforded the E, E-diene-aldehyde
14
where the E-selective Wittig
homologation occurred twice.
OH
Br
2. H
2
/Lindlar cat., EtOAc,
quinoline, 78%
TMS
Br
OH
CuI, NaI, K
2
CO
3
, 75%
TMS
OH
1. LiAlH
4
,reflux,
diethyl ether, 90%
2.Na
2
CO
3
, MeOH, 84%
1. CuI, NaI, K
2
CO
3
, DMF,
, 85%
OH
1. (+)-DET, Ti(O
i
Pr)
4
,

t
BuOOH, CH
2
Cl
2
, 90%
2. Dess-Marin periodinane,
NaHCO
3
, CH
2
Cl
2
, 92%
O
Ph
3
P
(S)
(S)
O
O
, DMF
3. toluene, 95ºC,
, 65%
2
3
5
8

Scheme 9. Synthesis of epoxy aldehyde 8 for total synthesis of 13S, 14S-epoxy-maresin

The synthesis of the key phosphonium salt intermediate 13 is shown in Scheme 10. After protecting the
carboxylic acid group of 4-pentynoic acid as OBO ester
15
, alkylation of the terminal alkyne was achieved
to afford 9. Alternative alkylation from the other side of the triple bond, namely the attempt to couple the
protected 3-butynol with OBO ester of 3-iodo-propanoic acid, was not successful. After cleavage of the
OBO ester and THP protection group at one step with acid after alkylation and subsequent
transesterification, the triple bond in 10 was reduced to a Z-double bond and the primary alcohol was then

51

tosylated and subsequently substituted by an iodine atom. The iodide was converted to the Wittig salt 13
(precussor of ylide) after refluxing with triphenylphosphine in toluene
8
.
O
O
O
OH
O
1, DCC, DMAP, CH
2
Cl
2
,
, 87% O
HO
I
THPO
3. n-BuLi, THF, HMPA,
, 85%
1. HCl, THF/H
2
O, 86%
2. MeOH, Et
3
N, 87%
O
O
HO
1. H
2
/Lindlar cat., EtOAc,
quinoline, 88%
2. TsCl, CH
2
Cl
2
,
Et
3
N, 99%
O
O
1. NaI, acetone, reflux, 98%
2. PPh
3
, toluene, reflux, 92%
TsO
O
O
Ph
3
P
I
2. BF
3
.
OEt
2
, CH
2
Cl
2
, 90%
THPO
9
10
14
13

Scheme 10. Synthesis of Wittig salt 13 for total synthesis of 13S, 14S-epoxy-maresin

3.2.3. Assembly of 13S, 14S-epoxy-maresin
With the epoxy aldehyde 8 and Wittig salt 13 prepared, a Z-selective Wittig reaction was carried out
(Scheme 11). The Wittig salt 13 was deprotonated by the non- nucleophilic base sodium
hexamethyldisilazide to form the ylide in situ, which was coupled with the aldehyde with a Z-double bond
predominantly generated. It is worth mentioning that the product epoxy methyl ester 1 was very
susceptible to epoxide ring-opening in acidic environment and it decomposed rapidly when in contact
with silica gel, which made reaction monitoring and purification challenging. A quick purification of the
reaction on a silica column treated with 10% triethylamine afforded the product 1, with no detection of E-

52

isomer, which might be produced at a trace amount and decomposed in purification. After removal of
eluent from purification, 1 was dissolved in 1% triethylamine-containing benzene and stored at -78⁰C to
avoid decomposition. 13S, 14S-epoxy maresin was obtained and used for biological assays after
hydrolysis of 1 in basic solution
16
. Due to the instability of 1 and 13S, 14S-epoxy maresin, the hydrolysis
was carried out when biological assays were immediately followed.
8
NaHMDS, THF, -78 ºC, 67%
(S)
(S)
O
O
(S)
(S)
O
O
O
Ph
3
P
I
COOMe
13
1
NaOH, acetone/H
2
O, rt, 1h
(S)
(S)
O
COOH
13S, 14S epoxy-Maresin

Scheme 11. Assembly of 13S, 14S-epoxy-maresin

The proton NMR spectra are useful for establishing the stereochemistry of the triene. In the olefinic
region of 1, four of the six triene protons are resolved and the coupling constants unambiguously prove
the 7Z, 9E, 11E structure (Figure 12).

53

Figure 12.
1
H NMR of olefinic region of 13S, 14S-epoxy-maresin methyl ester (1)

The synthesized 13S, 14S-epoxy-maresin methyl ester was hydrolyzed in a basic aqueous solution and
used immediately in bioassays. Results showed that the synthesized 13S, 14S-epoxy-maresin was
converted to maresin 1 in a highly specific manner under influence of macrophages
17
. In addition, this
13S, 14S-epoxy-maresin was found to down regulate the biosynthesis of leukotriene B
4
.
3.3. Conclusion
The total synthesis of 13S, 14S-epoxy maresin, the hypothesized epoxide-containing intermediate in
MaR1 biosynthesis, has been successfully achieved. This work of synthesis provides a facile and feasible
method for total synthesis of more similar epoxide-containing intermediates for DHA or EPA-derived
SPMs, and also the authentic compound which proves the postulated MaR1 biosynthetic pathway, and
help uncover additional biological mechanisms, such as suppressing the biosynthesis of LTB
4
,
contributing to the beneficial anti-inflammatory effects of ω-3 PUFAs.
3.4. Experimental Procedures
All reactions, unless otherwise noted, were carried in flame dried flasks under argon atmosphere. “Dried
and concentrated” refers to removal of residual water with anhydrous MgSO
4
, followed by evaporation of

54

the solvent on the rotary evaporator. THF was freshly distilled from sodium-benzophenone, benzene and
dichloromethane from CaH
2
and anhydrous DMF, EtOH, and MeOH were purchased from commercial
sources.
1
H and
13
C NMR spectra were recorded on a Varian Mercury 400 or 600 MHz using residual
1
H
or
13
C signals of deuterated solvents as internal standards. UV spectra were recorded on a Hewlett-
Packard 8350 instrument. HPLC analyses were performed on a Rainin dual pump HPLC system
equipped with a Phenomenex ODS column and an UV-VIS detector.

TMS
OH
2

6-(Trimethylsilyl)hexa-2,5-diyn-1-ol (2). Propargyl alcohol (0.4g, 6.98mmol) and 2.0g (3-bromoprop-
1-ynyl)trimethylsilane were mixed and dissolved in 40ml anhydrous DMF. CuI (4g, 31.4mmol), NaI
(3.1g, 31.4mmol) and K
2
CO
3
(2.9g, 31.4mmol) were added and the solution was stirred violently
overnight. Work up with NH
4
Cl aqueous solution and extract with ether. After removing the solvent the
mixture was purified on a silica column with 10% EtOAc/hexanes to afford the product (1.1g, 95%).
1
H
NMR (400 MHz, CDCl
3
) δ
H
4.25 (m, 2H), 3.25 (m, 2H), 0.15 (s, 9H).
13
C NMR (400 MHz, CDCl
3
) δ
C

99.5, 85.6, 79.8, 79.0, 51.4, 11.1, 0.01.

OH
3

(E)-Hex-2-en-5-yn-1-ol (3). Transfer 6 ml of 1.0M THF solution of LiAlH
4
into a flame-dried flask and
cooled to 0
⁰
C. Cannulate alcohol 2 (1g, 6.01mmol) in 3.3 ml ether into the solution and let the reaction
slowly warm up to r.t. and then reflux overnight. Work up with NH
4
Cl aqueous solution and extract with
ether. After removing the solvent the mixture was purified on a silica column with 10% EtOAc/hexanes

55

to afford the hydrogenated product, which was then dissolved in MeOH and 300 mg Na
2
CO
3
was added.
The slurry mixture was stirred overnight. Filtrate the mixture and after MeOH removal the mixture was
purified on a silica column with 10% EtOAc/hexanes to afford the product (0.4g, 69% for two steps).
1
H
NMR (400 MHz, CDCl
3
) δ
H
5.96 (m, 1H), 5.72 (m, 1H), 4.16 (m, 2H), 2.98 (m, 2H), 2.12 (t, J = 2.8 Hz,
1H).
13
C NMR (400 MHz, CDCl
3
) δ
C
131.3, 125.8, 70.6, 66.0, 63.2, 21.5.

OH
4

(E)-undeca-2-en-5,8-diyn-1-ol (4). Alcohol 3 (0.4g, 4.2 mmol) and 1-bromo-2-pentyne (2.1 ml, 20.5
mmol) were mixed and dissolved in 10ml anhydrous DMF. CuI (4g, 31.4mmol), NaI (3.1g, 31.4mmol)
and K
2
CO
3
(2.9g, 31.4mmol) were added and the solution was stirred violently overnight. Work up with
NH
4
Cl aqueous solution and extract with ether. After removing the solvent the mixture was purified on a
silica column with 10% EtOAc/hexanes to afford the product (0.61 g, 90%).
1
H NMR (600 MHz, CDCl
3
)
δ
H
. 5.90 (m, 1H), 5.69 (m, 1H), 4.14 (m, 2H), 3.14 (m, 2H), 2.95 (m, 2H), 2.17 (m, 2H), 1.37 (m, 1H),
1.12 (t, J = 7.8 Hz, 3H).
13
C NMR (600 MHz, CDCl
3
) δ
C
130.8, 126.8, 82.2, 73.6, 63.4, 21.9, 14.0, 12.5,
9.9.
OH
5

(2E,5Z,8Z)-undeca-2,5,8-trien-1-ol (5). Alcohol 4 (0.3 g, 1.85 mmol) was dissolved in 5ml EtOAc and 1
drop of quinolone was added. After adding 100 mg Lindlar catalyst the mixture was stirred violently
under H
2
atmosphere and the reaction was monitored by TLC. After the starting material disappeared, the
suspension was filtered and EtOAc was removed under vacuum. The mixture was purified on a silica
column with 8% EtOAc/hexanes to afford the hydrogenated product (0.17g, 56%).
1
H NMR (400 MHz,

56

CDCl
3
) δ
H
5.68 (m, 2H), 5.45-5.25 (m, 4H), 4.10 (m, 2H), 2.84-2.76 (m, 4H), 2.07 (m, 2H), 0.97 (t, J =
7.5 Hz, 3H).
13
C NMR (400 MHz, CDCl
3
) δ
C
132.2, 131.2, 129.5, 127.1, 126.9, 63.8, 30.1, 25.6, 20.7,
14.4.
OH
O
6

((2S,3S)-3-((2Z,5Z)-Octa-2,5-dienyl)oxiran-2-yl)methanol (6). Flame-dry a 10 ml flask with 40 mg 40
Å molecular sieve powder in it and add 1 ml of DCM. Cool down to -20⁰C and add (+)-Diethyl tartrate
(0.01 ml), Ti(O
i
Pr)
4
(0.02 ml) and t-BuOOH (0.28 ml) sequentially and stir for 1 hour. Cannulate allyl
alcohol 5 (0.16g, 0.96 mmol) dissolved in 0.5 ml DCM into the solution and allow the reaction to proceed
for 3 hours. Filter the mixture and after DCM removal the mixture was purified on a silica column with
25% EtOAc/hexanes to afford the product (0.15g, 87%).
1
H NMR (400 MHz, CDCl
3
) δ
H
5.60-5.25 (m,
4H), 4.06 (t, J = 6.8 Hz, 1H), 3.90 (d, J = 12.4 Hz, 1H), 3.62 (d, J = 12.4 Hz, 1H), 2.95 (m, 2H), 2.75 (m,
2H), 1.60 (m, 4H), 0.95 (t, J = 7.5 Hz, 3H).
13
C NMR (400 MHz, CDCl
3
) δ
C
132.1, 131.6, 128.9, 123.5,
64.5, 61.8, 58.5, 30.8, 25.7, 19.3, 13.9.
O
O
7

(2R,3S)-3-((2Z,5Z)-Octa-2,5-dienyl)oxirane-2-carbaldehyde (7). Alcohol 6 (80 mg, 0.44 mmol) was
dissolved in 8 ml DCM, then 380 mg Dess-Martin periodinane and 380 mg NaHCO
3
were added and the
slurry solution was stirred for 1 hour. Work up the reaction with 1:1 NaHCO
3
/Na
2
S
2
O
3
saturate solution
and after DCM removal, the mixture was purified on a silica column with 20% EtOAc/hexanes to afford

57

the aldehyde (70 mg, 90%).
1
H NMR (400 MHz, CDCl
3
) δ
H
9.03 (d, J = 6.4 Hz, 1H), 5.62-5.35 (m, 4H),
3.28 (td, J = 5.2 and 2.0 Hz, 1H), 3.18 (dd, J = 6.4 and 2.0 Hz, 1H), 2.80 (m, 2H), 2.52-2.35 (m, 4H), 0.97
(t, J = 7.6 Hz, 3H).
13
C NMR (400 MHz, CDCl
3
) δ
C
199.6, 132.3, 128.6, 126.3, 121.9, 59.3, 56.9, 31.3,
26.9, 20.7, 14.5.
O
O
8

(2E,4E)-5-((2S,3S)-3-((2Z,5Z)-Octa-2,5-dienyl)oxiran-2-yl)penta-2,4-dienal (8). Aldehyde 7 (150 mg,
0.83 mmol) was dissolved in 4 ml toluene and (triphenylphosphoranylidene)acetaldehyde (510 mg, 1.68
mmol) was added and the solution was stirred at 90⁰C for 2 hours. Remove toluene under vacuum and
purify the mixture on a silica column with 20% EtOAc/hexanes to afford the product (120 mg, 63%).
1
H
NMR (400 MHz, CDCl
3
) δ
H
9.57 (d, J = 7.6 Hz, 1H), 7.08 (ddd, J = 10.8, 15.2 and 0.8 Hz, 1H), 6.63 (dd,
J = 10.8 and 15.2 Hz, 1H), 6.17 (dd, J = 8.0 and 15.2 Hz, 1H), 5.98 (dd, J = 7.2 and 15.2 Hz, 1H), 5.55 (m,
1H), 5.41 (m, 2H), 5.29 (m, 1H), 3.26 (dd, J = 2.0 and 7.6 Hz, 1H), 2.97 (td, J = 5.6 and 2.4 Hz, 1H),
2.53-2.33 (m, 2H), 2.07 (m, 2H), 0.97 (t, J = 7.5 Hz, 3H).
13
C NMR (400 MHz, CDCl
3
) δ
C
193.7, 150.1,
141.3, 132.5, 132.3, 132.0, 131.1, 126.6, 122.8, 60.8, 56.9, 31.7, 29.7, 22.8, 14.3.
O
O
O
THPO
9

4-Methyl-1-(6-(tetrahydro-2H-pyran-2-yloxy)hex-3-ynyl)-2,6,7-trioxa-bicyclo[2.2.2]octane (9).
Dissolve acetylene (0.6 g, 3.3 mmol, preparation in Chapter 2) in 5 ml anhydrous THF and cool to -78⁰C.
Add 1.46 ml n-BuLi (2.5M solution in THF) dropwise and warm to -35⁰C and stir for 30 minutes. THP
protected 2-iodoethanol (1.1 g, 4.3 mmol) dissolved in 0.8 ml THF and 2.3 ml HMPA was then

58

cannulated into the solution mentioned above and stir at -20⁰C to r.t. overnight. Work up with NH
4
Cl
aqueous solution and extract with ether. After removing the solvent the mixture was purified on a silica
column with 20% EtOAc/hexanes to afford the product (0.95 g, 95%).
1
H NMR (400 MHz, C
6
D
6
) δ
H

4.52 (m, 1H), 3.90 (m 2H), 3.77 (m, 2H), 3.50 (s, 6H), 2.67 (m, 2H), 2.40 (m, 2H), 2.30 (m, 2H), 1.66 (s,
3H), 1.50 (m, 2H), 1.35-1.22 (m, 4H).
13
C NMR (400 MHz, C
6
D
6
) δ
C
108.6, 98.4, 81.0, 77.4, 72.5, 66.4,
61.5, 37.4, 31.9, 30.9, 29.9, 25.9, 20.8, 19.5, 13.9.

Methyl 7-hydroxyhept-4-ynoate (10). The ortho ester 9 (0.35 g, 1.1 mmol) was dissolved in 20 ml
MeOH and p-toluenesulfonic acid monohydrate (0.02 g, 0.1 mmol) was added. Stir at r.t. for 1 hour. Add
1 ml of water and stir for another 30 minutes. Remove the solvent under vacuum. Dissolve the dried
residue in 30 ml anhydrous MeOH and 8 drops of Et
3
N was added and the solution was stirred overnight
at room temperature. Remove the solvent under vacuum and purify the mixture on a silica column with 25%
EtOAc/hexanes to afford the product (0.13g, 78% for two steps).
1
H NMR (400 MHz, CDCl
3
) δ
H
3.69 (s,
3H), 3.66 (t, J = 6.0 Hz, 2H), 2.54-2.44 (m, 4H), 2.39 (m, 2H).
13
C NMR (400 MHz, CDCl
3
) δ
C
172.75,
80.73, 77.85, 61.38, 51.96, 33.86, 23.31, 14.97.

(Z)-Methyl 7-hydroxyhept-4-enoate (11). Acetylene 10 (100 mg, 0.64 mmol) was dissolved in 5ml
EtOAc and 1 drop of quinolone was added. After adding 50 mg Lindlar catalyst the mixture was stirred
violently under H
2
atmosphere and the reaction was monitored by
1
H NMR (as the product has exactly the

59

same R
f
value as the starting material). After the starting material disappeared, the suspension was
filtered and EtOAc was removed under vacuum. The mixture was purified on a silica column with 20%
EtOAc/hexanes to afford the hydrogenated product (90 mg, 90%).
1
H NMR (400 MHz, CDCl
3
) δ
H
5.55-
5.41 (m, 2H), 3.70-3.55 (s and m, 5H), 2.43-2.30 (m, 4H), 1.60 (m, 2H).
13
C NMR (400 MHz, CDCl
3
) δ
C

173.72, 130.80, 172.21, 62.19, 51.67, 33.86, 30.84, 22.82.

(Z)-Methyl 7-(tosyloxy)hept-4-enoate (14). Alcohol 11 (50 mg, 0.32 mmol) was dissolved in 0.7 ml
DCM. Et
3
N (0.16 mmol) and p-toluenesulfonyl chloride (90 mg) were added sequentially and the reaction
was allowed to stir overnight at room temperature. After removing the solvent under vacuum, the mixture
was purified on a silica column with 10% EtOAc/hexanes to afford the tosylate (94 mg, 95%).
1
H NMR
(400 MHz, CDCl
3
) δ
H
7.80 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H), 5.45 (m, 1H), 5.30 (m, 1H),
4.02 (t, J = 6.8 Hz, 2H), 3.65 (s, 3H), 2.45 (s, 3H), 2.42 (m, 2H), 2.35-2.27 (m, 4H).
13
C NMR (400 MHz,
CDCl
3
) δ
C
173.47, 144.87, 133.33, 131.44, 129.96, 128.06, 124.72, 69.72, 51.73, 33.84, 27.19, 22.94,
21.79.

(Z)-Methyl 7-iodohept-4-enoate (12). Tosylate 14 (94 mg, 0.30 mmol) was dissolved in 4 ml anhydrous
acetone. NaI (168 mg, 1.12 mmol) and K
2
CO
3
(120 mg, 0.87 mmol) were added and the reaction was
allowed to reflux for 2 hours. Work up the reaction with saturated Na
2
S
2
O
3
aqueous solution and extract
with ether. After removing the solvent under vacuum the mixture was purified on a silica column with 5%

60

EtOAc/hexanes to afford the product (68 mg, 85%).
1
H NMR (400 MHz, CDCl
3
) δ
H
5.52 (m, 1H), 5.39
(m, 1H), 3.67 (s, 3H), 3.14 (t, J = 7.2 Hz, 2H), 2.65 (m, 2H), 2.35 (m, 4H).
13
C NMR (400 MHz, CDCl
3
)
 C
173.5, 130.2, 129.5, 51.7, 33.9, 31.4, 23.1, 5.3.

(Z)-(7-Methoxy-7-oxohept-3-en-1-yl)triphenylphosphonium iodide (13). Dissolve iodide 12 (120 mg,
0.45 mmol) in 1 ml toluene and add PPh
3
(117 mg, 0.45 mmol) and reflux overnight. After removing
toluene under vacuum, 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 for another 2~4 times until no PPh
3
can be detected in the supernatant by TLC.
Remove the solvent and the product was afforded as very thick colorless oil (140 mg, 60%).
1
H NMR
(400 MHz, CD
3
OD) δ
H
7.95-7.72 (m, 15H), 5.54 (m, 1H), 5.46 (m, 1H), 3.62 (s, 3H), 3.50 (m, 2H), 2.48
(m, 2H), 2.36 (m, 2H), 2.42 (m, 2H).
13
C NMR (400 MHz, CD
3
OD) δ
C
174.0, 136.4, 136.3, 134.9, 134.8,
132.1, 131.6, 131.5, 127.4, 120.2, 119.3, 54.8, 26.4, 21.5, 21.3, 14.6.

(4Z,7Z,9E,11E)-methyl 12-((2S,3S)-3-((2Z,5Z)-octa-2,5-dien-1-yl)oxiran-2-yl)dodeca-4,7,9,11-
tetraenoate (1). Phosphonium salt 13 (91 mg, 0.17 mmol) was dried under vacuum and P
2
O
5
overnight.

61

Under Argon atmosphere, the phosphonium salt was dissolved in 0.6 anhydrous THF and cooled to -78ºC.
NaHMDS (0.17 ml, 0.17 mmol) in 1.0 M THF solution was added dropwise and the reaction was stirred
for 30 minutes and warm to 0ºC for 20 minutes, during which a bright orange color solution was
produced. Cool to -78ºC again and cannulate in aldehyde 8 (20 mg, 0.086 mmol) dissolved in 0.8 ml
anhydrous THF and let the reaction stir for 1 hour. Quickly remove the solvent under vacuum and
purified the mixture on a silica column with 5% EtOAc/5% Et
3
N/hexanes eluent to afford the product (10
mg, 33%). Store the product at -78ºC in benzene with 1% Et
3
N. UV: λ
max
= 280nm (in hexane).
1
H NMR
(600 MHz, C
6
D
6
) δ
H
6.53 (dd, J = 11.2 and 14.8 Hz, 1H), 6.38 (dd, J = 11.2 and 15.6 Hz, 1H), 6.10 (dd, J
= 10.8 and 14.8 Hz, 1H), 6.01 (dd, J = 11.2 and 11.2 Hz, 1H), 5.55-5.25 (m, 8H), 3.33 (s, 3H), 3.00 (dd, J
= 7.8 and 1.8 Hz, 1H), 2.89 (m, 2H), 2.75 (m, 2H), 2.72 (td, J = 5.4 and 1.8 Hz, 1H), 2.33-2.18 (m, 4H),
2.12 (t, J = 7.2 Hz, 2H), 2.00 (m, 2H), 0.90 (t, J = 7.5 Hz, 3H).
13
C NMR (600 MHz, C
6
D
6
) δ
C
172.7,
134.3, 132.5, 132.4, 132.3, 132.2, 131.4, 131.3, 131.1, 129.2, 128.9, 128.8, 124.0, 65.9, 60.1, 57.7, 65.9,
60.1, 57.7, 33.9, 26.5, 26.0, 23.1, 20.9, 15.6, 14.5.

3.5. References
1. Goodarzi, K.; Goodarzi, M.; Tager, A. M.; Luster, A. D.; and von Andrian, U. H. Leukotriene B
4

and BLT1 control cytotoxic effector T cell recruitment to inflamed tissues. Nat. Immunol. 2003, 4,
965-973.
2. Ott, V. L.; Cambier, J. C.; Kappler, J.; Marrack, P.; and Swanson, B. J. Mast cell–dependent
migration of effector CD8
+
T cells through production of leukotriene B
4
. Nat. Immunol. 2003, 4,
974-981.
3. Tager, A. M.; Bromley, S. K.; Medoff, B. D.; Islam, S. A.; Bercury, S. D.; Friedrich, E. B.;
Carafone, A. D.; Gerszten, R. E.; Luster, A. D. Leukotriene B
4
receptor BLT1 mediates early
effector T cell recruitment. Nat. Immunol. 2003, 4, 982-990.

62

4. Fabre, J. E.; Goulet, J. L.; Riche, E.; Nguyen, M.; Coggins, K.; Offenbacher, S.; and Koller, B. H.
Transcellular biosynthesis contributes to the production of leukotrienes during inflammatory
responses in vivo. J. Clin. Invest. 2002, 109, 1373-1380
5. Haeggstro¨m, J. Z. Leukotriene A
4
Hydrolase/Aminopeptidase, the Gatekeeper of Chemotactic
Leukotriene B
4
Biosynthesis. J Bio Chem 2004, 279, 50639-50642.
6. Orning, L.; Gierse, J. K.; Fitzpatrick, F. A. The bifunctional enzyme leukotriene A
4
hydrolase is
an arginine aminopeptidase of high efficiency and specificity. J. Biol. Chem. 1994, 269, 11269-
11273.
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. Kosaki, Y.; Ogawa, N.; Kobayashi, Y. Total synthesis of resolvin E2. Tetrahedron Letters 2010,
51, 1856-1859.
9. Gao, Y.; Klunder, J. M.; Hanson, R.M.; Ko, S. Y.; Masamune, H.; Sharpless, K. B. Catalytic
asymmetric epoxidation and kinetic resolution: modified procedures including in situ
derivatization. J. Am. Chem. Soc. 1987, 109, 5765-5780.
10. Altundas, R.; Mahadevan, A.; Razdan, R. K. A synthetic route to anandamide analogues carrying
a substituent at the terminal carbon and an acetylene group in the end pentyl chain. Tetrhedron
Letters 2004, 45, 5449-5491.
11. Sydnes, L. K.; Holmelid, B.; Kvernenes, O. H.; Valdersnes, S.; Hodne, M.; Boman, K.
Stereospecific synthesis of allylic and homoallylic alcohols from functionalized propargylic
alcohols. Archive for organic chemistry 2008, 14, 242.
12. Jin, J.; Weinreb, S. M. Application of a stereospecific intramolecular allenylsilane imino ene
reaction to dnantioselective total synthesis of the 5,11-methanomorphanthridine class of
amaryllidaceae alkaloids. J. Am. Chem. Soc. 1997, 119, 5773-5784.

63

13. S. D. Meyer; S. L. Schreiber. Acceleration of the Dess-Martin Oxidation by Water. J. Org. Chem.,
1994, 59, 7549-7552.
14. Yang, R. Total synthesis of novel anti-inflammatory lipid mediators. Ph.D. dissertation 2006.
15. Corey, E. J.; Raju, N. A new general synthetic route to bridged carboxylic ortho esters.
Tetrahedron Letters 1983, 24, 5571.
16. Rector, C. L.; Murphy, R. C. Determination of leukotriene A
4
stabilization by S100A8/A9
proteins using mass spectrometry. J Lipid Res. 2009, 50, 2064-2071.
17. Dalli, J.; Zhu, M.; Vlasenko, N. A.; Deng, B.; Haeggström, J. Z.; Petasis, N. A.; Serhan, C. N.
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 Journal,
2013, 27, 2573-2583.

64

Chapter 4. Total Synthesis of Deuterium Labeled DHA and Hydroxy-DHAs

4.1. Introduction
The enzymatic pathways that principally process the available ω-3 and ω-6 PUFAs into bioactive
metabolites are the cyclooxygenases (COXs) and the lipoxygenases (LOs). COXs are heme-containing
enzymes with peroxidase activity that play a primary role in prostanoid synthesis
1
, whereas the LOs are
non–heme iron dioxygenases that insert molecular oxygen with regional specificity into PUFAs and
generate more down-stream biologically active products
2
. Docosahexaenoic acid has been known to be
enzymatically converted by platelets, basophils, and liver microsomes into metabolites containing
conjugated dienes with allylic hydroxyl groups, namely hydroxydocosahexaenoic acids (HDHA), and
they can function as key signaling molecules, or intermediates to generate other signaling molecules
3
. For
example, DHA was reported to be oxidized to form 11- and 14-hydroxydocosahexaenoic acid (HDHA)
in platelets, 4- and 7-HDHA in basophils and 17-, 16-, 14-, 13-, 11-, l0-, 8-, and 7-HDHA in liver
4
.
The biosynthetic pathway of HDHAs involves enzymatic oxidation of DHA to conjugated dienes with
allylic hydroperoxy groups, which are reduced in vivo by peroxidases to allylic alcohols
5-8
(shown in
Figure 13, using 7S-HDHA as an example). It is of great importance to identify these metabolites in
biological fluids and study their functions and metabolic pathways and synthesis of a series of authentic
standard HDHAs as biomarkers is necessary
9
.
On the other hand, deuterium labeling greatly helps confirming the assignment of MS/MS ions and the
fragmentation mechanisms for structure elucidation and identification the DHA-derived compounds,
combing with matching of LC retention times and UV spectra
10-11
. For example, with help of deuterium
labeled HDHAs, a series of HDHAs have been identified in human neutrophils and blood, trout head-
kidney, and stroke-injury murine, and these HDHAs include 20-, 14-, 7- and 4-HDHA
10
.

65

(S)
COOH
OOH
COOH
(S)
COOH
OH
5-LOX
enzymatic reduction

Figure 13. Biosynthesis of 7S-HDHA

This chapter details the total synthesis of deuterium labeled DHA (6) and 20R-HDHA (1), 20S-HDHA (2),
14S-HDHA (3), 7S-HDHA (4) and 4S-HDHA (5) (Figure 14). As LOs specifically insert a hydroperoxy
group with S configuration, the synthesis targets 2-5 were designed as S-HDHA. Other isomer, such as R-
HDHA (1), can also be formed when certain enzymes such as acetylated COX-2 is involved in the
oxidation process (detailed in 1.3).
D
3
C
(R)
COOH
HO
1
D
3
C
(S)
COOH
HO
2
D
3
C
(S)
COOH
OH
3
(S)
COOH
OH
D
3
C
4
D
3
C
(S)
HO COOH
5
COOH
D
3
C
6

Figure 14. Deuterium labeled DHA and hydroxy-DHAs prepared by total synthesis
4.2. Results

4.2.1. Total Synthesis of 22d
3
, 20R-HDHA (1) and 22d
3
, 20S-HDHA (2)

66

Retrosynthetic analysis of 1 and 2 is shown in Figure 15. Due to relatively higher instability and difficulty
to construct, the Z-double bond in the diene moiety is the last C-C bond formed from a coupling reaction
and a subsequent Z-selective hydrogenation. The stereogenic center-contained building block has its
chirality from an enantiomerically pure epoxide and the stereo configuration is retained through a series
of conversions. The other Z-double bond rich building block is constructed via repeated couling-
hydrogenation-deprotection cycles. The terminal deuterium methyl group has its origin from the
deuterated methyl Grignard reagent, which regioselectively opens the expoxide ring in glycidols.
D
3
C
COOH
1 and 2
HO
D
3
C
COOMe
HO
D
3
C
OTBDPS
I
COOMe
COOMe
TMS
Br
O
OH
D
3
C Mg
I

Figure 15. Retrosynthetic analysis of 22d
3
, 20R-HDHA (1) and 22d
3
, 20S-HDHA (2)

Total synthetic of 1 and 2 is shown in Scheme 12. The TBS-protected (S)-glycidol underwent a ring-
opening by deuterated methyl Grignard reagent in the presence of Cu (I) bromide
12
. After protection of
the secondary alcohol in 23 with a more bulky tert-butyldiphenyl silyl group, the primary hydroxyl group
was selectively deprotected, and a subsequent Swern oxidation and Takai olefination yielded the vinyl
iodide product, whose silyl ester protection group was removed to afford 13. Starting from (R)-glycidol,
the vinyl iodide 14, enantiomer of 13, was prepared following the same synthetic route. On the other hand,

67

compound 25, which was a key building unit and used repeatedly in this chapter, was produced after
coupling TMS propargyl bromide with propargyl alcohol and a subsequent bromination reaction
13
. Two
cycles of Cu(I) mediated coupling
14
-Lindlar hydrogenation
15
-TMS group removal were carried out to
afford the terminal alkyne 15 for the final coupling step. It is worth mentioning that the TMS group
protected the terminal alkyne from been reduced under Lindlar hydrogenation condition, but the
protection was weak as the steric hindrance TMS provided was small, which decided that very careful
monitoring of the hydrogenation process was needed to avoid excessive over-hydrogenation. A more
bulky protection group than TMS, such as tert-butyldiisopropylsilyl ether group, was not successful in
this case as although it increases the steric selectivity, its removal afterwards requires harsher conditions
which harm the delicate ene-yne moiety.
O
(S)
OH
D
3
C OTBS
OH
D
3
C OH
OTBDPS
D
3
C
OH
I
1) TBS-Cl, Imidazole,
DMAP,rt, CH
2
Cl
2
,97%
Br
TMS
Br
TMS
1)
D
3
C
COOMe
HO
D
3
C
(R)
COOH
HO
O
(R)
OH
D
3
C
OH
I
D
3
C
COOMe
HO
D
3
C
(S)
COOH
HO
2
7
8
13
15
14
17
23
24
25
13 + 15
2) CD
3
MgI, CuBr.Me
2
S,
THF, -78
o
C, 89%
1) TBDPS-Cl, Imidazole,
DMAP,rt, CH
2
Cl
2
,98%
2) CSA, MeOH/CH
2
Cl
2
,
rt.,86%
1) DMSO, (COCl)
2
, Et
3
N,
-78
o
C, CH
2
Cl
2
,98%
2) CHI
3
, CrCl
2
, 0
o
C, THF, 58%
3) TBAF, THF, rt, 88%
COOMe
COOMe CuI, NaI, K
2
CO
3
, 88%
,
OH
2) PPh
3
, NBS, 0
o
C,
CH
2
Cl
2
,60%
1)
CuI, NaI, K
2
CO
3
, 85%
,
COOMe
3) MeOH, Na
2
CO
3
,rt.,98%
2) H
2
/Lindlar catalyst, quinoline,
rt, EtOAc, 65%
1) 25, CuI, NaI, K
2
CO
3
, 85%
3) MeOH, Na
2
CO
3
,rt.,98%
2) H
2
/Lindlar catalyst, quinoline,
rt, EtOAc, 65%
Pd(PPh
3
)
4,
CuI, rt, C
6
H
6,
88%
1) Zn(Cu/Ag), rt, MeOH/H
2
O,77%
2) NaOH, rt, MeOH/H
2
O, 100%
14 + 15
Pd(PPh
3
)
4,
CuI, rt, C
6
H
6,
88%
1) Zn(Cu/Ag), rt, MeOH/H
2
O,77%
2) NaOH, rt, MeOH/H
2
O, 100%
1

Scheme 12. Total synthesis of 22d
3
, 20R-HDHA (1) and 22d
3
, 20S-HDHA (2)

68

With 13 and 15 prepared, a Sonogarshira coupling
16
was carried out to construct the backbone of 1. A
subsequent hydrogenation using zinc achieved high Z-selectivity
17-18
and protected the delicate conjugated
diene from further over-reduction. The methyl ester was hydrolyzed in the presence of NaOH in methanol
and water to afford 1, and 2 was produced via the same route from coupling of 14 and 15. It is worth
mentioning that the hydroxyl group in 13 and 14 were deprotected before the Sonogarshira coupling,
instead of after the coupling which was more commonly adopted for synthesis of other lipid mediators in
this thesis, for the reason that the 13-cis double bond can easily migrate to a conjugated position to the
ene-yne formed from Sonogashira coupling, when conventional silyl ether deprotecting reagents such as
tert-butylammonium floride was used.
4.2.2. Total Synthesis of 22d
3
, 14S-HDHA (3)
Retrosynthetic analysis of 3 is similar with 1 and 2 and shown in Figure 16. The deuterated methyl group
in 3 is from 2,2,2-trideuterium iodoethane and introduced into the building block via alkynylation.
COOMe
TMS
Br
D
3
C
COOH
OH
3
D
3
C
COOMe
OH
COOMe
D
3
C
OTBDPS
I
D
3
C
I
OTBS TMS
O
OTBS

Figure 16. Retrosynthetic analysis of 22d
3
, 14S-HDHA (3)

69

Total synthesis of 3 (shown in Scheme 13) starts from synthesis of 22, which is a key building piece for
22d
3
, 14S-HDHA (3), 22d
3
, 14S-HDHA (4), 22d
3
, 14S-HDHA (5) and 22d
3
-DHA (6). After protection of
the primary hydroxyl group of propargyl alcohol, the terminal alkyne proton was extracted by n-butyl
lithium in the presence of HMPA and the carbon anion generated in situ substituted the iodine atom in
2,2,2-trideuterium iodoethane. TBS-protected (R)-glycidol was opened by lithium TMS-acetylene, and
after protecting the secondary hydroxyl group as a bulky tert-butyldiphenylsilyl ether, the trimethylsilyl
group was cleaved in the presence of sodium carbonate in methanol. The unmasked alkyne was coupled
to the propargyl bromide 22 in a key coupling reaction mediated by Cu (I) and sodium iodide. The
primary alcohol was selectively deprotected and the two triple bonds were reduced to two double bonds
simultaneously. The alcohol was then oxidized to aldehyde and finally converted to vinyl iodide 16 after
Takai olefination and silyl ether removal.
n-BuLi, THF, HMPA,
-78
o
C, 89% I D
3
C
D
3
C
Br
OTBDPS
D
3
C
OH
OH
D
3
C
I
D
3
C
COOMe
OH
D
3
C
COOH
OH
3
9
16
22
26
16 + 17
1)
OTBS
,
2) TBAF, THF, rt, 88%
3) PPh
3
, NBS, 0
o
C,
CH
2
Cl
2
,40%
TMS
,n-BuLi, BF
3
.
Et
2
O,
2) TBDPS-Cl, Imidazole, DMAP, rt.,89%
3) MeOH, Na
2
CO
3
,rt.,98%
4) 22,CuI, NaI, K
2
CO
3
, 88%
1)
-78
o
C, THF,84%
O
OTBS
17
COOMe
5) H
2
/Lindlar catalyst, quinoline,
rt, EtOAc, 65%
6) CSA, MeOH/CH
2
Cl
2
,
rt.,86%
1) DMSO, (COCl)
2
, Et
3
N,
-78
o
C, CH
2
Cl
2
,98%
2) CHI
3
, CrCl
2
, 0
o
C, THF, 58%
3) TBAF, THF, rt, 80%
Pd(PPh
3
)
4,
CuI, rt, C
6
H
6,
80%
1) Zn(Cu/Ag), rt, MeOH/H
2
O,67%
2) NaOH, rt, MeOH/H
2
O, 100%

Scheme 13. Total Synthesis of 22d
3
, 14S-HDHA (3)

70

Building block 16 was then coupled with 17, which was prepared as in 5.2.1, and subsequent
hydrogenation and hydrolysis afford 22d
3
, 14S-HDHA (3) as the final product.
4.2.3. Total Synthesis of 22d
3
, 7S-HDHA (4)
Retrosynthetic analysis of 4 and introduction of deuteated methyl group into 4 are similar with 4.2.2 and
shown in Figure 17. The Z-double bond rich building block wass constructed via repeated coupling-
hydrogenation-deprotection cycles.
COOH
OH
D
3
C
4
COOMe
OH
D
3
C
COOMe
OTBDPS
I
D
3
C
COOMe
O
OH
D
3
C
I
OTBS
TMS
TMS
Br

Figure 17. Retrosynthetic analysis of 22d
3
, 7S-HDHA (4)

With the preparation of 22 as in 4.2.2, two cycles of Cu(I) mediated coupling-Lindlar hydrogenation-
TMS group removal were carried out to afford the terminal alkyne 18 for the final coupling step (Scheme
14). It is worth mentioning that many intermediates involved are volatile and unstable, the six steps in the
two cycles were performed as quickly as possible and purifications did not need to be perfect to
compensate for the low isolated yields. Compound 19 was obtained from deprotection of vinyl iodide 22
prepared in Chapter 2, and then coupled to 18 under Sonogashira condition. Subsequent hydrogenation
and hydrolysis afford 22d
3
, 7S-HDHA (4) as the final product.

71

CD
3
TMS
TMS
Br
I
COOMe
OH
COOMe
OH
D
3
C
COOMe
OH
D
3
C
4
10
18
19
18+19
D
3
C
Br
22
,CuI, NaI, K
2
CO
3
, 78% 1)
2) H
2
/Lindlar catalyst, quinoline, rt, EtOAc, 55%
3) MeOH, Na
2
CO
3
,rt.,98%
,CuI, NaI, K
2
CO
3
, 68% 4)
5) H
2
/Lindlar catalyst, quinoline, rt, EtOAc, 44%
6) MeOH, Na
2
CO
3
,rt.,98%
I
COOMe
OTBDPS
22 (Chapter 2)
TBAF, THF, rt, 86%
Pd(PPh
3
)
4,
CuI, rt, C
6
H
6,
80%
1) Zn(Cu/Ag), rt, MeOH/H
2
O,67%
2) NaOH, rt, MeOH/H
2
O, 100%

Scheme 14. Total Synthesis of 22d
3
, 7S-HDHA (4)

4.2.4. Total Synthesis of 22d
3
, 4S-HDHA (5)
Retrosynthetic analysis of 5 and introduction of deuterated methyl group into 5 are similar with 4.2.3 and
shown in Figure 18. The Z-double bond rich building block wass constructed via repeated couling-
hydrogenation-deprotection cycles. The stereogenic center-contained building block has its chirality from
an enantiomerically pure lactone and the stereo configuration is retained through a series of conversions.

72

D
3
C
I
OTBS
TMS
D
3
C
HO COOH
5
D
3
C
O
O
O
O
I
D
3
C
O
O
O
OH
TMS
Br

Figure 18. Retrosynthetic analysis of 22d
3
, 4S-HDHA (5)

With 22 prepared as in 4.2.2, two cycles of Cu(I) mediated coupling-Lindlar hydrogenation-TMS group
removal were carried out to afford the terminal alkyne 21 for the final coupling step (shown in Scheme
15). On the other hand, the starting material five-membered lactone was opened in acidic condition and
the carboxylic acids were esterified. After selective reduction of the ester group adjacent to the secondary
alcohol by sodium boron hydride
12
, the 1, 2-diol was protected by two TBS groups. The primary alcohol
was then selectively deprotected and was then oxidized to aldehyde and finally converted to vinyl iodide
20 after Takai olefination and silylether removal. It is worth mentioning that after silylether removal with
tert-butylammonium floride, a five-membered ring lactone was formed simultaneously.

73

TMS
5
20+21
D
3
C
Br
22
,CuI, NaI, K
2
CO
3
, 78% 1)
2) H
2
/Lindlar catalyst, quinoline, rt, EtOAc, 55%
3) MeOH, Na
2
CO
3
,rt.,98%
25,CuI, NaI, K
2
CO
3
, 77% 4)
5) H
2
/Lindlar catalyst, quinoline, rt, EtOAc, 54%
6) MeOH, Na
2
CO
3
,rt.,98%
1) Pd(PPh
3
)
4,
CuI, rt, C
6
H
6,
80%
2) Zn(Cu/Ag), rt, MeOH/H
2
O,37%
1) NaOH, rt, MeOH/H
2
O, 100%
CD
3
21
I
O
O
O
OH O
O
20
1) HCl, MeOH, reflux, 98%
2) NaBH
4
, MeOH, 87%
3) TBS-Cl, Imidazole, DMAP, rt.,86%
OTBS
O
O
HO
1) DMSO, (COCl)
2
, Et
3
N,
-78
o
C, CH
2
Cl
2
,98%
2) CHI
3
, CrCl
2
, 0
o
C, THF, 58%
3) TBAF, THF, rt, 88%
D
3
C
O
O
11
4) CSA, MeOH/CH
2
Cl
2
, 0
o
C, 68%
D
3
C
HO
COONa

Scheme 15. Total Synthesis of 22d
3
, 4S-HDHA (5)

After 20 and 21 were prepared they were then coupled under Sonogarshira condition to form 11. However
we noticed that when 11 was hydrogenated under the condition which worked fine for the other cases
mentioned in 4.2.1, 4.2.2 and 4.2.3, the reaction failed to give the hydrogenated lactone or 4-hydroxyl
methyl ester, probably due to the labile nature of the C-O bond at C
4
. Alternatively, we hydrolyzed the
lactone under basic condition and the ene-yne moiety in the Na salt was successfully hydrogenated,
however with a yield lower than average.
4.2.5. Total Synthesis of 22d
3
-DHA (6)
Retrosynthetic analysis of 6 and introduction of deuteated methyl group into 6 are similar with 4.2.3 and
shown in Figure 19.

74

D
3
C
I
OTBS
TMS
Br
COOH
D
3
C
6
COOMe
D
3
C
COOMe
D
3
C
Br
COOMe

Figure 19. Retrosynthetic analysis of 22d
3
-DHA(6)

With building block 15 prepared as in 4.2.1 and 22 prepared as in 4.2.2, 15 and 22 are coupled using the
Cu (I) mediated coupling reaction. Subsequent hydrogenation using Lindlar catalyst and hydrolysis afford
22d
3
-DHA (6) as the final product.
15
COOMe
D
3
C
Br
22
15+22
2) NaOH, rt, MeOH/H
2
O, 100%
CuI, NaI, K
2
CO
3
, 88%
COOMe
D
3
C
1) H
2
/Lindlar catalyst, quinoline, rt, EtOAc, 55%
COOH
D
3
C

Scheme 16. Total Synthesis of 22d
3
-DHA (6)

4.3. Conclusion

75

The total synthesis of C
22
deuterated DHA and five C
22
deuterated monohydroxyl-DHAs has been
successfully achieved. This work of total synthesis establishes facile and feasible synthetic routes to DHA
and monohydroxy-DHAs, and also provides a series of isotope-labeled compounds which can assist
exploration of metabolic pathways of DHA. These synthetic routes can also be used to prepare DHA and
monohydroxy DHAs loaded with or conjugated to imaging atoms or groups such as fluorescent
chromophore or radioactive isotopes, which can assist uncovering of cellular receptors involved in the
DHA signaling pathways.
4.4. Experimental Procedures
All reactions, unless otherwise noted, were carried in flame dried flasks under argon atmosphere. “Dried
and concentrated” refers to removal of residual water with anhydrous MgSO
4
, followed by evaporation of
the solvent on the rotary evaporator. THF was freshly distilled from sodium-benzophenone, benzene and
dichloromethane from CaH
2
and anhydrous DMF, EtOH, and MeOH were purchased from commercial
sources.
1
H and
13
C NMR spectra were recorded on a Varian Mercury 400 or 600 MHz using residual
1
H
or
13
C signals of deuterated solvents as internal standards. UV spectra were recorded on a Hewlett-
Packard 8350 instrument. HPLC analyses were performed on a Rainin dual pump HPLC system
equipped with a Phenomenex ODS column and an UV-VIS detector.
D
3
C OH
OTBDPS
24

4d
3
, (R)-2-(tert-butyldiphenylsilyloxy)butan-1-ol (24). To a solution of TBS-protected protected
primary alcohol (0.20 g, 0.46 mmol) (prepared as referenced in a previous thesis, but from deuterium
labeled starting material CD
3
MgI) in a 1:1 mixture of CH
2
Cl
2
:MeOH (15 mL) was added
camphorsulfonic acid (86.3 mg, 0.37 mmol) at room temperature. The progress of the reaction was
monitored by TLC. The reaction was over by an hour, it was then quenched with Et
3
N (0.30 mL, 2.30

76

mmol). The solvent was evaporated to dryness to give a crude mixture, which was then purified on a
silica gel column using 15% EtOAc/hexane to afford the primary alcohol 14 (0.13 g, 85%).
D
3
C
OTBDPS
I
27

5d
3
, (R)-tert-butyl(1-iodopent-1-en-3-yloxy)diphenylsilane (27). At 0
°
C, to 350mg (2.84mmol)
chromium chloride suspended in 3.0 ml THF, 100 mg (0.31mmol) aldehyde (prepared as referenced in a
previous thesis, but from deuterium labeled 24) and 560mg (1.42mmol) iodoform in 10ml THF was
added and stirred for 3 hours at 0
o
C. Workup with ether and brine and extract with diethyl ether. Remove
the solvent under vaccum and ran chromatography to purify the mixture (silica, 1% ethyl acetate in
hexanes) and 27 was obtained (78 mg, 56%).
1
H NMR (400 MHz, CDCl
3
) δ
H
7.65 (m, 4H), 7.40 (m, 6H),
6.44 (dd, J = 14.4 and 6.8 Hz, 1H), 5.92 (dd, J = 14.4 and 1.1 Hz, 1H), 4.04 (m, 1H), 1.46 (m, 2H), 1.06
(s, 9H).
13
C NMR (400 MHz, CDCl
3
) δ
C
148.21, 136.05, 135.97, 133.85, 129.84, 129.81, 127.70, 127.67,
77.36, 53.57, 29.79, 27.15, 19.52.
OH Cl
30

4-Chlorobut-2-yn-1-ol (30). To a solution of diol (6.0g, 0.07 mol) in 8 ml dry benzene, 6.0 g pyridine
was added dropwise over 1.5 hour with vigorous stirring. Cool to 0⁰C and slowly add 5.6 ml SOCl
2
and
stir overnight. Work up the reaction with saturated aqueous NaHCO
3
solution and extract with diethyl
ether. Remove the solvent under vacuum and the mixture was purified on a silica column using 20% ethyl
acetate in hexanes to afford the product (5.6 g, 77%).
1
H NMR (400 MHz, CDCl
3
) δ
H
4.37 (dt, J = 2.0 and
0.4 Hz, 2H), 3.98 (t, J = 2.0 Hz, 2H).

77

Br
TMS
25

(6-Bromohexa-1,4-diynyl)trimethylsilane (25). At room temperature, 1.88 g (19.2 mmol, 1.9 ml) TMS-
acetylene, 1.0 g (9.6 mmol) chloride 30, 3.6 g (19.2 mmol) CuI, 2.6 g (19.2 mmol) potassium carbonate,
2.9 g (19.2 mmol) sodium iodide in 30ml DMF was stirred overnight. Workup with saturated NH
4
Cl
aqueous solution. Keep extracting the aqueous layer with hexanes until no product was left in the aqueous
layer which was confirmed by TLC. The organic layer was combined and was rinsed with brine twice.
Dried over magnesium sulfate, and evaporate the solvent. The crude product was purified on silica
column using 20% EtOAc/hexanes to afford the coupling product (impure). Dissolve the coupling
product in 20 ml dry dichloromethane and add 2.5 g (9.6 mmol) PPh
3
.Cool to 0⁰C and slowly add 1.7 g
(9.6 mmol) NBS. Stir at 0⁰C for 1 hour. Work up the reaction with saturated aqueous NaHCO
3
solution
and extract with diethyl ether. Remove the solvent under vacuum and the mixture was purified on a silica
column using 1% ethyl acetate in hexanes to afford the product (1.4 g, 67% for two steps).
1
H NMR (400
MHz, CDCl
3
) δ
H
3.91 (t, J = 2.3 Hz, 2H), 3.28 (t, J = 2.3 Hz, 2H), 0.16 (s, 9H).
13
C NMR (400 MHz,
CDCl
3
) δ
C
98.88, 85.93, 81.05, 75.90, 65.79, 11.37, 0.01.
COOMe
17

(4Z,7Z)-methyl undeca-4,7-dien-10-ynoate (17). At room temperature, 1.1 g (20 mmol, ) methyl-pent-4-
ynoate, 2.3 g (10 mmol) bromide 25, 3.8 g (20 mmol) CuI, 2.7 g (20 mmol) potassium carbonate, 3.0 g
(20 mmol) sodium iodide in 30ml DMF was stirred overnight. Workup with saturated NH
4
Cl aqueous
solution. The organic layer was combined and was rinsed with brine twice. Evaporate the solvent and the
crude product was purified on a silica column using 3% EtOAc/hexanes to afford the coupling product,

78

which was dissolved in 20 ml EtOAc and kept under hydrogen atmosphere. Lindlar catalyst and 1 drop
quinoline were added and stirred vigorously. The reaction was carefully monitored by TLC every 15
minutes. Filtrate the mixture through celite and evaporate the solvent, then the crude product was purified
on a silica gel column using 3% EtOAc/hexanes to afford the diene, which was dissolved in 10 ml MeOH
and 500 mg Na
2
CO
3
was added. Stir vigorously overnight. Filtrate the mixture and evaporate MeOH, then
the crude product was purified on a silica gel column using 3% EtOAc/hexanes to afford the pure product
(0.65 g, 34% for three steps).
COOMe
15

(4Z,7Z,10Z,13Z)-methyl heptadeca-4,7,10,13-tetraen-16-ynoate (15). At room temperature, 0.38 g (2
mmol) 17, 0.91 mg (4 mmol) bromide 25, 0.76 g (4 mmol) CuI, 0.55 g (4 mmol) potassium carbonate, 0.6
g (4 mmol) sodium iodide in 10 ml DMF was stirred overnight. Workup with saturated NH
4
Cl aqueous
solution. The organic layer was combined and was rinsed with brine twice. Evaporate the solvent and the
crude product was purified on a silica column using 3% EtOAc/hexanes to afford the coupling product,
which was dissolved in 10 ml EtOAc and kept under hydrogen atmosphere. Lindlar catalyst and 1 drop
quinoline were added and stirred vigorously. The reaction was carefully monitored by TLC every 15
minutes. Filtrate the mixture through celite and evaporate the solvent, then the crude product was purified
on a silica gel column using 3% EtOAc/hexanes to afford the tetraene, which was dissolved in 10 ml
MeOH and 500 mg Na
2
CO
3
was added. Stir vigorously overnight. Filtrate the mixture and evaporate
MeOH, then the crude product was purified on a silica gel column using 3% EtOAc/hexanes to afford the
pure product (160 mg, 29% for three steps).
1
H NMR (400 MHz, CDCl
3
) δ
H
5.42-5.22 (m, 8H), 3.57 (s,
3H), 2.87 (m, 2H), 2.77-2.70 (m, 6H), 2.33-2.23 (m, 4H).
13
C NMR (400 MHz, CDCl
3
) δ
C
173.66,
130.22, 129.42, 128.76, 128.32, 128.23, 128.06, 127.54, 124.19, 82.70, 68.29, 51.71, 34.16, 25.79, 25.74,
25.69, 22.96, 17.03.

79

D
3
C
OTBS
28

Tert-butyldimethyl(5d
3
-pent-2-ynyloxy)silane (28). Iodoethane-2,2,2-d
3
(1.45 g, 9.1 mmol, 0.75 ml)
was dissolved in 40 ml anhydrous THF and cooled to -78⁰C. 1.3M n-BuLi solution (18.2 mmol, 14 ml)
was added dropwise. Warm up to -35⁰C and TBS-protected propargyl alcohol (3.1g, 18.2 mmol)
dissolved in 10 ml HMPA and 5 ml THF was cannulated into the reaction and stir for 35 minutes. After
that the reaction was warmed to -20⁰C and stir overnight, during which time the reaction was warmed to
room temperature. Work up the reaction with saturated aqueous NH
4
Cl solution and extract with diethyl
ether. Remove the solvent under vacuum and the mixture was purified on a silica column using 1% ethyl
acetate in hexanes to afford the product (1.6 g, 88%).
1
H NMR (400 MHz, CDCl
3
) δ
H
4.29 (t, J = 2.2 Hz,
2H), 2.19 (t, J = 2.1 Hz, 2H), 0.90 (s, 9H), 0.10 (s, 6H).
D
3
C
OH
29

5d
3
, Pent-2-yn-1-ol (29). TBS-protected 28 (1.6 g, 7.9 mmol) was dissolved in 20 ml THF and 1.0 M
THF solution was added (15.8 ml, 15.8 mmol). Stir at room temperature for 2 hours. Remove THF under
vacuum and the mixture was purified on a silica column using 20% ethyl acetate in hexanes to afford the
product (520 mg, 76%).
1
H NMR (400 MHz, CDCl
3
) δ
H
4.22 (m, 2H), 2.19 (m,2H).
D
3
C
Br
22

5d
3
, 1-Bromopent-2-yne (22). Dissolve 29 (520 mg, 6.0 mmol) in 10 ml dry dichloromethane and add
1.6 g (6.0 mmol) PPh
3
.Cool to 0⁰C and slowly add 1.0 g (6.0 mmol) NBS. Stir at 0⁰C for 1 hour. Work up
the reaction with saturated aqueous NaHCO
3
solution and extract with diethyl ether. Remove the solvent

80

under vacuum and the mixture was purified on a silica column using 1% ethyl acetate in hexanes to afford
the product (780 mg, 87%).
1
H NMR (400 MHz, CDCl
3
) δ
H
3.92 (t, J = 2.4 Hz, 2H), 2.24 (m, 2H).
13
C
NMR (400 MHz, CDCl
3
) δ
C
89.60, 74.76, 15.86, 12.56.

(S,1E,5Z,8Z)-1-iodoundeca-1,5,8-trien-3-ol (16). At 0
°
C, to 350 mg (2.9 mmol) chromium chloride
suspended in 5 ml THF, 150mg (0.36 mmol) aldehyde (prepared as in Chapter XX, using bromide 22 as
the starting material) and 400 mg (1.0 mmol) iodoform in 5 ml THF was added and stirred for 3 hours at
0
o
C. Workup with ether and brine and extract with diethyl ether. After removing the solvent under
vacuum the mixture was purified on a silica column with 1% EtOAc/hexanes and 16 was obtained (115
mg, 60%).
1
H NMR (400 MHz, CDCl
3
) δ
H
4.22 (m, 2H), 2.19 (m,2H). 7.65 (m, 4H), 7.40 (m, 6H), 6.49
(dd, J = 12.0 Hz and 4.0 Hz, 1H), 6.00 (dd, J = 16.0 Hz and 4.0 Hz, 1H), 5.48 – 5.18 (m, 4H), 4.12 (m,
1H), 2.60 (m, 2H), 2.25 (m, 2H), 2.00 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H).
13
C-NMR (400 MHz, CDCl
3
) δ
C
136.1, 136.0, 135.4, 133.9, 133.6, 132.2, 131.0, 130.4, 129.9, 129.9, 128.0, 127.8, 127.7, 127.0, 124.2,
77.1, 75.8, 35.4, 34.8, 31.8, 27.1, 26.7, 25.8, 25.5, 22.8, 14.3.
CD
3
18

13d
3
, (4Z,7Z,10Z)-trideca-4,7,10-trien-1-yne (18). At room temperature, 1.4 g (10 mmol)
trimethyl(penta-1,4-diynyl)silane, 0.75 g (5.0 mmol) bromide 22, 1.9 g (10 mmol) CuI, 1.4 g (10 mmol)
potassium carbonate, 1.5 g (10 mmol) sodium iodide in 20 ml DMF was stirred overnight. Workup with
saturated NH
4
Cl aqueous solution. The organic layer was combined and was rinsed with brine twice.

81

Evaporate the solvent and the crude product was purified on a silica column using 0.5% EtOAc/hexanes
to afford the coupling product, which was dissolved in 10 ml EtOAc and kept under hydrogen atmosphere.
Lindlar catalyst and 1 drop quinoline were added and stirred vigorously. The reaction was carefully
monitored by TLC every 15 minutes. Filtrate the mixture through celite and evaporate the solvent, then
the crude product was purified on a silica gel column using 0.5% EtOAc/hexanes to afford the diene,
which was dissolved in 10 ml MeOH and 300 mg Na
2
CO
3
was added. Stir vigorously overnight. Filtrate
the mixture and evaporate MeOH, then the crude product was purified on a silica gel column using 0.1%
EtOAc/hexanes to afford the product, which was then dissolved in 5 ml DMF and 1.9 g (10 mmol) (3-
bromoprop-1-ynyl)trimethylsilane, 1.9 g (10 mmol) CuI, 1.4 g (10 mmol) potassium carbonate, 1.5 g (10
mmol) sodium iodide were added and stir at room temperature overnight. Workup with saturated NH
4
Cl
aqueous solution. The organic layer was combined and was rinsed with brine twice. Evaporate the solvent
and the crude product was purified on a silica column using 0.5% EtOAc/hexanes to afford the coupling
product, which was dissolved in 10 ml EtOAc and kept under hydrogen atmosphere. Lindlar catalyst and
1/2 drop quinoline were added and stirred vigorously. The reaction was carefully monitored by TLC
every 15 minutes. Filtrate the mixture through celite and evaporate the solvent, then the crude product
was purified on a silica gel column using 0.5% EtOAc/hexanes to afford the triene, which was dissolved
in 5 ml MeOH and 200 mg Na
2
CO
3
was added. Stir vigorously overnight. Filtrate the mixture and
evaporate MeOH, then the crude product was purified on a silica gel column using 0.1% EtOAc/hexanes
to afford the product (88 mg, 10% for 6 steps).
1
H NMR (400 MHz, CDCl
3
) δ
H
5.45-5.35 (m, 6H), 2.97
(m, 2H), 2.82 (m, 4H), 1.98 (t, J = 2.8 Hz, 1H).
13
C NMR (400 MHz, CDCl
3
) δ
C
130.33, 129.20, 127.92,
127.24, 127.02, 124.13, 82.75, 77.36, 29.87, 25.68, 20.47, 17.04, 14.22.
I
O
O
20

82

(S)-5-(2-iodovinyl)-dihydrofuran-2(3H)-one (20). TBS-protected 4-hydroxyl vinyl iodide (prepared as
referenced in a previous thesis) (40 mg, 0.10 mmol) was dissolved in 5 ml THF and 1.0 M THF solution
was added (0.20 ml, 0.20 mmol). Stir at room temperature for 2 hours. Remove THF under vacuum and
the mixture was purified on a silica column using 10% ethyl acetate in hexanes to afford the product (20
mg, 80%).
1
H NMR (400 MHz, CDCl
3
) δ
H
7.22 (dd, J = 7.2 and 8.8 Hz, 1H), 7.06 (J = 1.2 and 14.4 Hz,
1H), 5.65 (m, 1H), 2.27 (m, 2H), 2.05 (m, 2H).
CD
3
21

16d
3
, (4Z,7Z,10Z,13Z)-hexadeca-4,7,10,13-tetraen-1-yne (21). At room temperature, 1.4 g (10 mmol)
trimethyl(penta-1,4-diynyl)silane, 0.75 g (5.0 mmol) bromide 22, 1.9 g (10 mmol) CuI, 1.4 g (10 mmol)
potassium carbonate, 1.5 g (10 mmol) sodium iodide in 20 ml DMF was stirred overnight. Workup with
saturated NH
4
Cl aqueous solution. The organic layer was combined and was rinsed with brine twice.
Evaporate the solvent and the crude product was purified on a silica column using 0.5% EtOAc/hexanes
to afford the coupling product, which was dissolved in 10 ml EtOAc and kept under hydrogen atmosphere.
Lindlar catalyst and 1 drop quinoline were added and stirred vigorously. The reaction was carefully
monitored by TLC every 15 minutes. Filtrate the mixture through celite and evaporate the solvent, then
the crude product was purified on a silica gel column using 0.5% EtOAc/hexanes to afford the diene,
which was dissolved in 10 ml MeOH and 300 mg Na
2
CO
3
was added. Stir vigorously overnight. Filtrate
the mixture and evaporate MeOH, then the crude product was purified on a silica gel column using 0.1%
EtOAc/hexanes to afford the product, which was then dissolved in 5 ml DMF and 2.3 g (10 mmol)
bromide 25, 1.9 g (10 mmol) CuI, 1.4 g (10 mmol) potassium carbonate, 1.5 g (10 mmol) sodium iodide
were added and stir at room temperature overnight. Workup with saturated NH
4
Cl aqueous solution. The
organic layer was combined and was rinsed with brine twice. Evaporate the solvent and the crude product
was purified on a silica column using 0.5% EtOAc/hexanes to afford the coupling product, which was

83

dissolved in 10 ml EtOAc and kept under hydrogen atmosphere. Lindlar catalyst and 1/2 drop quinoline
were added and stirred vigorously. The reaction was carefully monitored by TLC every 15 minutes.
Filtrate the mixture through celite and evaporate the solvent, then the crude product was purified on a
silica gel column using 0.5% EtOAc/hexanes to afford the triene, which was dissolved in 5 ml MeOH and
200 mg Na
2
CO
3
was added. Stir vigorously overnight. Filtrate the mixture and evaporate MeOH, then the
crude product was purified on a silica gel column using 0.1% EtOAc/hexanes to afford the product (100
mg, 12% for 6 steps).
1
H NMR (400 MHz, CDCl
3
) δ
H
5.50-5.40 (m, 8H), 3.00-2.90 (m, 2H), 2.85-2.75
(m, 6H), 2.10-2.02 (m, 2H), 1.99 (t, J = 2.8 Hz, 1H).
D
3
C
COOMe
HO
7

22d
3
, (R,4Z,7Z,10Z,13Z,18E)-methyl-20-hydroxydocosa-4,7,10,13,18-pentaen-16-ynoate (7). TBDPS-
protected 27 (30 mg, 0.066 mmol) was dissolved in 5 ml THF and 1.0 M THF solution was added (0.13
ml, 0.13mmol). Stir at room temperature for 4 hours. Remove THF under vacuum and the mixture was
purified on a silica column using 10% ethyl acetate in hexanes to afford the product 13. 20mg (0.093
mmol) vinyl iodide 13, 20mg (0.074 mmol) alkyne 15, 6mg (0.005mmol) Pd(PPh
3
)
4
, 1.9mg (0.01mmol)
CuI, and 0.14ml (1 mmol) Et
3
N were added in 1.5ml benzene and stirred at room temperature overnight.
Worked up with saturated ammonium chloride aqueous solution and ether. The organic layer was
combined and washed with brine. Evaporated the solvent and purified the mixture on a silica column
using 10-20% EtOAc/hexanes to afford the pure product (26 mg, 87%). λ
max
= 228 nm (in MeOH).
1
H
NMR (400 MHz, CDCl
3
) δ
H
6.06 (dd, J = 15.6 and 6.4 Hz, 1H), 5.68 (dtd, J = 15.9, 2.2 and 1.4 Hz, 1H),

84

5.50-5.35 (m, 8H), 4.05 (m, 1H), 3.67 (s, 3H), 3.09 (m, 2H), 2.85 (m, 6H), 2.37 (m, 6H).
13
C NMR (400
MHz, CDCl
3
) δ
C
173.73, 144.56, 129.95, 129.45, 128.72, 128.31, 128.28, 128.07, 127.67, 124.49, 110.56,
88.94, 78.44, 73.81, 51.74, 34.17, 29.77, 25.81, 25.75, 25.73, 22.97, 18.01.

D
3
C
COOMe
HO
8

22d
3
, (S,4Z,7Z,10Z,13Z,18E)-methyl-20-hydroxydocosa-4,7,10,13,18-pentaen-16-ynoate (8). Prepare
similarly as 7, from vinyl iodide 14 (enantiomer of 13) and alkyne 15. λ
max
= 228 nm (in MeOH).
1
H
NMR (400 MHz, CDCl
3
) δ
H
6.06 (dd, J = 15.6 and 6.4 Hz, 1H), 5.68 (dtd, J = 15.9, 2.2 and 1.4 Hz, 1H),
5.50-5.35 (m, 8H), 4.05 (m, 1H), 3.67 (s, 3H), 3.09 (m, 2H), 2.85 (m, 6H), 2.37 (m, 6H).
13
C NMR (400
MHz, CDCl
3
) δ
C
173.73, 144.56, 129.95, 129.45, 128.72, 128.31, 128.28, 128.07, 127.67, 124.49, 110.56,
88.94, 78.44, 73.81, 51.74, 34.17, 29.77, 25.81, 25.75, 25.73, 22.97, 18.01.
COOMe
OH
9

85

(S,4Z,7Z,12E,16Z,19Z)-methyl 14-hydroxydocosa-4,7,12,16,19-pentaen-10-ynoate (9). TBDPS-
protected 16 (50 mg, 0.093 mmol) was dissolved in 5 ml THF and 1.0 M THF solution was added (0.19
ml, 0.19 mmol). Stir at room temperature for 4 hours. Remove THF under vacuum and the mixture was
purified on a silica column using 10% ethyl acetate in hexanes to afford the product 31. Then coupled
product 9 was prepared similarly as 7, from vinyl iodide 31 and acetylene 17. λ
max
= 228 nm (in MeOH).
1
H NMR (400 MHz, CDCl
3
) δ
H
6.08 (dd, J = 16.0 and 6.0 Hz, 1H), 5.70 (d, J = 16.0 Hz, 1H), 5.61-5.35
(m, 8H), 4.18 (m, 1H), 3.67 (s, 3H), 3.08 (m, 2H), 2.80 (m, 4H), 2.39-2.29 (m, 6H).
13
C NMR (400 MHz,
CDCl
3
) δ
C
173.69, 143.92, 132.45, 132.23, 129.87, 128.80, 128.42, 126.78, 124.53, 124.32, 110.63,
89.12, 77.36, 71.77, 51.74, 35.15, 34.11, 29.85, 25.87, 25.66, 22.95, 18.01, 14.39.
COOMe
OH
D
3
C
10

22d
3
, (S,4Z,8E,13Z,16Z,19Z)-methyl 7-hydroxydocosa-4,8,13,16,19-pentaen-10-ynoate (10). Prepare
similarly as 7, from vinyl iodide 19 (prepared as in Chapter 2) and acetylene 18. λ
max
= 229 nm (in
MeOH).
1
H NMR (600 MHz, CDCl
3
) δ
H
6.02 (dd, J = 15.8 and 5.8 Hz, 1H), 5.66 (d, J = 15.9 Hz, 1H),
5.48-5.20 (m, 8H), 4.13 (m, 1H), 3.60 (s, 3H), 3.03 (m, 2H), 2.75 (m, 4H), 2.36-2.25 (m, 6H).
13
C NMR
(600 MHz, CDCl
3
) δ
C
173.83, 143.96, 135.23, 135.15, 131.43, 130.65, 128.24, 128.17, 126.02, 124.47,
110.51, 89.11, 78.41, 71.58, 51.80, 35.16, 33.77, 29.85, 25.69, 22.92, 21.20, 18.01, 14.35.

86

D
3
C
O
O
11

22d
3
, (S)-5-((1E,6Z,9Z,12Z,15Z)-octadeca-1,6,9,12,15-pentaen-3-ynyl)-dihydrofuran-2(3H)-one (11).
Prepare similarly as 7, from vinyl iodide 20 and acetylene 21. UV: λ
max
= 229 nm (in MeOH).
1
H NMR
(400 MHz, CDCl
3
) δ
H
6.02 (dd, J = 15.8 and 6.4 Hz, 1H), 5.78 (dq, J = 15.8 and 2.0 Hz, 1H), 5.55-5.25
(m, 8H), 4.95 (m, 1H), 3.10 (m, 2H), 2.88-2.72 (m, 4H), 2.57-2.51 (m, 2H), 2.15-1.95 (m, 6H).

COOMe
CD
3
12

22d
3
, (4Z,7Z,10Z,13Z)-methyl docosa-4,7,10,13-tetraen-16,19-diynoate (12). At room temperature, 20
mg (0.073 mmol) alkyne 15, 55 mg (0.36 mmol) bromide 22, 70 mg (0.36 mmol) CuI, 50 mg (0.36 mmol)
potassium carbonate, 54 mg (0.36 mmol) sodium iodide in 2 ml DMF was stirred overnight. Workup with
saturated NH
4
Cl aqueous solution. The organic layer was combined and was rinsed with brine twice.
Evaporate the solvent and the crude product was purified on a silica column using 5% EtOAc/hexanes to
afford the coupling product.
1
H NMR (400 MHz, CDCl
3
) δ
H
6.06 (dd, J = 15.6 and 6.4 Hz, 1H), 5.68 (dtd,
J = 15.9, 2.2 and 1.4 Hz, 1H), 5.50-5.35 (m, 8H), 4.05 (m, 1H), 3.67 (s, 3H), 3.09 (m, 2H), 2.85 (m, 6H),
2.37 (m, 6H).
13
C NMR (400 MHz, CDCl
3
) δ
C
173.73, 144.56, 129.95, 129.45, 128.72, 128.31, 128.28,
128.07, 127.67, 124.49, 110.56, 88.94, 78.44, 73.81, 51.74, 34.17, 29.77, 25.81, 25.75, 25.73, 22.97,
18.01.

87

D
3
C
COOMe
HO
1

22d
3
, (R,4Z,7Z,10Z,13Z,16Z,18E)-methyl-20-hydroxydocosa-4,7,10,13,16,18-hexaenoate (1). 200mg
Zn dust was weighed in N
2
glove box and 3 ml degassed water was added. Argon was bubbled through
the mixture for 15 min. Then 50 mg Cu(OAc)
2
was added and stirred for 20 min. After that, 50mg AgNO
3
was added and stirred for another 30 min. The precipitate was rinsed by water (2 x 3mL), MeOH (2 x
3mL), acetone (2 x 3mL), and ether (2 x 3mL) and dried under in vacuo. Weigh 100 mg powder prepared
in this way and transferred into a 25 ml flask. 0.5ml of degassed water was added in, then add in 1.0 mg
acetylene 7 dissolved in 0.5 ml MeOH. The suspension was stirred at room temperature for 4 hours.
Filtrate off the solids and concentrate the solution under vacuum. The product was purified on a HPLC
preparatory column using 22% water/MeOH as eluent (0.80mg, 80%). UV: λ
max
= 237 nm (in MeOH).
1
H NMR (400 MHz, CDCl
3
) δ
H
6.52 (ddt, J = 15.2, 11.1 and1.2 Hz, 1H), 6.00 (ddt, J = 12.3, 10.8 and 1.9
Hz, 1H), 5.69 (dd, J = 15.2 and 6.7 Hz, 1H), 5.45-5.34 (m, 9H), 4.10 (m, 1H), 3.67 (s, 3H), 2.97 (m, 2H),
2.84 (m, 6H), 2.42-2.36 (m, 6H).
13
C NMR (600 MHz, CDCl
3
) δ
C
173.74, 136.45, 130.32, 129.47, 128.69,
128.37, 128.27, 128.24, 128.22, 128.05, 127.83, 125.67, 74.21, 51.74, 34.17, 30.09, 26.27, 25.82, 25.75,
22.96, 22.81, 14.27.

88

D
3
C
COOMe
HO
2

22d
3
, (S,4Z,7Z,10Z,13Z,16Z,18E)-methyl-20-hydroxydocosa-4,7,10,13,16,18-hexaenoate (2). Prepare
similarly as 1, from acetylene 8. UV: λ
max
= 237 nm (in MeOH).
1
H NMR (400 MHz, CDCl
3
) δ
H
6.52
(ddt, J = 15.2, 11.1 and1.2 Hz, 1H), 6.00 (ddt, J = 12.3, 10.8 and 1.9 Hz, 1H), 5.69 (dd, J = 15.2 and 6.7
Hz, 1H), 5.45-5.34 (m, 9H), 4.10 (m, 1H), 3.67 (s, 3H), 2.97 (m, 2H), 2.84 (m, 6H), 2.42-2.36 (m, 6H).
13
C NMR (600 MHz, CDCl
3
) δ
C
173.74, 136.45, 130.32, 129.47, 128.69, 128.37, 128.27, 128.24, 128.22,
128.05, 127.83, 125.67, 74.21, 51.74, 34.17, 30.09, 26.27, 25.82, 25.75, 22.96, 22.81, 14.27.
COOMe
OH
3

(S,4Z,7Z,10Z,12E,16Z,19Z)-methyl 14-hydroxydocosa-4,7,10,12,16,19-hexaenoate (3). Prepare
similarly as 1, from acetylene 9. UV: λ
max
= 237 nm (in MeOH).
1
H NMR (600 MHz, CDCl
3
) δ
H
6.56
(ddt, J = 15.2, 11.1 and 1.3 Hz, 1H), 6.00 (dd, J = 10.9 and 10.9 Hz, 1H), 5.73 (dd, J = 15.2, 6.4 Hz, 1H),
5.60-5.25 (m, 9H), 4.23 (m, 1H), 3.67 (s, 3H), 2.96 (m, 2H), 2.88-2.72 (m, 4H), 2.42-2.32 (m, 4H), 2.06
(m, 2H), 0.96 (t, J = 7.5 Hz, 3H).
13
C NMR (600 MHz, CDCl
3
) δ
C
171.30, 135.92, 132.36, 131.75,

89

130.38, 129.40, 128.63, 128.11, 128.06, 127.84, 126.93, 125.58, 124.85, 72.14, 51.77, 41.51, 36.24, 34.15,
26.30, 22.99, 20.85, 18.92, 14.35.
COOMe
OH
D
3
C
4

22d
3
, (S,4Z,8E,10Z,13Z,16Z,19Z)-methyl 7-hydroxydocosa-4,8,10,13,16,19-hexaenoate (4). Prepare
similarly as 1, from acetylene 10. UV: λ
max
= 237 nm (in MeOH).
1
H NMR (400 MHz, CDCl
3
) δ
H
6.56
(ddt, J = 15.2, 11.0 and 1.2 Hz, 1H), 6.00 (dd, J = 9.2 and 9.2 Hz, 1H), 5.73 (dd, J = 15.2 and 6.3 Hz, 1H),
5.55-5.25 (m, 9H), 4.24 (m, 1H), 3.67 (s, 3H), 2.98 (m, 2H), 2.82 (m, 4H), 2.44-2.30 (m, 8H).
D
3
C
HO
COONa
5

22d
3
, Sodium (S,5E,7Z,10Z,13Z,16Z,19Z)-4-hydroxydocosa-5,7,10,13,16,19-hexaenoate (5). Dissolve
acetylene 11 (1.0 mg, 0.003 mmol) in 0.5 ml MeOH and add 0.5 ml 1M NaOH aqueous solution. Monitor
the hydrolysis by HPLC until completion and purify the mixture using HPLC. Sodium salt thus obtained
was hydrogenated and product 5 was obtained similarly as 1 from the hydrolyzed 11.
1
H NMR (400 MHz,
CD
3
OD) δ
H
6.57 (dd, J = 15.2 and 11.2 Hz, 1H), 6.00 (dd, J = 11.6 and 11.6 Hz, 1H), 5.73 (dd, J = 15.6

90

and 7.2 Hz, 1H), 5.43-5.30 (m, 9H), 4.16 (m, 1H), 3.67 (s, 3H), 2.98 (m, 2H), 2.82 (m, 4H), 2.44-2.30 (m,
8H), 2.97 (m, 2H), 2.85 (m, 6H), 2.10-1.90 (m, 6H).

22d
3
, (4Z,7Z,10Z,13Z,16Z,19Z)-methyl docosa-4,7,10,13,16,19-hexaenoate (6). Diyne 12 was
dissolved in 2 ml EtOAc and kept under hydrogen atmosphere. Lindlar catalyst and 1/10 drop quinoline
were added and stirred vigorously. The reaction was very carefully monitored by TLC every 5 minutes.
Filtrate the mixture through celite and evaporate the solvent, then the crude product was purified on a
silica gel column using 5% EtOAc/hexanes to afford 6. UV: λ
max
= 202 nm (in MeOH).
1
H NMR (400
MHz, CD
3
OD) δ
H
5.46-5.32 (m, 12H), 3.67 (s, 3H), 2.87-2.77 (m, 10H), 2.43-2.34 (m, 6H).
13
C NMR
(600 MHz, CDCl
3
) δ
C
173.69, 131.02, 129.46, 128.94, 128.72, 128.42, 128.39, 128.37, 128.27, 128.24,
128.23, 128.02, 127.16, 51.71, 38.89, 34.18, 30.52, 29.08, 25.79, 23.91, 23.14, 22.96, 21.20.
4.5. References
1. SanGiovanni, J. P.; Chew, E. Y. The role of ω-3 long-chain polyunsaturated fatty acids in health
and disease of the retina. Prog. Retin. Eye Res. 2005, 24 ,87-138.
2. C. D. Funk. Prostaglandins and leukotrienes: Advances in eicosanoid biology. Science 2001, 294,
1871-1875.
3. VanRollins, M.; Murphy R. C. Autooxidation of docosahexaenoic acid: analysis of ten isomers
of hydroxydocosahexaenoate. J. Lipid. Res. 1984, 25, 507-517.
4. Hamberg, M.; B. Samuelsson. Oxygenation of unsaturated fatty acids by the vesicular gland of
sheep. J. Biol. Chem. 1967, 242, 5344-5354.
COOMe
CD
3
6

91

5. Bazan, N. G.; Birkle, D. L.; Reddy, T. S. Docosahexaenoic acid (22:6, n-3) is metabolized to
lipoxygenase reaction products in the retina. Biochem. Biophys. Res. Commun. 1984, 125, 741-
747.
6. Yergey, J. A.; Kim, H.-Y.; and Salem, N., Jr. High-performance liquid
chromatography/thermospray mass spectrometry of eicosanoids and novel oxygenated
metabolites of docosahexaenoic acid. Anal. Chem. 1986, 58, 1344-1348.
7. Lee, T. H.; Mencia-Huerta, J. M.; Shih, C.; Corey, E. J.; Lewis, R. A.; Austen, K. F. Effects of
exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of 5-
lipoxygenase pathway products by ionophore-activated human neutrophils. J. Clin. Invest. 1984,
74, 1922–1933.
8. Whelan, J.; Reddanna, P.; Nikolaev, V.; Hildenbrandt, G. R.; and Reddy, T. S. Biological
Oxidation Systems 1990, 2, 765-778.
9. Reynaud, D.; Thickitt, C. P.; Pace-Asciak, C. R. Facile preparation and structural determination
of monohydroxy derivatives of docosahexaenoic acid (HDoHE) by α-tocopherol-directed
autoxidation. Ana. Biochem. 1993, 214, 165-170.
10. Hong, S.; Lu, Y.; Yang, R.; Gotlinger, K. H.; Petasis, N.; Serhan, C. N. Resolvin D1, Protectin
D1, and related docosahexaenoic acid-derived products: analysis via electrospray/low energy
tandem mass spectrometry based on spectra and fragmentation mechanisms. J Am Soc Mass
Spectrom. 2007, 18, 128-144.
11. VanRollins, M.; Baker, R. C.; Sprecher, H. W.; Murphy, R. C. Oxidation of docosahexaenoic
acid by rat liver microsomes. J. Biol. Chem. 1984, 269, 5776-5783.
12. Uddin, J. Design and synthesis of novel anti-inflammatory lipid mediators and anticancer small
molecules. Ph.D. dissertation 2008.
13. Yang, R. Total synthesis of novel anti-inflammatory lipid mediators. Ph.D. dissertation 2006.

92

14. Altundas, R.; Mahadevan, A.; Razdan, R. K. A synthetic route to anandamide analogues carrying
a substituent at the terminal carbon and an acetylene group in the end pentyl chain. Tetrhedron
Letters 2004, 45, 5449-5491.
15. Jin, J.; Weinreb, S. M. Application of a stereospecific intramolecular allenylsilane imino ene
reaction to dnantioselective total synthesis of the 5,11-methanomorphanthridine class of
amaryllidaceae alkaloids. J. Am. Chem. Soc. 1997, 119, 5773-5784.
16. Nicolaou, K. C.; Bulger, P. G.; Sarlah, D. Palladium-catalyzed cross-coupling reactions in total
synthesis. Angew. Chem. Int. Ed. 2005, 44, 4442-4489.
17. Boland, W.; Schroer, N.; Sieler, C.; Feigel, M. Sterospecific syntheses and spectroscopic
properties of ssomeric 2,4,6,8-undecatetraenes. New hydrocarbons from the marine brown alga
giffordia mitchellae. Part IV. Helv. Chim. Acta 1987, 70, 1025-1040.
18. Chemin, D.; Linstrumelle, G. A short stereocontrolled synthesis of leukotriene B
4
. Tetrahedron
1992, 48, 1943-1952.

93

Chapter 5. Total Synthesis of Aspirin-Triggered Neuroprotectin D1/Protection D1 (AT-
NPD1/PD1) and its Stereoisomers

5.1. Introduction
Neuroprotectin D1 (NPD1) is the first identified DHA-derived oxygenated mediator that elicits potent
neuroprotective effects at nanomolar concentrations in the brain and retina
1
. It is found in murine
ischemic stroke and is a potent regulator of PMN infiltration, reducing stroke-mediated tissue damage
2
. It
has been found that NPD1 is made on-demand in the nervous system and retinal pigment epithelial cells
in response to oxidative stress, which activates pro-survival signaling via regulation of gene expression
and other processes
3
. Since this potent chemical mediator has a broader range of activities in the immune,
cardiovascular and renal systems, for non-neuronal local biosynthesis and actions it is also termed
protectin D1 (PD1)
4
. The complete stereochemistry and anti-inflammatory actions of NPD1/PD1
(10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z hexanenoic acid) were unambiguously established
and its immunoregulatory roles were demonstrated
5
. Notably, recent results indicate that NPD1/PD1 is
also renoprotective
6
, induces corneal nerve regeneration
7
and stimulates cardiac and neural stem cell
differentiation at nanomolar potencies
8
.
Aside from NPD1/PD1, another potent stereoisomer has been discovered in inflammatory exudates and
human leukocytes, namely aspirin-triggered neuroprotectin D1/protectin D1 [AT-(NPD1/PD1)], which
displays potent protective bioactions comparable to NPD1/PD1 in vitro and in vivo
5
. It has been
demonstrated that AT-(NPD1/PD1) treatment reduced PMN recruitment in murine peritonitis, decreased
transendothelial PMN migration as well as enhanced efferocytosis of apoptotic human PMN by
macrophages
9
.
This chapter details the total synthesis of NPD1/PD1 (1) and AT-(NPD1/PD1) (2), which features a
highly stereo-controlled and convergent strategy to afford milligram scales of enantiomerically pure

94

compounds for bioactivity studies of NPD1/PD1 and AT-(NPD1/PD1), as well as structural elucidation of
AT-(NPD1/PD1). Importantly, we also desire to explore the structure-activity relationship of NPD1/PD1
isomers which may help identifying simpler and therefore more synthetically accessible leads for clinical
tests. Based on these considerations, a series of other NPD1/PD1 stereoisomers (3-6) were prepared via
total synthesis (shown in Figure 20).
(R)
(S)
COOMe
OH
(S)
OH
COOMe
(R)
OH
(S)
OH
COOMe
(R)
OH
(R)
OH
COOMe
(R)
OH
(R)
(R)
COOMe
OH
OH
OH
(R)
COOMe
(R)
OH
OH
1
2
3
4
5
6

Figure 20. Stereoisomers of NPD1/PD1 prepared by total synthesis
5.2. Results
5.2.1. Retrosynthetic analysis of AT-NPD1/PD1 stereoisomers
The retrosynthetic analysis of NPD1/PD1 stereoisomers and analogs is shown in Figure 21. For all
isomers and analogs, the last C-C bonds generated are the 15-double bonds (E for 5 and 6, Z for the others)
and compounds containing 15-Z double bond are all converted from 15-alkynes. The diene-ynes or trienes
(for 5 and 6) are constructed using corresponding coupling methods from two basic building blocks, each
of which contains a stereogenic center and can be varied to afford different stereoisomers. The synthesis
of both building blocks involves a key epoxide ring-opening reaction by terminal alkynes, which extends
the length of backbones and retains stereogenic configurations of the commercially available
enantiomerically pure glycidols. Starting from (R)- and/or (S)-glycidol, different chirality combinations at
C
10
and C
17
in NPD1/PD1 stereoisomers and analogs can be achieved in a timely efficient way.

95

R
1
O
O
R
1
= Si
t
BuMe
2

R
2
= Si
t
BuPh
2
R
1
O
O
OH
COOMe
OH
COOMe
OH
OH
OH
COOMe
OH
COOMe
OH
OH
OH
COOMe
OH
O
OR
2
O
COOMe
OR
2
(1 and 2)
(3 and 4)
(5 and 6)
OR
2
OH
Br
COOH
OR
1

Figure 21. Retrosynthetic analysis of NPD1/PD1 stereoisomers

5.2.2. Synthesis of building blocks of AT-NPD1/PD1 stereoisomers
Synthesis of building block 13 and 14 is shown in Scheme 17 (A). The synthesis originates from
protection of (R)- or (S)-glycidol as silyl ethers, and in the presence of the Lewis acid boron trifluoride
etherate, TBS-protected glycidols are opened by alkynyl lithium
10
, which is generated in situ by treating

96

TBDMS-protected propargyl alcohol with n-butyl lithium. After protection of the secondary alcohol
generated from the ring-opening reaction, the two primary hydroxyl group were selectively deprotected
11
and the more reactive propargyl alcohol is converted to propargyl bromide
12
, which is coupled with
methyl pent-4-ynoate in the presence of copper (I)
13
. Hydrogenation with Lindlar catalyst
14
afforded two
Z-double bonds simultaneously and a subsequent Swern oxidation
15
yields the aldehyde 13 and 14 for
later conversions. Similarly, the TBDMS-protected glycidols are opened by butyne, and after protecting
the secondary hydroxyl group as a bulky tert-butyldiphenylsilyl ether, the primary alcohol is selectively
deprotected and the acetylene is reduced to cis-alkene with Lindlar catalyst. The alcohol is then oxidized
to aldehyde 15 and 16 for subsequent reactions (shown in Scheme 17 (B)).
O
OH
COOMe
Br
HO
TBDPSO
13: (10R)
14: (10S)
1)TBDMS-Cl, Imidazole, 4-DMAP, rt,
CH
2
Cl
2
,97%
2)
n-BuLi, BF
3
.
Et
2
O, -78
o
C, THF, 63%
3) TBDPS-Cl, Imidazole, 4-DMAP, rt,
CH
2
Cl
2
,97%
4) CSA, CH
2
Cl
2
/MeOH,81%
5) PPh
3
, NBS, 0
o
C,CH
2
Cl
2
,60%
NaI, CuI, K
2
CO
3
, rt, DMF,75%
O
COOMe
OTBDPS
4) H
2
/Lindlar catalyst, quinoline, rt,
EtOAc,68%
5) DMSO, (COCl)
2
, Et
3
N, -78
o
C,
CH
2
Cl
2
,98%
1) TBDMS-Cl, Imidazole, 4-DMAP, rt,
CH
2
Cl
2
,97%
3) CSA, CH
2
Cl
2
/MeOH, 86%
7: (2S)
8: (2R)
9: (2R)
10: (2S)
OTBDMS
,
2) ,
1)TBDMS-Cl, Imidazole, 4-DMAP, rt,
CH
2
Cl
2
,97%
2)
BF
3
.
Et
2
O,-78
o
C, THF, 84%
,
n-
BuLi,
3)TBDPS-Cl, Imidazole, 4-DMAP, rt,
CH
2
Cl
2
, 89%
1) CSA, CH
2
Cl
2
/MeOH, 86%
2) H
2
/Lindlar catalyst, quinoline, rt,
EtOAc,68%
3) DMSO, (COCl)
2
, Et
3
N, -78
o
C,
CH
2
Cl
2
,98%
OTBDPS
O
15: (2R)
16: (2S)
O
OH
7: (2S)
8: (2R)
OTBDPS
11: (2R)
12: (2S)
OTBDMS
(A)
(B)

Scheme 17. Synthesis of aldehyde 13-16 for total synthesis of NPD1/PD1 stereoisomers and analogs

The aldehyde 13-16 were converted to vinyl iodide or terminal alkynes 17-24 which were direct building
blocks for the final coupling steps. Aldehyde 13 and 14 can either be converted to E, E- dienyl iodide 17
and 18 via a Wittig homologation
17
followed by a Takai olefination
16
, or directly to E-vinyl iodide 19 and
20 via a Takai olefination (Scheme 18 (A)). For aldehyde 15 and 16, they can either be converted to ene-
yne 23 or 24 via a Wittig homologation and a two-step Corey-Fuchs reaction
18
, or directly to terminal
alkyne 21 and 22 via Corey-Fuchs reaction (Scheme 18 (B)).

97

13: (10R)
14: (10S)
O
COOMe
OTBDPS
OTBDPS
O
15: (2R)
16: (2S)
19: (7R)
20: (7S)
17: (7R)
18: (7S)
CHI
3
, CrCl
2
, 0
o
C, THF, 56%
2) CHI
3
, CrCl
2
, 0
o
C, THF, 58%
, toluene, 90
o
C, 2h, 80%
PPh
3
O
1)
, toluene, 90
o
C, 2h, 78%
PPh
3
O
1)
2) CBr
4
, PPh
3
, 0
o
C, 1h, 89%
3) LDA, THF, -78
o
C, 80%
1) CBr
4
, PPh
3
, 0
o
C, 1h, 88%
2) LDA, THF, -78
o
C, 83%
23: (7R)
24: (7S)
21: (7R)
22: (7S)
(A)
(B)
COOMe
OTBDPS
I
COOMe
OTBDPS
I
OTBDPS OTBDPS

Scheme18. Synthesis of building block 17-24 for total synthesis of NPD1/PD1 stereoisomers and analogs

5.2.3. Assembly and spectroscopic study of AT-NPD1/PD1 stereoisomers and analogs
With the building blocks 17-24 prepared, the various stereo-isomers of NPD1/PD1 can be assembled in a
timely-efficient way. Variations at C
10
and C
17
chirality are achieved using building blocks with
corresponding chirality, and variations of triene E/Z arrangment at C
11
-C
15
are achieved by carrying out
corresponding coupling reactions
19
. To be more specific, isomers 1 and 2 were obtained from
hydrogenation of 15-ynes (25 or 26), which were prepared from the coupling of dienyl iodide 17 and
terminal alkyne (21 or 22) (Scheme 19 (A)), and isomers 3 and 4 were generated from the hydrogenation
of 13-yne
20-21
, which was obtained from the coupling of vinyl iodide (20 or 21) and ene-yne 23 (Scheme
19 (B)). In contrast, isomer 5 and 6 are synthesized directly from the coupling of dienyl iodide and
terminal alkyne (17 or 18) followed by silylether protecting group removal, without the hydrogenation
step (Scheme 19 (C)).

98

3: (10S,11E,13Z,15E,17R)
4: (10R,11E,13Z,15E,17R)
1) 22 or 21, Pd(PPh
3
)
4,

CuI, rt, C
6
H
6.
2) TBAF, THF, rt.
1) 23, Pd(PPh
3
)
4,
CuI, rt, C
6
H
6.
2) TBAF, THF, rt.
1) Zn(Cu/Ag), rt, MeOH/H
2
O
1) 21, Cp
2
ZrHCl, ZnCl
2
,Pd(PPh
3
)
4
,rt.
2) TBAF, THF, rt.
(A)
(B)
(C)
25: (10R,11E,13E,17S)
26: (10R,11E,13E,17R)
27: (10S,11E,15E,17R)
28: (10R,11E,15E,17R)
17: (10R)
COOMe
OTBDPS
I
COOMe
OH
OH
COOH
OH
OH
2) NaOH, rt, MeOH/H
2
O
1: (10R,11E,13E,15Z,17S)
2: (10R,11E,13E,15Z,17R)
20: (10S)
19: (10R)
COOMe
OTBDPS
I
COOMe
OH
OH
1) Zn(Cu/Ag), rt, MeOH/H
2
O
2) NaOH, rt, MeOH/H
2
O
COOH
OH
OH
3) NaOH, rt, MeOH/H
2
O
18: (10S)
17: (10R)
COOMe
OTBDPS
I
COOH
OH
5: (10S,11E,13E,15E,17R)
6: (10R,11E,13E,15E,17R)
OH

Scheme19. Assembly of NPD1/PD1 Stereoisomer 1-6

The proton NMR spectra are effective to prove the stereochemistry of the triene in these molecules. As an
example, in the olefinic region of isomer 2 [AT-(NPD1/PD1)], five of the six triene protons are resolved
and the coupling constants unambiguously proves the 11E, 13E, 15Z structure (shown in Figure 22). The
1
H NMR olefinic regions of stereoisomer 2, 4 and 6 were also compared and their differences were shown
to be significant (Figure 23).

99

Figure 22.
1
H NMR of olefinic region of NPD1/PD1 methyl ester (1)

Figure 23.
1
H NMR comparison of olefinic region of NPD1/PD1 stereoisomers

100

5.2.4. Structure elucidation/biosynthetic pathway study of AT-NPD1/PD1 and bioactivity
exploration of AT-NPD1/PD1 stereoisomers
The LC-MS-MS profiles of AT-NPD1/PD1 prepared by above synthesis were compared with endogenous
AT-NPD1/PD1 in inflammation exudates, and the (10R, 11E, 13E, 15Z, 17R) (isomer 2) was confirmed
to match the endogenous compound (first peak shown in aspirin-treated inflammation exudate shown in
Figure 24 (A), synthetic sample in Figure 24 (B) and co-injection in Figure 24 (C)). Hence the structural
elucidation of AT-NPD1/PD1 was successfully achieved.

Figure 24. LC-MS/MS matching: comparisons for biologic and synthetic AT-(NPD1/PD1)
9

101

Based upon above findings, a new biosynthetic pathway of DHA to produce AT-(NPD1/PD1) is proposed
and compared with the hypothesized biosynthetic pathway of NPD1/PD1
9
(Scheme 20). While
NPD1/PD1 is formed through a tandem phospholipase A
2
(PLA
2
)-15-lipoxygenase (15-LO) action on free
DHA, via a 16S,17S-DHA epoxide, COX-2 is involved in synthesis of AT-(NPD1/PD1) in the presence
of aspirin. In this case, aspirin acetylated COX-2 produces lipoxygenase-like product 17-HpDHA but
with the oxygen insertion in the R configuration rather than S as is the case with lipoxygenases
intermediate. Subsequent enzymatic conversion from 17R-HpDHA to a 16R, 17R-DHA epoxide, followed
by an enzymatic hydrolysis, affords the AT-(NPD1/PD1) as the 17-epimer of NPD1/PD1.
(R) OH
(R)
COOH
OH
(S)
(R)
COOH
OH
OH
(R)
(R)
O
COOH
(S)
(S)
COOH
O
(R) OOH
COOH
(S) OOH
COOH
COOH
AT-NPD1/PD1
NPD1/PD1
15-LO
COX-2
Aspirin
DHA
17R-HpDHA
17S-HpDHA

Scheme 20. Biosynthesis of NPD1/PD1 and AT-(NPD1/PD1)

With NPD1/PD1 and AT-(NPD1/PD1) synthesized in milligram scale, our collaborators were able to
explore more biological activities of both molecules. Results showed that NPD1/PD1 rescues neurons and
primary human retinal pigment epithelial cells from death by attenuating induced proteotoxic stress and
apoptosis
20
. As with AT-(NPD1/PD1), results show that AT-(NPD1/PD1) displays potent protective

102

bioactions comparable to NPD1/PD1 in vitro and in vivo, reducing both PMN infiltration and enhancing
the removal of apoptotic PMN by macrophages
9
. In addition to the confirmation of the biosynthesis
synthetic AT-NPD1, in either its sodium salt or as a methyl ester, was able to attenuate cerebral ischemic
injury
21
which leads to a novel approach for pharmaceutical intervention and clinical translation.
5.3. Conclusion
The total synthesis of AT-NPD1/PD1 and a series of its stereoisomers have been achieved successfully.
Based on the work of synthesis which produced enantiomerically pure authentic compounds, the complete
stereochemistry assignment of AT-NPD1/PD1 has been achieved. NPD1/PD1 and AT-NPD1/PD1 were
showed to be potent anti-inflammatory and pro-resolving SPMs which may help the development of
theraputics for the treatment of inflammation, neurologic diseases and more.
5.4. Experimental Procedures
All reactions, unless otherwise noted, were carried in flame dried flasks under argon atmosphere. “Dried
and concentrated” refers to removal of residual water with anhydrous MgSO
4
, followed by evaporation of
the solvent on the rotary evaporator. THF was freshly distilled from sodium-benzophenone, benzene and
dichloromethane from CaH
2
and anhydrous DMF, EtOH, and MeOH were purchased from commercial
sources.
1
H and
13
C NMR spectra were recorded on a Varian Mercury 400 or 600 MHz using residual
1
H
or
13
C signals of deuterated solvents as internal standards. UV spectra were recorded on a Hewlett-
Packard 8350 instrument. HPLC analyses were performed on a Rainin dual pump HPLC system
equipped with a Phenomenex ODS column and an UV-VIS detector.

103

(R) O
COOMe
OTBDPS
13

(R,4Z,7Z)-methyl 10-((tert-butyldiphenylsilyl)oxy)-11-oxoundeca-4,7-dienoate (13). Prepared as in
the referenced previous thesis
12
and NMR data was confirmed to be identical.
(R)
COOMe
OTBDPS I
17

(R,4Z,7Z,11E,13E)-methyl 10-((tert-butyldiphenylsilyl)oxy)-14-iodotetradeca-4,7,11,13-tetraenoate
(17). Prepared as in the referenced previous thesis
12
and NMR data was confirmed to be identical.
(S)
COOMe
I
20
OTBDPS

(S,4Z,7Z,11E)-methyl 10-((tert-butyldiphenylsilyl)oxy)-12-iodododeca-4,7,11-trienoate (20). At 0
°
C,
to 350mg (2.84mmol) chromium chloride suspended in 3.0 ml THF, 140mg (0.30mmol) enantiomer of
aldehyde 13 (prepared in the identical way as 13 but from the enantiomeric glycidol) and 560mg
(1.42mmol) iodoform in 10ml THF was added and stirred for 3hrs at 0
o
C. Workup with ether and brine.
Ran the column (silica, 3% ethyl acetate in hexanes) and 20 was obtained (105mg, 60%).
1
H NMR (400
MHz, CDCl
3
) δ
H
7.65 (m, 4H), 7.38 (m, 6H), 6.47 (dd, J = 6.8 and 14.4 Hz, 1H), 5.97 (d, J = 14.4 Hz,

104

1H), 5.35 (m, 4H), 4.12 (m, 2H), 3.66 (s, 3H), 2.65 (m, 2H), 2.40-2.30 (m, 4H), 2.23 (m, 2H), 1.06 (s, 9H).
13
C NMR (400 MHz, CDCl
3
) δ
C
173.6, 147.9, 136.0, 130.5, 130.0, 129.9, 129.3, 128.0, 127.8, 124.5,
75.7, 53.6, 34.1, 31.7, 27.1, 22.8, 14.3.
O
(R)
OTBDPS
15

(R,Z)-2-((tert-butyldiphenylsilyl)oxy)hept-4-enal (15). Prepared as in the referenced previous thesis
12

and NMR data was confirmed to be identical.
(R)
OTBDPS
21

(R,Z)-tert-butyl(oct-5-en-1-yn-3-yloxy)diphenylsilane (21). Prepared as in the referenced previous
thesis
12
and NMR data was confirmed to be identical.
(R)
OTBDPS
23

Tert-butyl((R,3E,7Z)-deca-3,7-dien-1-yn-5-yloxy)diphenylsilane (23). Vinyl iodide (50mg, 0.10 mmol)
was dissolved in pyrrolidine (1 ml) and 5mg Pd(PPh
3
)
4
and 0.1 ml TMS acetylene was added. The
reaction was stirred for 30 minutes. After removing pyrrolidine, the mixture was purified on a silica
column with 1% EtOAc/hexanes. The product was dissolved in MeOH and 200 mg Na
2
CO
3
was added

105

and the reaction was stirred overnight. The slurry solution was filtered and purified on a silica column
with 1% EtOAc/hexanes to afford the product (30mg, 78% for two steps).
1
H-NMR (400 MHz, CDCl
3
) δ
H
7.57 (m, 4H), 7.30 (m, 6H), 6.14 (dd, J = 4.0 and 16.0 Hz, 1H), 5.50 (dd, J = 16.0 and 2.0 Hz, 1H), 5.29
(m, 1H), 5.11 (m, 1H), 4.12 (m, 1H), 2.76 (d, J = 2.4 Hz, 1H), 2.10 (m, 2H), 1.70 (m, 2H), 0.97 (s, 9H),
0.75 (t, J = 7.5 Hz, 3H).
13
C-NMR (400 MHz, CDCl
3
) δ
C
147.4, 136.0, 134.4, 129.9, 128.5, 127.7, 123.2,
108.4, 82.2, 77.5, 73.2, 35.5, 27.2, 20.7, 19.5, 14.2.
(R)
(R)
COOMe
OH
OH
26

(4Z,7Z,10R,11E,13E,17R,19Z)-methyl 10,17-dihydroxydocosa-4,7,11,13,19-pentaen-15-ynoate (26).
50mg (0.081mmol) vinyl iodide 17, 50mg (0.14mmol) alkyne 21, 6mg (0.005mmol) Pd(PPh
3
)
4
, 1.9mg
(0.01mmol) CuI, and 0.14ml (1mmol) NEt
3
were added in 1.5ml benzene and stirred at room temperature
overnight. Worked up with saturated ammonium chloride aqueous solution and ether. The organic layer
was combined and washed with brine. Evaporated the solvent and purified the mixture on a silica column
using 3.5% EtOAc/hexanes to afford 60 mg impure product. To 60 mg protected product from last step in
4ml THF, added 0.37ml (0.37mmol) 1.0 M TBAF. The mixture was stirred at room temperature
overnight. Workup with NH
4
Cl solution and extracted with ether, then wash the organic layer with brine.
Evaporated the solvent and purified the mixture on a silica column using 35% EtOAc/hexanes to afford
the product 26 as colorless oil (19 mg, 65% for two steps).
1
H-NMR (400 MHz, CDCl
3
) δH 6.56 (dd, J =
15.6 and 10.8 Hz, 1H), 6.29 (dddd, J = 15.3, 10.8, 1.4 and 0.7 Hz, 1H), 5.83 (ddt, J = 15.2, 5.9 and 0.8 Hz,
1H), 5.62-5.38 (m, 7H), 4.53 (m, 1H), 4.23 (m, 1H), 3.67 (s, 3H), 2.83 (m, 2H), 2.56-2.46 (m, 2H), 2.42-
2.30 (m, 6H), 2.09 (m, 2H), 0.97 (t, J = 7.5 Hz, 3H).

106

(R)
(R)
COOMe
OH
OH
2

(4Z,7Z,10R,11E,13E,15Z,17R,19Z)-methyl 10,17-dihydroxydocosa-4,7,11,13,15,19-hexaenoate (2).
200mg Zn dust was weighed in N
2
glove box and 3mL degassed water was added. Argon was bubbled
through the mixture for 15min. Then 50mg Cu(OAc)
2
was added and stirred for 20min. After that, 50mg
AgNO
3
was added and stirred for another 30min. The precipitate was rinsed by water (2 x 3mL), MeOH
(2 x 3mL), acetone (2 x 3mL), and ether (2 x 3mL) and dried under in vacuo. Weigh 100mg powder
prepared in this way and transferred into a 25ml flask. 0.5ml of degassed water was added in, then added
in 1.0 mg acetylene 26 dissolved in 0.5ml MeOH. The suspension was stirred at room temperature for 4
hrs and the progress of reaction was monitored by HPLC. Filtrate off the solids and concentrate the
solution in vacuo. The product was purified on a HPLC preparatory column using 30% water/MeOH as
eluent (0.80mg, 80%).
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.51(dd, J = 14.0 and 11.4Hz, 1H), 6.26 (m, 2H),
6.09 (dd, J = 11.0 and 11.0 Hz, 1H), 5.79 (dd, J = 14.4 and 6.0Hz), 5.62-5.30(m, 7H), 4.60(m, 1H),
4.23(m, 1H), 3.67(s, 3H), 2.83(m, 2H), 2.44-2.22(m, 8H), 2.07(m, 2H), 0.96(t, J=7.5Hz, 3H).
13
C NMR
(400Hz, CDCl
3
): δ
C
173.63, 136.47, 135.31, 133.77, 133.45, 131.19, 130.30, 130.01, 129.00, 128.01,
127.76, 124.84, 123.47, 71.79, 67.72, 51.62, 35.28, 33.90, 25.74, 22.77, 22.60, 20.72, 14.05.

(S)
COOMe
(R)
OH
27
OH

107

(4Z,7Z,10S,11E,15E,17R,19Z)-methyl 10,17-dihydroxydocosa-4,7,11,15,19-pentaen-13-ynoate (27).
Prepared similarly as 26, from vinyl iodide 20 and alkyne 23.
1
H-NMR (600 MHz, CDCl
3
) δ
H
6.08-6.03
(m, 2H), 5.78-5.73 (m, 2H), 5.52-5.42 (m, 2H), 5.36-5.20 (m, 4H), 4.16-4.10 (m, 2H), 3.57 (s, 3H), 2.73
(m, 2H), 2.30-2.18 (m, 8H), 1.99-1.93 (m, 2H), 0.87 (t, J = 7.5 Hz, 3H).
13
C-NMR (600 MHz, CDCl
3
) δ
C
173.8, 144.9, 144.8, 136.0, 131.8, 129.1, 128.3, 124.6, 123.2, 110.2, 110.1, 88.4, 88.3, 71.7, 71.6, 51.0,
35.1, 34.1, 29.9, 25.9, 23.0, 20.9, 14.3.
(S)
COOMe
(R)
OH
3
OH

(4Z,7Z,10S,11E,13Z,15E,17R,19Z)-methyl 10,17-dihydroxydocosa-4,7,11,13,15,19-hexaenoate (3).
Prepared similarly as 2, from acetylene 27.
1
H-NMR (400 MHz, CDCl
3
) δ
H
6.72 (m, 2H), 5.98 (m, 2H),
5.76 (m, 2H), 5.60-5.61 (m, 2H), 5.46-5.33 (m, 4H), 4.25 (m, 2H), 3.66 (s, 3H), 2.83 (m, 2H), 2.42-2.30
(m, 8H), 2.08-2.04 (m, 2H), 0.96 (t, J = 7.5 Hz, 3H).
13
C-NMR (600 MHz, CDCl
3
) δ
C
173.8, 136.9, 136.7,
135.5, 131.4, 129.2, 129.1, 129.0, 128.2, 125.6, 125.5, 125.1, 123.7, 72.0, 71.9, 51.7, 35.6, 35.4, 34.1,
25.9, 23.0, 20.9, 14.3.
(S)
OH
COOMe
(R)
OH
5

(4Z,7Z,10S,11E,13E,15E,17R,18Z,21Z)-methyl 10,17-dihydroxytetracosa-4,7,11,13,15,18,21-
heptaenoate (5). To a 0.2ml THF suspension of 19mg (0.074mmol) zirconocene chloride hydride

108

(Schwartz's reagent) was added alkyne 21 (26mg, 0.074mmol) dissolved in 0.3ml THF. The suspension
was heated up to 50°C and maintained for 40 minutes. Cooled to room temperature and 0.05ml
(0.096mmol, 1.9M in 2-methyltetrahydrofuran) ZnCl
2
was added and stirred for 10 minutes. Then vinyl
iodide 18 (32mg, 0.052mmol) dissolved in 0.2ml THF was added through cannula, together with 3mg
(10%) Pd(PPh
3
)
4
catalyst. Stir at room temperature for 3 hours. Work up the reaction with saturated
NH
4
Cl solution and extract with ether, then wash the organic layer with brine. Remove the solvents in
vacuo and purified the mixture on a silica column using 3.5% EtOAc/hexanes to afford 20mg impure
protected 5. To 20 mg impure protected product from last step in 4ml THF, added 0.12ml (0.12mmol) 1.0
M TBAF. The mixture was stirred at room temperature for 4 hours. Workup with NH
4
Cl solution and
extracted with ether, then wash the organic layer with brine. Evaporated the solvent and purified the
mixture on a silica column using 35% EtOAc/hexanes to afford the product 5, which was further purified
on a HPLC preparatory column using 28% water/MeOH as eluent (0.50 mg, 34% for two steps).
1
H-
NMR (600 MHz, CDCl
3
) δ
H
6.30-6.17 (m, 4H), 5.76-5.72 (m, 2H), 5.60-5.50 (m, 2H), 5.45-5.31 (m, 4H),
4.21 (m, 2H), 3.67 (s, 3H), 2.83 (m, 2H), 2.42-2.28 (m, 8H), 2.07 (m, 2H), 1.24 (t, J = 7.5 Hz, 3H).
13
C-
NMR (600 MHz, CDCl
3
) δ
C
173.8, 136.0, 135.9, 135.6, 132.4, 132.4, 131.3, 130.5, 130.4, 129.2, 128.2,
125.1, 123.7, 72.1, 72.0, 51.8, 35.5, 35.4, 34.1, 25.9, 22.9, 20.9, 14.3.
(R)
OH
COOMe
(R)
OH
6

(4Z,7Z,10R,11E,13E,15E,17R,18Z,21Z)-methyl 10,17-dihydroxytetracosa-4,7,11,13,15,18,21-
heptaenoate (6). Prepared similarly as 5 from acetylene 21 and enantiomer of vinyl iodide 18.
1
H-NMR
(600 MHz, CDCl
3
) δ
H
6.30-6.17 (m, 4H), 5.76-5.72 (m, 2H), 5.60-5.50 (m, 2H), 5.45-5.31 (m, 4H), 4.21

109

(m, 2H), 3.67 (s, 3H), 2.83 (m, 2H), 2.42-2.28 (m, 8H), 2.07 (m, 2H), 1.24 (t, J = 7.5 Hz, 3H).
13
C-NMR
(600 MHz, CDCl
3
) δ
C
173.8, 136.0, 135.9, 135.6, 132.4, 132.4, 131.3, 130.5, 130.4, 129.2, 128.2, 125.1,
123.7, 72.1, 72.0, 51.8, 35.5, 35.4, 34.1, 25.9, 22.9, 20.9, 14.3.

References:
1. Mukherjee, P. K.; Marcheselli, V.L.; Barreiro, S.; Hu, J.; Bok, D.; Bazan, N. G. Neurotrophins
enhance retinal pigment epithelial cell survival through neuroprotectin D1 signaling. Proc. Natl.
Acad. Sci. USA. 2007, 104, 13152-13157.
2. Marcheselli, V. L.; Hong, S.; Lukiw, W. J.; Tian, X. H.; Gronert, K.; Musto, A.; Hardy, M.;
Gimenez, J. M.; Chiang, N.; Serhan, C. N.; Bazan, N. G. J. Biol. Chem. 2003, 278, 43807-43817.
3. Mukherjee, P. K.; Marcheselli, V. L.; Serhan, C. N.; Bazan, N. G. Neuroprotectin D1: a
docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from
oxidative stress. Proc. Natl. Acad. Sci. USA 2004, 101, 8491-8496.
4. Serhan, C. N.; Chiang, N.; van Dyke, T. E. Resolving inflammation: dual anti-inflammatory and
pro-resolution lipid mediators. Nat. Rev. Immunol. 2008, 8, 349-361.
5. Serhan, C. N.; Gotlinger, K.; Hong, S.; Lu, Y.; Siegelman, J.; Baer, T.; Yang, R.; Colgan, S. P.;
Petasis, N.A. Anti-inflammatory actions of neuroprotectin D1/protectin D1 and its natural
stereoisomers: assignments of dihydroxy-containing docosatrienes. J Immunol 2006, 176, 1848-
1859.
6. Hassan, I. R.; Gronert, K. Acute changes in dietary omega-3 and omega-6 polyunsaturated fatty
acids have a pronounced impact on survival following ischemic renal injury and formation of
renoprotective docosahexaenoic acid-derived protectin D1. J. Immunol. 2009, 182, 3223-3232.

110

7. Cortina, M. S.; He, J.; Li, N.; Bazan, N. G.; Bazan, H. E. Neuroprotectin D1 synthesis and
corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA. Invest
Ophthalmol Vis Sci. 2010, 51, 804-810.
8. Yanes, O.; Clark, J.; Wong, D. M.; Patti, G. G.; Sánchez-Ruiz, A.; Benton, H.P.; Trauger, S. A.;
Desponts, C.; Ding, S.; Siuzdak, G. Metabolic oxidation regulates embryonic stem cell
differentiation. Nat Chem Biol. 2010, 6, 411-417.
9. Serhan, C. N.; Fredmana, G.; Yang, R.; Karamnova, S.; Belayeva, L. S.; Bazan, N. G.; Zhu, M.;
Winkler, J. W.; Petasis, N. A. Novel pro-resolving aspirin-triggered DHA pathway. Chemistry
&Biology, 2011, 18, 976-987.
10. Mohr, P.; Tamm, C. Stereoselective synthesis of functionalized erythro/1,3-diols. Tetrhedron
Letters 1987, 28, 391.
11. Nicolaou, K. C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M. R. V.;
Yang, Z. Total syntheses of epothilones A and B via a macrolactonization-based strategy. J. Am.
Chem. Soc. 1997, 119, 7974.
12. Yang, R. Total synthesis of novel anti-inflammatory lipid mediators. Ph.D. dissertation 2006.
13. Altundas, R.; Mahadevan, A.; Razdan, R. K. A synthetic route to anandamide analogues carrying
a substituent at the terminal carbon and an acetylene group in the end pentyl chain. Tetrhedron
Letters 2004, 45, 5449-5491.
14. Jin, J.; Weinreb, S. M. Application of a stereospecific intramolecular allenylsilane imino ene
reaction to dnantioselective total synthesis of the 5,11-methanomorphanthridine class of
amaryllidaceae alkaloids. J. Am. Chem. Soc. 1997, 119, 5773-5784.
15. Mancuso, A. J.; Huang, S. L.; Swern, D. Oxidation of long-chain and related alcohols to
carbonyls by dimethyl sulfoxide "activated" by oxalyl chloride. J. Org. Chem. 1978, 43, 2480.
16. Takai, K.; Nitta, K.; Utimoto, K. Simple and selective method for aldehydes (RCHO) .fwdarw.
(E)-haloalkenes (RCH:CHX) conversion by means of a haloform-chromous chloride system. J.
Am. Chem. Soc. 1986, 108, 7408-7410.

111

17. For a review see: Petasis, N. A. In Science of Synthesis: Alkenes; Meijere, A. d. Ed.; Thieme-
Verlag: Stuttgart, 2009; pp. 161-246.
18. Corey, E. J.; Fuchs, P. L. A synthetic method for formyl→ethynyl conversion (RCHO→RC≡CH
or RC≡CR’) Tetrahedron Letters 1972, 13, 3769-3772.
19. Nicolaou, K. C.; Bulger, P. G.; Sarlah, D. Palladium-catalyzed cross-coupling reactions in total
synthesis. Angew. Chem. Int. Ed. 2005, 44, 4442-4489.
20. Calandria, J. M.; Mukherjee, P. K.; Vaccari, J. C. R.; Zhu, M.; Petasis, N. A.; Bazan, N. G.
Ataxin-1 poly-Q-induced proteotoxic stress and apoptosis are attenuated in neural cells by
docosahexaenoic acid-derived neuroprotectin D1. J. Bio. Chem. 2012, 287, 23726-23739.
21. Bazan, N. G.; Eady, T.; Khoutorova, L.; Atkins, K.; Hong, S.; Lu, Y.; Zhang, C.; Jun, B.;
Obenaus, A.; Fredman, G.; Zhu, M.; Winkler, J. W.; Petasis N. A.; Serhan, C. N.; Belayev, L.
Novel Aspirin-triggered Neuroprotectin D1 rescues the penumbra after experimental ischemic
stroke. Experimental Neurology 2012, 236, 122-130.

112

Chapter 6. Total Synthesis of Benzo-lipoxin A
4
Analogs

6.1. Introduction
Lipoxins are endogenous lipoxygenase-derived eicosanoids biosynthesized from arachidonic acid and are
generated during inflammatory, hypersensitivity, and vascular events, that display vasodiatory, anti-
inflammatory, and pro-resolution activity, and lipoxin A
4
(LXA
4
) (structure shown in Figure 26), a major
member of mammalian lipoxins, is a short-lived, trihydroxytetraene eicosanoid with potent anti-
inflammatory activities
1-2
. Importantly, in the presence of aspirin, cyclooxygenase-2 (COX-2) retains the
enzymatic capacity to generate 15-epimers of native lipoxins, namely aspirin-triggered lipoxins (ATLs),
in which the hydroxyl group at the carbon-15 is in the R rather than the S configuration, and ATLs retain
many of the bioactivities of native lipoxins
3-4
. As native lipoxins and ATL undergo inactivation in vivo
through prostaglandin dehydrogenase-mediated oxidation β-oxidation and ω-oxidation
5
, stable LXA
4

analogs have been synthesized
15
and 15-epi-16-(para-fluoro)-phenoxy-LXA
4
(ATLa) (shown in Figure 26)
have been shown to be active in vivo in multiple models of inflammatory diseases and has been
established as the benchmark of lipoxin analogs, demonstrating potent anti-inflammatory actions as well
as resistance to inactivation
6-11
.
Despite the potent activity of the ATLa, it is still cleared quickly in vivo
12
and more stable LXA
4
analogs
which retain both the anti-inflammatory and immunomodulatory actions need to be introduced. As the Z-
double bond in the tetraene moiety plays a key role retaining the bioactivities of LXA
4
and ATLa and thus
cannot be replaced by a E-double bond which simplifies its organic synthesis, it is preferable to replace
the tetraene unit of native LXA
4
with a benzo-fused ring system, which not only increases the thermal and
in vivo stability but also enables highly convergent and efficient syntheses. The novel benzo-LXA
4

analogs, prepared in our laboratory
13-14
, have already been demonstrated to have impressive anti-
inflammatory activity in multiple models.

113

LXA
4
exerts its anti-inflammatory effects through signals generated by binding to a high affinity, G
protein-coupled LXA
4
receptor, ALXR
16-17
and since the detailed three-dimensional molecular structure of
ALXR is not known, the design of LXA
4
analogs has been substrate-driven
13-14
. It has also been noted that
15-epi-LXA
4
is much less susceptible to enzymatic inactivation than native LXA
4
but exhibit, in most
cases, equal potencies in vitro
5
. Based on the considerations, additional benzo-LXA
4
analogs (1) were
prepared (Figure 25) with varied 3D molecular shapes where the relative positions of the two substituents
on the benzene ring change (analog 2, 5 and 6, where the two substituents are at meta-, ortho- of benzene
ring and 2,5-postion of thiophene ring, respectively), and with enzymatically stable moieties which resist
in vivo oxidation (analog 3 and 4, with a para-fluorophenoxy tail and fluorobenzene core respectively).
O
OH
HO OH
OH
LXA
4
O
OH
HO OH
HO
R
1
O
OMe
HO OH
HO
O
OMe
O
HO
OH
HO
F
O
OMe
HO
OH
HO
S
O
OMe
HO
OH
HO
2 3
6
5
HO OH
O
OMe
HO
F
4
O
OH
HO OH
O
15-epi-16-(para-fluoro)-phenoxy-LXA
4
(ATLa)
F
OH
O
OH
HO OH
OH
AT-LXA
4

Figure 25. Lipoxin A
4
and epi-benzo-lipoxin A
4
analogs prepared via total synthesis
6.2. Results

114

6.2.1. Retrosynthetic analysis of benzo-lipoxin A
4
analogs
The retrosynthetic analysis of epi-benzo-lipoxin A
4
methyl ester analogs (1) is shown in Figure 26. The
two alkenyl substituents on the phenyl ring can be prepared in a convergent way and coupled to a
bromophenyl bronic acid in varied order. Building blocks with varied terminal functional groups for the
coupling reactions were prepared and it is shown herein the mostly adopted strategy which has proved
successful in multiple cases. The carboxylester-containing building block can be prepared as a terminal
alkene or vinyl iodide, both of which are can prepared from enantiomerically pure D-deoxyribose, and
extension of the carbon chain is achieved via a Wittig reaction. On the other hand, the monohydroxy-
containing building block can be prepared as a vinyl borane, which is typically prepared from a terminal
alkyne containing a third stereogenic center.
O
OMe
HO OH
HO
R
TBSO
R
X
O
OMe
TBSO OTBS
Br
B(OH)
2
O
OH
OH
HO
Ph
3
P
O
OMe
X = I or H
TBSO
R
(RO)
2
B
1

Figure 26. Retrosynthetic analysis of epi-benzo-lipoxin A
4
methyl ester analogs

6.2.2. Synthesis of building blocks of benzo-lipoxin A
4
methyl ester analogs
Synthesis of the carboxylester-containing building block started from a Wittig coupling
23
between a
commercially available ylide and D-deoxyribose
24
, which contains two stereogenic centers, and the E-
double bond produced from the Wittig reaction was hydrogenated to a single bond subsequently. After
protection of three hydroxyl groups as silyl ethers, the primary alchohol was selectively deprotected
25
. A

115

Swern oxidation
26
of the alcohol and Takai olefination
27
of the resulting aldehyde afforded vinyl iodide 8
as the dihydroxy-containing building block (Scheme 21 (A)).
O
HO
OH
OH
1)
Ph
3
P
O
OMe
THF, reflux, 99%
2) H
2
, Pd/C, 100%
3) TBS-Cl, imidazole,
DMAP, 95%
1) CSA, MeOH/CH
2
Cl
2
,
0
o
C, 87%
2) DMSO, (COCl)
2
, NEt
3
,
-78
o
C, 88%
3) CrCl
2
, CHI
3
, 0
o
C, 65%
O
OMe
TBSO OTBS
I
O
OMe
TBSO OTBS
TBSO
(A)
HO F
O
HO
1)
CsF, DMF, 90%
F O HO
OTBS
2) TBS-Cl, imidazole,
DMAP, 95%
3) CSA, MeOH/CH
2
Cl
2
,
0
o
C, 67%
1)Dess-Marin periodinane,
NaHCO
3
, CH
2
Cl
2
, 92%
2) CBr
4
, PPh
3
, 0
o
C, 1h, 88%
3) LDA, THF, -78
o
C, 83%
O
OTBS
F
(B)
(C)
HO
TBS-Cl, imidazole,
DMAP, 98%
TBSO
7
8
12
13
14
(D)
8
O
O
BH
60
o
C, 80%
O
OTBS
F
15
B
O
O
12
O
O
BH
60
o
C, 70%
OTBS
16
B
O
O

Scheme 21. Synthesis of building blocks of epi-benzo-lipoxin A
4
methyl ester analogs

Ring-opening of (S)-glycidol by para-fluorophenol in the presence of CsF introduced the para-
fluorophenoxy into the monohydroxy-containing building block
28
. After protection of two hydroxyl
groups as silyl ethers, the primary alchohol was selectively deprotected. Oxidation of alcohol 14 using
Dess-Martin perodinane and a two-step Corey-Fuchs reaction
29
afforded termina alkyne 8 (Scheme 21
(B)). On the other hand, enantiomerically pure S-octyn-3-ol was protected as silyl ether 12 and used in
subsequent coupling reactions (Scheme 21 (C)). Both 8 and 12 underwent hydroboration with
catecholborane to afford vinyl borane 15 and 16, both of which were directly used for the following
coupling reaction without purification
24
(reaction yields were estimated from
1
H NMR spectra).

116

6.2.3. Assembly of benzo-lipoxin A
4
methyl ester analogs
With the building blocks 7, 15, 16 and a series of bromophenyl bronic acids in hand, epi-benzo-lipoxin A
4

methyl ester 2-6 were assembled via analogous procedures (Scheme 22). A Suzuki coupling was carried
out between vinyl iodide 7 and 3-bromophenylboronic acid and 9 was produced and subsequently used in
a second Suzuki coupling reaction with 16 to afford the TBS-protected precursor of 2, and with 15 to
afford the TBS-protected precursor of 3. Both TBS-protected precursors were treated with tert-
butylammonium fluoride to have their hydroxyl groups deprotected, and freshly made diazomethane
converted a mixture of free carboxylic acids, which was formed in the deprotection reaction and mixed
with methyl esters, to methyl ester 2 and 3 with good combined yields. Analog 4 and 6 were prepared
similarly as 2, while 5-bromothiophene-2-boronic acid and 3-bromo-5-fluorophenylboronic acid were
used to replace 3-bromophenylboronic acid. It is worth mentioning that the first Suzuki coupling reaction
generally gave low to moderate yields, due to occurrence of multiple side coupling reactions, and the
second Suzuki coupling reaction generally gave excellent yields.

117

1) 16, K
2
CO
3
, Pd(PPh
3
)
4
, H
2
O, 95%
2) TBAF, then CH
2
N
2
, 87%
K
3
PO
4
, Pd(PPh
3
)
4
, 38%
O
OMe
TBSO OTBS
Br
B(OH)
2
Br
HO OH
O
OMe
HO
2
O
OMe
O
HO OH
HO
F
O
OMe
HO OH
HO
S
3
6
HO OH
O
OMe
HO
F
4
2) TBAF, then CH
2
N
2
, 84%
9
9
7
K
3
PO
4
, Pd(PPh
3
)
4
, 35%
7
S
B(OH)
2
Br
TBSO OTBS
O
OMe
S
Br
11
2) TBAF, then CH
2
N
2
, 81%
K
3
PO
4
, Pd(PPh
3
)
4
, 35%
7
Br
TBSO OTBS
O
OMe
F
10
2) TBAF, then CH
2
N
2
, 45%
Br
F
B(OH)
2
1) 16, K
2
CO
3
, Pd(PPh
3
)
4
, H
2
O, 90%
1) 16, K
2
CO
3
, Pd(PPh
3
)
4
, H
2
O, 87%
1) 15, K
2
CO
3
, Pd(PPh
3
)
4
, H
2
O, 80%
1) 16, K
2
CO
3
, Pd(PPh
3
)
4
, H
2
O, 95%
2) TBAF, then CH
2
N
2
, 87%
K
3
PO
4
, Pd(PPh
3
)
4
, 38%
O
OMe
TBSO OTBS
5
17
7
B(OH)
2
Br
Br
O
OMe
HO OH
HO

Scheme 22. Assembly of epi-benzo-lipoxin A
4
methyl ester analog 2-6
6.3. Conclusion
The design and total synthesis of novel benzo-LXA
4
analogs have been successfully achieved. This work
of total synthesis provided a series of benzo-LXA
4
analogs featuring enzymatic inactivation resistant
structures and various molecular shapes for optimal binding to their cellular receptors in signaling
pathways and to establish structure-activity relationships. The actions of these analogs were investigated
13
.

118

6.4. Experimental Procedures
All reactions, unless otherwise noted, were carried in flame dried flasks under argon atmosphere. “Dried
and concentrated” refers to removal of residual water with anhydrous MgSO
4
, followed by evaporation of
the solvent on the rotary evaporator. THF was freshly distilled from sodium-benzophenone, benzene and
dichloromethane from CaH
2
and anhydrous DMF, EtOH, and MeOH were purchased from commercial
sources.
1
H and
13
C NMR spectra were recorded on a Varian Mercury 400 or 600 MHz using residual
1
H
or
13
C signals of deuterated solvents as internal standards. UV spectra were recorded on a Hewlett-
Packard 8350 instrument. HPLC analyses were performed on a Rainin dual pump HPLC system
equipped with a Phenomenex ODS column and an UV-VIS detector.
TBSO OTBS
O
O I
7

(5S,6R)-methyl 5,6-bis((tert-butyldimethylsilyl)oxy)-8-iodooct-7-enoate (7). At 0
°
C, to 570 mg (4.6
mmol) chromium chloride suspended in 3 ml THF, 250mg (0.59 mmol) aldehyde (prepared as in a
referenced previous thesis
24
) and 650 mg (1.65 mmol) iodoform in 2 ml THF was added and stirred for 3
hours at 0
o
C. Workup with ether and brine. Ran the column (silica, 5% ethyl acetate in hexanes) and 7
was obtained (180mg, 57%).
1
H NMR (400 MHz, CDCl
3
) δ
H
6.70 (dd, J = 12.0 Hz and 4.0 Hz, 1H), 6.38
(dd, J = 16.0 Hz and 4.0 Hz, 1H), 3,98 (m, 1H), 3.65 (s, 3H), 3.33 (m, 1H), 2.25 (m, 2H), 1.68-1.44 (m,
6H), 1.0 (s, 18H), 0.08 (s, 12H).
13
C NMR (400 MHz, CDCl
3
) δ
C
173.1, 142.4, 82.1, 79.9, 75.8, 51.9,
33.9, 32.2, 25.8, 19.3, 18.5, -5.3.

119

(S)-Tert-butyl((1-(4-fluorophenoxy)but-3-yn-2-yl)oxy)dimethylsilane (8). Carbon tetrabromide (90mg,
0.27mmol) was dissolved in 0.5ml CH
2
Cl
2
and cooled to 0°C and triphenylphosphine (141mg, 0.54mmol)
was added in via canula in 1ml CH
2
Cl
2
solution. Stirred at 0°C for 1 hour and aldehyde (80mg, 0.27mmol)
(prepared as in a referenced previous thesis) was added in via canula in 1ml CH
2
Cl
2
solution. Stirred at
0°C for 1 hr. Work up with sodium bicarbonate solution, extract with ether. Evaporated the solvent and
purified the mixture on a silica column using 1% EtOAc/hexanes to afford the dibromo olefin (90mg, a
little impure). At -78°C, the dibromo olefin was dissolved in 2 ml anhydrous THF and lithium
diisopropylamide in THF solution (2.0M, 0.30 ml) was added dropwise. Stirred at -78°C for 1hour. Work
up with NH
4
Cl solution and extract with ether. Evaporated the solvent and purified the mixture on a silica
column using 1% EtOAc/hexanes to afford the product 8 as colorless oil (42 mg, 53% for two steps).
1
H
NMR (400 MHz, CDCl
3
) δH 6.70 (dd, J = 12.0 Hz and 4.0 Hz, 1H), 6.38 (dd, J = 16.0 Hz and 4.0 Hz,
1H), 3,98 (m, 1H), 3.65 (s, 3H), 3.33 (m, 1H), 2.25 (m, 2H), 1.68-1.44 (m, 6H), 1.0 (s, 18H), 0.08 (s,
12H).
13
C NMR (400 MHz, CDCl3) δ
C
173.1, 142.4, 82.1, 79.9, 75.8, 51.9, 33.9, 32.2, 25.8, 19.3, 18.5, -
5.3.

(R)-tert-butyldimethyl(oct-1-yn-3-yloxy)silane (12). To a mixture of imidazole (0.55 g, 8.1 mmol),
TBDMS-Cl (1.2 g, 8.1 mmol), and DMAP (0.04 g, 0.33 mmol) in dry CH
2
Cl
2
(10 mL) at 0
°
C was added
(R)-oct-1-yn-3-ol (0.84 g, 6.7 mmol). The reaction mixture was warmed to room temperature, and stirred
for overnight. The reaction mixture was quenched with a saturated aqueous solution of NH
4
Cl, extracted

120

with ether, washed with brine, dried over MgSO
4
, and concentrated under reduced pressure to give a
crude product. The crude product was purified on silica column using 1% EtOAc/hexane as the eluent to
give 12 (1.6 g, 99%) as a colorless oil.
1
H NMR (400 MHz, CDCl
3
) δ
H
6.70 (dd, J = 12.0 Hz and 4.0 Hz,
1H), 6.38 (dd, J = 16.0 Hz and 4.0 Hz, 1H), 3,98 (m, 1H), 3.65 (s, 3H), 3.33 (m, 1H), 2.25 (m, 2H), 1.68-
1.44 (m, 6H), 1.0 (s, 18H), 0.08 (s, 12H).
13
C NMR (400 MHz, CDCl
3
) δ
C
173.1, 142.4, 82.1, 79.9, 75.8,
51.9, 33.9, 32.2, 25.8, 19.3, 18.5, -5.3.
Br
TBSO OTBS
O
O
9

(5S,6R)-methyl 8-(3-bromophenyl)-5,6-bis((tert-butyldimethylsilyl)oxy)oct-7-enoate (9). Alcohol In a
flame-dried flask, (3-bromophenyl)boronic acid (16 mg, 0.083 mmol), K
3
PO
4
(52 mg,0.25 mmol),
Pd(PPh
3
)
4
(5 mg) was dissolved in 1 ml anhydrous DMF. Vinyl iodide 7 (45 mg, 0.083 mmol) was
dissolved in 1 ml DMF and cannulated to the solution. The reaction was allowed to stir at 65
o
C for 5
hours. Work up with NH
4
Cl aqueous solution and extract with ether. Evaporated the solvent and purified
the mixture on a silica column using 1.5% EtOAc/hexanes to afford the product 9 as colorless oil (17 mg,
37%).
1
H NMR (400 MHz, CDCl
3
) δ
H
7.48 (m, 1H), 7.35 (m, 1H), 7.17 (m, 1H), 6.41 (dd, J = 16.1 and
0.8 Hz, 1H), 6.17 (dd, J = 16.0 and 7.0 Hz, 1H), 4.11 (m, 1H), 3.66 (m and s, 4H), 2.31 (t, J = 7.4 Hz, 2H),
1.73 (m, 2H), 1.55 (m, 2H), 0.91 (s, 9H), 0.86 (s, 9H), 0.08 (s, 3H), 0.04 (s, 3H), 0.02 (s, 3H), 0.01 (s, 3H).

121

(5S,6R)-Methyl 8-(3-bromo-5-fluorophenyl)-5,6-bis((tert-butyldimethylsilyl)oxy)oct-7-enoate (10).
Prepared similarly as 6, starting from vinyl iodide 7 and (3-bromo-5-fluorophenyl)boronic acid.
1
H NMR
(400 MHz, CDCl
3
) δ
H
7.10 (m, 1H), 6.99 (m, 1H), 6.39 (d, J = 16.0 Hz, 1H), 6.19 (dd, J = 15.9, 6.8 Hz,
1H), 4.14-4.09 (m, 2H), 3.66 (m and s, 4H), 2.31 (t, J = 7.4 Hz, 2H), 1.73 (m, 2H), 1.55 (m, 2H), 0.91 (s,
9H), 0.86 (s, 9H), 0.08 (s, 3H), 0.04 (s, 3H), 0.02 (s, 3H), 0.01 (s, 3H).
TBSO OTBS
O
O
S
Br
11

(5S,6R)-Methyl 8-(5-bromothiophen-2-yl)-5,6-bis((tert-butyldimethylsilyl)oxy)oct-7-enoate (11).
Prepared similarly as 9, starting from vinyl iodide 7 and (5-bromothiophen-2-yl)boronic acid.
1
H NMR
(400 MHz, CDCl
3
) δ
H
5.60.
13
C NMR (400 MHz, CDCl
3
) δ
C
132.1.
1
H NMR (400 MHz, CDCl
3
) δ
H
6.70
(dd, J = 12.0 Hz and 4.0 Hz, 1H), 6.38 (dd, J = 16.0 Hz and 4.0 Hz, 1H), 3,98 (m, 1H), 3.65 (s, 3H), 3.33
(m, 1H), 2.25 (m, 2H), 1.68-1.44 (m, 6H), 1.0 (s, 18H), 0.08 (s, 12H).
13
C NMR (400 MHz, CDCl
3
) δ
C

173.1, 142.4, 82.1, 79.9, 75.8, 51.9, 33.9, 32.2, 25.8, 19.3, 18.5, -5.3.
HO OH
O
O
HO
2

(5S,6R)-methyl 5,6-dihydroxy-8-(3-((R,E)-3-hydroxyoct-1-en-1-yl)phenyl)oct-7-enoate (2).
Catecholborane (31 mg, 0.262 mmol, 28μl) and acetylene 12 (63 mg, 0.262 mmol) was mixed in a pear-
shape flask and stir overnight at 65
°
C. To the white muddy substance, 5 mg Pd (PP
3
)
4
and 25 mg K
2
CO
3

was added and aryl bromide 9 (32 mg, 0.0525 mmol) was cannulated to the mixture after dissolved in 1

122

ml 1,4-dioxane. Add 2 ml degassed water into the mixture and the reaction was allowed to go overnight at
80
°
C. Work up with NH
4
Cl aqueous solution and extract with ether. Evaporated the solvent and purified
the mixture on a silica column using 1% EtOAc/hexanes to afford 55 mg coupled product as colorless oil
(a little impure), which was dissolved in 2 ml THF and TBAF solution (0.45 ml, 1.0M, 0.45 mmol) was
added and the reaction was allowed to go for 2 hours. Work up with NH
4
Cl aqueous solution and extract
with ether. Add freshly prepared CH
2
N
2
diethyl ether solution into the combined ether layer to convert the
free acid to methyl ester. Evaporated the solvent and purified the mixture on a silica column using 5%
MeOH/ CH
2
Cl
2
to afford the product (6.9 mg, 34% for 3 steps), which can be further purified by HPLC if
necessary. UV: λ
max
= 245 nm (in MeOH).
1
H NMR (400 MHz, CDCl
3
) δ
H
6.70 (dd, J = 12.0 Hz and 4.0
Hz, 1H), 6.38 (dd, J = 16.0 Hz and 4.0 Hz, 1H), 3,98 (m, 1H), 3.65 (s, 3H), 3.33 (m, 1H), 2.25 (m, 2H),
1.68-1.44 (m, 6H), 1.0 (s, 18H), 0.08 (s, 12H).
13
C NMR (400 MHz, CDCl
3
) δ
C
173.1, 142.4, 82.1, 79.9,
75.8, 51.9, 33.9, 32.2, 25.8, 19.3, 18.5, -5.3.
HO OH
O
O
O
HO
F
3

(5S,6R)-methyl 8-(3-((S)-4-(4-fluorophenoxy)-3-hydroxybut-1-en-1-yl)phenyl)-5,6-dihydroxyoct-7-
enoate (3). Prepared similarly as 2, starting from aryl bromide 9 and acetylene 8. UV: λ
max
= 245 nm (in
MeOH).
1
H NMR (400 MHz, CDCl
3
) δ
H
6.70 (dd, J = 12.0 Hz and 4.0 Hz, 1H), 6.38 (dd, J = 16.0 Hz
and 4.0 Hz, 1H), 3,98 (m, 1H), 3.65 (s, 3H), 3.33 (m, 1H), 2.25 (m, 2H), 1.68-1.44 (m, 6H), 1.0 (s, 18H),
0.08 (s, 12H).
13
C NMR (400 MHz, CDCl
3
) δ
C
173.1, 142.4, 82.1, 79.9, 75.8, 51.9, 33.9, 32.2, 25.8, 19.3,
18.5, -5.3.

123

(5S,6R)-methyl 8-(3-fluoro-5-((R)-3-hydroxyoct-1-en-1-yl)phenyl)-5,6-dihydroxyoct-7-enoate (4).
Prepared similarly as 1, starting from aryl bromide 10 and acetylene 12. UV: λmax = 245 nm (in MeOH).
1
H NMR (400 MHz, CDCl
3
) δ
H
6.70 (dd, J = 12.0 Hz and 4.0 Hz, 1H), 6.38 (dd, J = 16.0 Hz and 4.0 Hz,
1H), 3,98 (m, 1H), 3.65 (s, 3H), 3.33 (m, 1H), 2.25 (m, 2H), 1.68-1.44 (m, 6H), 1.0 (s, 18H), 0.08 (s,
12H).
13
C NMR (400 MHz, CDCl
3
) δ
C
173.1, 142.4, 82.1, 79.9, 75.8, 51.9, 33.9, 32.2, 25.8, 19.3, 18.5, -
5.3.
HO OH
O
O
S
HO
6

(5S,6R)-methyl 5,6-dihydroxy-8-(5-((R)-3-hydroxyoct-1-en-1-yl)thiophen-2-yl)oct-7-enoate (6).
Prepared similarly as 2, starting from aryl bromide 11 and acetylene 12. UV: λmax = 245 nm (in MeOH).
1
H NMR (400 MHz, CDCl
3
) δ
H
6.70 (dd, J = 12.0 Hz and 4.0 Hz, 1H), 6.38 (dd, J = 16.0 Hz and 4.0 Hz,
1H), 3,98 (m, 1H), 3.65 (s, 3H), 3.33 (m, 1H), 2.25 (m, 2H), 1.68-1.44 (m, 6H), 1.0 (s, 18H), 0.08 (s,
12H).
13
C NMR (400 MHz, CDCl
3
) δ
C
173.1, 142.4, 82.1, 79.9, 75.8, 51.9, 33.9, 32.2, 25.8, 19.3, 18.5, -
5.3.

124

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Abstract (if available)

Abstract This dissertation reports research on the total synthesis of a series of DHA-derived specialized pro-resolving mediators (SPMs), which can be divided into four projects corresponding to four classes of DHA-derived SPMs, and the design and synthesis of synthetic lipoxin A4 analogs. ❧ Chapter 1 briefly explains the term “specialized pro-resolving mediator” (SPM) and reviews the background of research on ω-3 polyunsaturated fatty acids, as well as the identification, biosynthesis, total synthesis, anti-inflammatory/pro-resolving activities and actions of ω-3 polyunsaturated fatty acid-derived SPMs. It also briefly reviews some related research on the development of pharmaceuticals based on SPMs. In Chapter 2, the total synthesis of maresin 1 (MaR1), a most recently discovered DHA-derived SPM, as well as a series of its stereoisomers, are described. The synthesis helped the structure elucidation of MaR1 and the discovery of new bioactivities, as well as the structure-activity-relationships of MaR1 and its stereoisomers. In Chapter 3, a previously hypothesized but never isolated biosynthetic intermediate was prepared by total synthesis. The 13S, 14S-epoxy-maresin has helped prove the proposed biosynthesis of MaR1 and uncovered more unexplored biological processes this biosynthetic intermediate was involved in. In Chapter 4, a series of deuterium-labeled monohydroxy-DHAs and deuterium-labeled DHA were prepared by total synthesis. Work in this chapter not only produced a series of isotope-labeled DHA derivatives to assist the exploration of DHA metabolic pathways, but also provided facile and feasible routes to synthesize labeled DHA and its monohydroxy-E, Z-diene derivatives. In Chapter 5, a newly elucidated stereoisomer of NPD1/PD1, namely the aspirin-trigger NPD1/PD1, was prepared bytotal synthesis to help confirm its stereochemical assignments and explore its bioactivities. A series of other stereoisomers as well as structrual analogs were prepared to study the structure-activity-relationships of NPD1/PD1 and its isomers. In Chapter 6, a series of novel stable benzo-analogs of lipoxin A4 were prepared by total synthesis. These benzo-LXA4 analogs are expected to either exhibit higher in vivo stability or help to uncover an optimal structure for future analogs.

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Asset Metadata

Creator Zhu, Min (author)

Core Title Total synthesis of specialized pro-resolving lipid mediators and their analogs

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 11/30/2013

Defense Date 10/18/2013

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

Tag lipid mediator,OAI-PMH Harvest,total synthesis

Language English

Contributor Electronically uploaded by the author (provenance)

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

Creator Email min.mountainee@gmail.com,minzhu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-353055

Unique identifier UC11288219

Identifier etd-ZhuMin-2197.pdf (filename),usctheses-c3-353055 (legacy record id)

Legacy Identifier etd-ZhuMin-2197.pdf

Dmrecord 353055

Document Type Dissertation

Rights Zhu, Min

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