University of Southern California Dissertations and Theses

Synthesis of novel nucleotide analogues based on the traditional and nontraditional bioisosteres

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Synthesis of novel nucleotide analogues based on the traditional and nontraditional bioisosteres

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SYNTHESIS OF NOVEL NUCLEOTIDE ANALOGUES BASED ON
THE TRADITIONAL AND NONTRADITIONAL BIOISOSTERES
by

Anton Shakhmin

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)

2014

Copyright Anton Shakhmin

- 2 -

DEDICATION
To my first school chemistry teacher who opened to me the world of
Chemistry
Popova Eugenia Alekseevna

- 3 -

ACKNOWLEDGMENTS

First, I would like to express my gratitude to Prof. G. K. Surya Prakash for giving me
opportunity to pursue the doctoral degree performing research under his supervision. I would like
to sincerely thank Prof. Prakash for his kindness, understanding, unlimited support and guidance
throughout this academic endeavor.
Next, I would like to show my gratitude to my co-supervisor Dr. George A. Olah who
provided excellent support and advice during my study at the University of Southern California.
I also am very grateful to Dr. Robert Anizfeld for support during this research program,
maintaining research institute capabilities at high level and constant care for safety of everyone
in the lab.
I would like to extend my appreciation to the Dr. Peter Jones with whom I greatly
enjoyed the collaboration effort.
I also would like to express my thanks to Dr. Myron Goodman and Dr. Stan Lui for
biological evaluation of the prepared nucleotides within the collaborative project.
I would like to thank Dr. Mikhail Zibinsky who advised me throughout the program and
worked with me on the methodology for preparation of monofluoroalkenes and nucleotide
analogues project, Dr. Inessa Bychinskaya who worked with me on NTP analogs project, and
John-Paul Jones who worked with me on the nucleotide analogue project and helped with the
NMR instruments. I also would like to thank Dr. Ralf Haiges for helping me with X-ray analysis
of my samples and Dr. Keriann Oertell for bio-testing of nucleotide analogs. I would like to
acknowledge Thomas Mathew, Parag Jog, Alain Goeppert, Miklos Czaun for help and support in
my research projects. I am very thankful to Thomas Mathew for critical review of this
manuscript. My special thanks to friend and lab mates for support and helpful scientific
discussions Andrey Rudenko, Anne-Marie Finaldi, Arjun Narayanan, Alex Butkevich, Somesh
K. Ganesh, Aditya Kulkarni, Hema Krishnan, Nadezda Fomina, Ivan Krylov, Valeria Zakharova
I would like to express my warmest thanks to all past and present members of our research
group.
I am very grateful to my wife Ekaterina for endless support, help, patience and
understanding. I would like to thank my family members and friends constantly supporting me
all these years; without them this effort would have been hard to accomplish. Finally, I must
- 4 -

acknowledge the financial support of my work by NCI-NIH and Loker Hydrocarbon Research
Institute.

- 5 -

TABLE OF CONTENTS

DEDICATION 2
ACKNOWLEDGMENTS 3
1. Chapter 1 Synthesis of ( , ), ( , ) - bisdifluoromethylene NTP Analogs
1.1. INTRODUCTION 8
DNA polymerase β 8
Structure of DNA polymerase β 8
Structural modifications of nucleotide analogues 11
1.2. RESULTS AND DISCUSSION 12
Preparation of BM
F4
TPA 12
Preparation of benzoyl-protected 5‟-tosyl-nucleosides 12
Preparation of protected 5‟-tosyl guanosine 13
Coupling of BM
F4
TPA with protected nucleosides tosylates 14
1.3. CONCLUSIONS 15
1.4. EXPERIMENTAL PART 16
1.5. REPRESENTATIVE NMR SPECTRA
AND HPLC CHROMATOGRAMS 21
1.6. REFERENCES 36

2. Chapter 2. Investigations toward bioisosteric replacement of phosphoric
hydroxyl group with difluoromethyl motif in nucleotide analogues

2.1. INTRODUCTION 38
2.2. RESULTS AND DISCUSSION 40
Preparation of the difluoromethyl-containing phosphate isosteres 40
Preparation of nucleotide analogues with the difluoromethyl-modified
- 6 -

phosphate moiety 41
2.3. CONCLUSIONS 45
2.4. EXPERIMENTAL PART 46
2.5. REPRESENTATIVE NMR SPECTRA 55
2.6. REFERENCES 74

3. Chapter 3. Synthesis of monofluoroalkenes via Julia-Kosinski reaction

3.1. INTRODUCTION 76
3.2. RESULTS AND DISCUSSION 77
Preparation of monofluoroalkenes via Julia-Kocienski olefination reaction 77
Prepartion of monofluoromethyl 3,5-bis(trifluoromethyl)phenyl sulfone 78
3.3. CONCLUSIONS 81
3.4. EXPERIMENTAL PART 81
3.5. SPECTRAL DATA OF PRODUCTS 83
3.6. REFERENCES 90

4. Chapter 4. Bioisosteric replacement of α,- β,- and γ- phosphate of
trisphosphoric acid with the squaryl moiety. Synthesis of corresponding
nucleotide analogues and evaluation of their chemical properties.
4.1. INTRODUCTION 92
Preparation of cyclobutenediones 94
Application to organic synthesis and catalysis 96
Application to medicinal chemistry 97
Squaric acid as isosteric replacement of carboxylic acids and amino acids 99
Sqauric acid as natural isostares of guanidines and cyanoguanidines 100
Traditional and novel phosphate mimic 101
Squaric acid-structural motifs as a phosphate mimic 105
Design squaric acid based nucleotide analogues 107
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4.2. RESULTS AND DISCUSSION 108
Studies toward synthesis of squaric acid-based dNTP Analogues 103
Preparation of DNTP analogues based on
3,4
BM-β-SQTPA 114
Redesign of squaric acid based triphosphoric acid analogues 115
The investigation toward the synthesis of 2,4-substituted BMPA-β-SQ 116
Study toward incorporation squaryl moiety into γ-position
of triphosphoric acid 117
Study toward incorporation of squaryl moiety into α-position
of triphosphoric acid 121
Computational Investigations 124
4.3. CONCLUSIONS 126
4.4. EXPERIMENTAL PART 126
4.5. REPRESENTATIVE NMR SPECTRA 137
4.6. REFERENCES 159

5. Chapter 5 Investigations toward synthesis of phosphoramidates of
2'-deoxy- ψ-isocytidine
5.1. INTRODUCTION 164
5.2. RESULTS AND DISCUSSION 166
Preparation of the 2'-deoxy-ψ-isocytidine 166
Preparation of the phosphorochloridates 168
Synthetic study toward phosphoramidates of 2'-deoxy-ψ-isocytidine 169
5.3. CONCLUSIONS 178
5.4. EXPERIMENTAL PART 178
5.5. REPRESENTATIVE NMR SPECTRA 186
5.6. REFERENCES 199
BIBLIOGRAPHY 201
- 8 -

Chapter 1 Synthesis of
( , ), ( , ) - bisdifluoromethylene NTP Analogs
1.1 INTRODUCTION
DNA polymerase β
DNA polymerase β (pol β) is a well-known member of X family of DNA polymerases which
occurs in the nuclei of eukaryotic cells [1]. Nevertheless, its biological function in the cells has
not been completely determined. It is known that pol β similarly to other DNA polymerases,
catalyzes a nucleotidyl transfer reaction adding new nucleotides to a growing chain [2].
However, it appears that pol β is primarily responsible for maintaining genome integrity by
participating in base excision repair mechanism, which is essential for DNA maintenance,
replication, recombination, and drug resistance. It possesses the exceptional ability to repair
single-stranded DNA gaps smaller than 6 nucleotides. However, in contrast to other DNA
polymerases pol β does not have exonuclease (proofreading) activity. The deficiency of pol β
relates to hypersensitivity to alkylating agents, induced apoptosis, and chromosomal breaking
whereas overexpression of pol β has been associated with a number of cancer types. Thus, it is
crucial to maintain pol β expression strongly regulated. Polymerase β has relatively simple
structure among the family of DNA polymerases and therefore, it was studied most extensively
in order to acquire understanding of the mechanism of its action and develop new anticancer and
HIV treatment [1-4].
Structure of DNA polymerase β
Pol β exists as a single polypeptide chain containing 335 amino acids. It consists of two
domains, an 8 kDa domain and a 31 kDa domain (Figure 1.1) [1]. The 31 kD is the principal
polymerase domain that involves several subdomains: the fingers, the thumb, and the palm,
which are all important for catalysis. The palm subdomain demonstrates high affinity for
phosphate groups and thus is considered to play a significant role in pol β binding to double
strained gaped DNA backbone which is the substrate for pol β. Stabilization of the negatively
charged DNA backbone performed through the DNA binding channel containing the two
positively charged lysine and arginine side chains. Three main aspartate residues located in
- 9 -

polymerase active site and complexed with two magnesium ions are responsible for binding of
the incoming nucleotide. Asp
190
and Asp
192
mainly involved in positioning of the incoming
nucleotide whereas asp
256
mainly performs stabilization of transition state complex [1].
Magnesium ions play the most important role in the catalytic reaction, they are involved in
positioning, stabilization and activation of dNTP. One of the Mg
2+
metal ions specifically binds
to the negatively charged α-phosphate, whereas another magnesium ion predominantly
coordinates with β- and γ- phosphates of the ddNTP functioning as a bidentate ligand. However,
it also participates in the stabilization of the non-bridging α- phosphate oxygen. Van der Waals
interactions between sugar moiety of the incoming nucleotide and the pol β-residues promote
further positioning dNTP in the active site. Additional interactions occur between 3‟ carbon site
of the ribose and pol β-residues responsible for distinguishing between dNTP and NTP. Figure
1.1 represents the catalytic site of the pol β with residues (D190, D192 and D256) marked in red
and magnesium ions are colored magenta [1, 3, 5].

Figure 1.1. Structure of polymerase β [6].
During nucleotidyl transfer reaction 3' -hydroxyl group of the terminal nucleotide of primer
strand undergo nucleophilic attack on the α-phosphate of incoming dNTP producing
- 10 -

pyrophosphate and the primer strand extended by one nucleotide (Scheme 1.1). It appears that
during catalytic reaction there is no stabilization of the negative charge on the bridging oxygen
of α-phosphate which makes anhydride bond labile stimulating the nucleophilic substitution and
subsequent release of the pyrophosphate [5, 8, 9]. Figure 1.2 shows binding and positioning of
the incoming dNTP with respect to terminal nucleotide of primer strand, where 3‟hydroxyl group
is ready to attack α-phosphate of the incoming nucleotide [1, 5].
Scheme 1.1. Nucleotidyl transfer reaction [6].

Figure 1.2 Binding and positioning of the incoming dNTP in the active site of pol β [6].

- 11 -

Structural modifications of nucleotide analogues
Modified nucleotides are extensively used as tools for revealing enzymatic functions and
mechanisms, as well as potential antiviral and anticancer agents [9-11]. The triphosphate group
is the part of the nucleotide molecule that undergoes catalytic cleavage during nucleotidyl
transfer reaction; therefore modification of this fragment could reveal varieties of the enzyme
activities. The most commonly used modification of the triphosphate group is the substitution of
an oxygen atom for an „unnatural‟ atom or group in the (α, β) - and/or (β, γ) - phosphoanhydride
bonds. Replacement of the P-O-P bridging oxygen atom in a natural mononucleotide by an NH
or methylene group makes the corresponding nucleotide analogue completely non-hydrolizable.
Non-hydrolizable nucleotide analogues could not be cleaved in the nucleotidyl transfer reaction,
therefore, these analogues remain intact in stable ternary DNA complexes with the enzyme
making it useful probe for studying complex structure and function [12, 13].
In our attempt to design and synthesize triphosphate analogues in which bioisosteric
modifications will resemble the stereoelectronic properties of natural triphosphate the synthesis
of bis(difluoromethylene)triphosphonic acid (BM
F4
TPA) (1) was developed [13]. Moreover,
several α, β and β, γ-CF
2
-substituted analogues of deoxynucleotides 5‟-triphosphates (dNTPs) (2-
5) have been prepared (Figure 1.3). The X-ray crystal structure of a ternary complex of DNA
polymerase β with incoming analogue (α, β) - (β, γ) - bisCF
2
dTTP was obtained. The acquired
X-ray structure represents precatalytic state of the nucleotidyl transfer reaction and clearly
indicates that CF
2
bridging group prevents the dissociation of phosphoranhydride bond, trapping
the complex. Thus (α, β) - (β, γ) - bisCF
2
dNTP analogues successfully demonstrate their ability
to serve as suitable non-hydrolyzable mimics of the natural dNTP [13].
Base
O
OH
O P
O
F
2
C P
F
2
C P
O O
O O
O
O
O P
O
F
2
C P
F
2
C P
O O
O O
O
O
(Bu
4
N
+
)
5
1 2 (Base = Tymine)
3 (Base =Guanine;
4 (Base =Cytosine)
5 (Base =Adenine)

Figure 1.3. BM
F4
TPA [Bu
4
NH
+
]
5
1, and (α, β) - (β, γ) - bisCF
2
dNTP (2-5).

- 12 -

In perusal of the novel idea of the preparation of nucleotide analogues that may reveal
additional nuances of enzyme complex structure and function we attempted the synthesis of a
number of novel (α, β)- and (β, γ) - CF
2
-substituted analogues of nucleoside 5‟-triphosphates
(NTPs).
1.2 RESULTS AND DISCUSSION
Preparation of BM
F4
TPA
BM
F4
TPA 10 and its tetrabutyl ammonium salt 1 were prepared according to the procedure
developed by Prakash and co-workers. [13] (Scheme 1.3). LTMPA was proven to be more
advantageous in comparison to other lithium containing or other metallic bases in the
deprotonation of the diethyl difluoromethyl phosphonate and subsequent engagement of it in the
reactions as a nucleophile. After the coupling reaction of dimethylphosphoramidous dichloride 7
(1 equivalent) with diethyl difluoromethyl phosphonate 6 (two equivalents) in presence of
LTMPA, the resulting dimethyl amino-triphosphoric acid analogue was oxidized in situ with m-
CPBA. Subsequent hydrolysis with TMSBr and water afforded BM
F4
TPA, which was further
converted into tetrabutyl ammonium salt by treating it with 1 M solution of tetrabutylammonium
hydroxide.
Scheme 1.3. Preparation of BM
F4
TPA [Bu
4
N
+
]
5
.
P
O
EtO CF
2
H
OEt
+
P Cl
Cl
N
LTMPA, THF
EtO
P
C
F
2
P
C
F
2
P
OEt
O N O
EtO OEt
m-CPBA
EtO
P
C
F
2
P
C
F
2
P
OEt
O
N
O
EtO OEt
O
1. TMSBr
2. H
2
O
HO
P
C
F
2
P
C
F
2
P
OH
O
OH
O
HO OH
O
-
O
P
C
F
2
P
C
F
2
P
O
-
O
O
-
O
-
O O
-
O
(Bu
4
N
+
)
5
2
Bu
4
N
+
OH
-
6 7 8 9
10 1

Preparation of benzoyl-protected 5’-tosyl-nucleosides
For the preparation of protected tosylnucleosides 1-3, we used the synthetic protocol
described in Scheme 1.4 [14]. Nucleosides were first treated with TBDMSCl in pyridine, which
selectively protected 5‟-hydroxyl group. Reaction with benzoyl chloride gave access to
- 13 -

multibenzoylated nucleosides 14-16. After that 5‟-hydroxy group can be selectively deprotected
with very good yields using specific ratio of THF, H
2
O and TFAA = (4 : 1 : 1). Benzoyl
protected nucleosides were recrystallized from ethanol and purified using flash chromatography
on silica gel. Final treatment with p-toluenesulfonyl chloride (TsCl) in anhydrous pyridine for a
week at -24 °C provided desired protected nucleoside tosylates in good yields. Sometimes
considerable amount of reaction time was required to achieve maximum conversion. However,
they usually do not exceed 70 - 80%. The purification of the protected RNA tosylates was
performed using flash chromatography on silica with the EtOAc / Hexane mixture as the eluent.
Scheme 1.4. Synthesis of benzoyl-protected 5‟-tosyl-nucleosides 20-22.
Base
O
OH OH
HO
Si Cl
Pyridine
Base
O
OH OH
TBDMSO
BaseBz
n
O
OBz OBz
TBDMSO
TFA/H
2
O/THF
BaseBz
n
O
OBz OBz
HO
Pyridine
H
3
C S
O
O
Cl
BaseBz
n
O
OBz OBz
TsO
Cl
O
11-13 14-16
17-19
20-22

Preparation of protected 5’-tosyl guanosine
For preparation of protected guanosine tosylate, we had to employ an approach slightly
different from the one that we used for the other three RNA nucleosides (Scheme 1.5). This was
due to significantly reduced solubility of guanosine in organic solvents compare to other
nucleosides. As a result, we had to introduce a lipophilic protective groups on polar amino and
5‟-hydroxyl group of guanosine that would increase the solubility in organic solvents. Therefore
in the first step, 2-amino group of guanosine was protected by reaction with
N,N-dimethylformamide dimethylacetal in acetonitrile. This reaction requires almost two weeks
for completion, however, it enables to access the desired protected guanosine 23 with an yield of
about 98 % [15]. Following treatment with TBDPSCl [15] provided selective protection of the
5‟-hydroxyl group. Subsequent protection of the 3‟and 4‟ hydroxyl groups was carried out in situ
- 14 -

via reaction with benzoyl chloride producing fully protected guanosine 27. After complete
deprotection of TBDPS group by treatment with TBAF [16], tosylation of the protected
guanosine was accomplished adopting the general procedure described for other nucleosides.

Scheme 1.5. Preparation of protected Tosyl Guanosine.
NH
N
N
O
NH
2
N
O
OH OH
HO
N
O
O
MeOH
Si Cl
Ph
Ph
NH
N
N
O
N
N
O
OH OH
HO
N
Me
Me
DMAP, Py
NH
N
N
O
N
N
O
OH OH
O
N
Me
Me
Si
NH
N
N
O
N
N
O
OBz OBz
O
N
Me
Me
Si TBAF/THF/AcOH
NH
N
N
O
N
N
O
OBz OBz
HO
N
Me
Me
S
O
O
Cl
Py
NH
N
N
O
N
N
O
OBz OBz
TsO
N
Me
Me
25 26
27 20 24
Cl
O

Coupling of BM
F4
TPA with protected nucleosides tosylates
Scheme 1.6. Preparation of ( , ), ( , ) - bisdifluoromethylene NTP Analogs.
BasePG:
N
N
N
N
NBz
2
N
N
NHBz
O
NBz
N
O
O
Base:
N
N
N
N
NH
2
N
N
NH
2
O
NH
N
O
O
NH
N
N
N
O
N
NH
N
N
N
O
NH
2
N

BasePG
O
OBz OBz
TsO
P
C
F
2
C
F
2
P
O
P
O
O
O
O
O
O
O
+ (Bu
4
N
+
)
5
DMF
BasePG
O
OBz OBz
O
P
O
F
2
C
P
O
F
2
C
P
O
O
O O O
H
2
O/MeOH/NH
3
Base
O
OH OH
O
P
O
F
2
C
P
O
F
2
C
P
O
O
O O O
110
o
C/ 1h
(Bu
4
N
+
)
4
21-24 1 28-31 32-35
(Et
3
NH
+
)
4

Due to the strong electron withdrawing effect of difluoromethylene bridging groups,
BM
F4
TPA act as a very poor nucleophile. Conversion of BM
F4
TPA into tetrabutylammonium
- 15 -

salt significantly enhances its reactivity toward electrophilic centers. Thus the target nucleotide
analogues 32-25 can be accessed through reaction of benzoyl protected 5‟-tosyldeoxynucleosides
with tetrabutylammonium salts of BM
F4
TPA. The reaction mixture was dissolved in anhydrous
DMF and initially heated for one hour at 110 °C, followed by stirring several days at room
temperature. The progress of the reactions was monitored by HPLC using a weak ion-exchange
column (Tosoh Bioscience DEAE-5PW) with water / Et
3
NH
+
HCO
3
-
solution as the eluent. After
reaction reached maximum conversion, all nucleotide analogues were deprotected using water /
methanol solution of ammonium hydroxide. All RNA nucleotide analogues exhibit similar
retention times as deoxynucleotide analogues and can be eluted within 28 - 35 minutes. After
completion of the reaction, all volatiles were removed and crude mixtures were subjected to
HPLC purification on DEAE-5PW semi-preparative column using water / 1 M Et
3
NH
+
HCO
3
-
solution as the eluent. In the case of adenosine, cytidine and uridine, we were able to achieve
conversions close to 90 %, although in the case of the guanosine, conversion did not exceeded
20 % and yield was just 6 %.
Table 1.1. The yields of the prepared nucleosides 17-24 and (α, β), (β, γ) - bisdifluoromethylene
NTP analogs 32-35.
Nucleoside Benzoyl
protected
nucleoside
Yield (%) Protected
nucleoside
tosylate
Yield (%) BM
F4
TPA
nucleotide
analogue
Yield (%)
Adenosine 17 92 21 61 32 29
Cytidine 18 90 22 43 33 33
Uridine 19 8 23 55 34 22
Guanosine 20 47 24 59 35 6

1.3 CONCLUSIONS
The developed synthetic approach gave access to (α, β), (β, γ) - bisdifluoromethylene NTP
analogs, which are useful probes for studying different enzymes structure. All four RNA based
analogues (α, β), (β, γ) – bis(CF
2
) ATP (32), (α, β), (β, γ) – bis(CF
2
) CTP (33), (α, β), (β, γ) –
bis(CF
2
) UTP (34) and (α, β), (β, γ) – bis(CF
2
) GTP (35) were synthesized via electrophilic
tosylate substitution at 5‟position of ribonucleotide with acceptable yields. Purification of (α, β),
(β, γ) – bis(CF
2
) NTP analogues was performed using DEAE weak anion exchange column with
Et
3
NH
+
HCO
3
-
/ H
2
O solution as the eluent. Compounds were isolated in the form of their
- 16 -

triethylammonium salts. All compounds were fully characterized by
1
H,
19
F,
31
P NMR
spectroscopy and by HRMS analysis. In addition, non-hydrolysable properties of prepared
nucleotide analogues were confirmed by performing two single-turnovers gap filling assay.
Incorporation of dTTP, UTP, and bis(CF
2
) UTP analogue into a single-gapped DNA substrate
with dA at the templating position, using DNA pol β, indeed verified non-hydrolysable nature of
bis(CF
2
) UTP analogue. Additional biochemical assays involving incorporation of UTP or
bis(CF
2
) UTP by T7 RNA pol also showed resistance of 34 toward enzymatic hydrolysis. Both
experiments indicated that unlike natural UTP and dTTP substrates difluoromethylene groups of
the bisCF
2
UTP inhibit incorporation into DNA primer and cannot be employed by polymerases.
1.4 EXPERIMENTAL PART
General procedure for preparation of nucleotide analogues by using reaction of
(Bu
4
N
+
)
5
BM
F4
TPA salt with benzoyl protected nucleoside 5’- tosylates
A 10 mL flask kept under argon atmosphere containing 2 mL of dry DMF was charged with
100 mg (0.12 mmol) of 2‟-deoxy-5‟-tosylnucleoside and 91.5 mg (0.6 mmol) of
(Bu
4
N
+
)
5
BM
F4
TPA salt. The resulting mixture was stirred for one hour at 100 °C and then left at
room temperature for two days. The conversion was checked by
19
F NMR and HPLC on DEAE-
5PW weak ion-exchange column with 1 M Et
3
NH
+
HCO
3
-
/ H
2
O solution as the eluent. After
reaction achieved maximum conversion and most of the starting material was consumed, all
volatiles were removed under reduced pressure and the remains were suspended in H
2
O / MeOH
/ NH
4
OH (1:1:10) mixture and left at room temperature for three days. After deprotection, all
undissolved residue was filtered off and the filtrate was concentrated in vacuum, then re-
dissolved in water and subjected to HPLC purification on a preparative DEAE-5PW weak ion-
exchange column with water / Et
3
NH
+
HCO
3
-
solution as the eluent (0 to 60% gradient regime).
The buffer solution was removed by evaporation and desired nucleotide analogues were dried
under high vacuum.
General procedure for preparation of nucleosides-5’-tosylates 20-22 and 24
(500 mg) of Benzoyl protected ribonucleoside were dried three times by azeotropic
evaporation of pyridine on a rotary evaporator under vacuum. After that, the flask was filled with
inert gas and the ribonucleoside was redissolved in 25 mL of anhydrous pyridine. Subsequently,
- 17 -

equimolar solution of p-toluenesulfonyl chloride dissolved in dry pyridine was added to the
stirred solution of ribonucleoside precooled to 0 C. The reaction mixture was removed from the
ice and placed into the freezer (-24 °C) for 5 - 7 days. Then the flask was warmed to room
temperature, pyridine was evaporated by rotatory evaporation and the residue was subjected for
chromatographic separation on silica gel using CH
2
Cl
2
/ MeOH (1:10) as eluent. The yields of
5‟-tosylates (20-22 and 24) are shown in Table 1.1.
5’-Tosyl-N,N,2',3'-tetrabenzoyladenosine 21
N
N
N
N
NBz
2
O
OBz OBz
TsO

1
H NMR (400 MHz, CDCl
3
) δ: 2.34 (s, 3H), 4.44 (dd, J = 11 Hz, 4 Hz, 1H), 4.5 (dd, J = 11 Hz, 3
Hz, 1H), 4.66 (m, 1H), 5.93 (dd, J = 6 Hz, J = 4.8 Hz, 1H), 6.11 (dd, J= 6 Hz, 5.4 Hz, 1H), 6.44
(d, J = 5.4 Hz, 1H),7.22-7.56 (m,14H), 7.23-7.93 (m, 10H), 8.29 (s, 1H), 8.54 (s, 1H) ppm.
13
C
NMR (100 MHz, CDCl
3
) δ 21.8, 68.5, 71.5, 74.2, 81, 87.1, 127.9 128.2, 128.4, 128.6, 128.8,
128.8, 129, 129.7, 130, 130.1, 130.3, 132.3, 133.3, 134.1, 143.7, 145.8, 152.3, 152.6, 152.9,
165.2, 165.5, 172.5.
5’-Tosyl-2',3'-O, N4-tribenzoylcytidine 22
N
NHBz
O N
O
OBz OBz
TsO

1
H NMR (400 MHz, CDCl
3
) δ: 2.34 (s, 3H), 4.38 (dd, J = 11.2 Hz, 3.2 Hz, 1H), 4.48-4.55 (m,
2H), 5.58-5.64 (m, 2H), 6.35-6.36 (m, 1H), 7.18-7.56 (m, 12H), 7.78-7.88 (m, 8H).
13
C NMR
(100 MHz, CDCl
3
) δ 21.7, 68.2, 70.9, 74.4, 80.7, 97.5, 127.6, 127.9, 128.4, 128.45, 128.5, 129,
129.6, 129.7, 129.8, 129.9, 130.2, 144.7, 145.6, 154.7, 162.7, 165.1, 165.2, 166.5.

- 18 -

5’-Tosyl-2',3'-O,N3-tribenzoyluridine 23
NBz
O
O N
O
OBz OBz
TsO

1
H NMR (400 MHz, CDCl
3
) δ: 2.44 (s, 3H), 4.4 (dd, J = 11.2 Hz, 3.2 Hz, 1H), 4.49 (dd, J = 11.2
Hz, 2.4 Hz, 1H), 4.55 (m, 1H), 5.6 (m, 1H), 5.7 (m, 1H), 5.87 (d, J = 8 Hz, 1H), 6.35 (d, J =
6 Hz, 1H), 7.24-7.63 (m, 12H), 7.66 (d, J = 8 Hz, 1H), 7.8-7.93 (m, 7H), ppm.
13
C NMR (100
MHz, CDCl
3
) δ 21.7, 68.6, 71.2, 73.5, 80.6, 87.5, 103.4,127.9, 128.1, 128.4, 128.5, 128.6, 129.2,
129.7, 129.8, 130.3, 130.5, 131.1, 132, 133.8, 133.9, 135.2, 139.7, 145.8, 149.4, 161.7, 165.2,
165.3, 168.4.
N-[(dimethylamino)methylene]- 5’-tosyl, 2',3'- dibenzoylguanosine 20
NH
N
N
O
N
N
O
OBz OBz
HO
N
Me
Me

1
H NMR (400 MHz, CDCl
3
) δ: 2.86 (s, 3H), 3.07 (s, 3H), 3.8-4.02 (m 2H), 4.5 (m, 1H), 6.1 (m,
1H), 6.17 (m, 1H), 6.4 (m, 1H), 7.2-7.48 (m, 6H), 7.67-7.9 (m, 5H), 8.52 (s, 1H), 10.4 (s, 1H).
13
C NMR (100 MHz, CDCl
3
) δ: 35, 41.3, 61.6, 71.8, 73.6, 84.1, 87.8, 121.1, 128.4, 128.5, 128.9,
129.6, 129.7, 133.5, 133.6, 138.2, 149.8, 157.5, 158.2, 158.7, 165, 165.4.
N-[(dimethylamino)methylene]-, 2',3'- dibenzoylguanosine 24
NH
N
N
O
N
N
O
OBz OBz
TsO
N
Me
Me

1
H NMR (400 MHz, CDCl
3
) δ: 2.2 (s, 3H), 3.03 (s, 3H), 3.18 (s, 3H), 4.27 (dd, J = 11.6 Hz,
4 Hz, 1 H), 4.4 (dd, J=11.6 Hz, 2.4 Hz, 1H), 4.5 (m, 1H), 6.0 (d, J=2.4 Hz 1H), 6.2 (m, 1H), 6.3
- 19 -

(m, 1H), 7-7.5 (m, 10H), 7.58 (s, 1H), 7.75-7.8 (m, 4H), 8.7 (s, 1H), 10.16 (s, 1H).
13
C NMR
(100 MHz, CDCl
3
) δ: 21.6, 35.3, 41.3, 67.6, 69.8, 73.9, 78.7, 87.9, 121, 127.6, 128.3, 128.4,
128.5, 128.6, 129.6, 129.7, 131.9, 133.7, 133.9, 145.3, 149.7, 157.3, 158.3, 158.8, 165, 165.2.
(α, β), (β, γ) – Bis(CF
2
) ATP 32
O
OH OH
O
N
N
N
N
NH
2
P
O
O
F
2
C P
F
2
C P
O O
O O
O [Et
3
NH
+
]
4

1
H NMR (400 MHz, D
2
O): 1.05 (t, J = 7.6 Hz, 27H, Et
3
NH
+
), 2.97 (q, J = 7.6 Hz, 18H,
Et
3
NH
+
)4.07-4.12 (m, 2H), 4.15-4.19 (m, 1H), 4.25-4.42 (m, 2H), 4.33-4.38 (m, 1H), 4.55-4.59
(m, 1H), 5.92 (d, J = 6.4 Hz, 1H) 8.03 (s, 1H), 8.36 (s, 1H) ppm.
19
F NMR (376 MHz, D
2
O): -
119.2 (t, J = 72.2 Hz), - 120.3 (t, J=83.8Hz) ppm.
31
P NMR (162 MHz, D2O): 1.8-4.8 (m, 2P),
10.9-13.2 (m, 1P) ppm. HRMS: calculated for [M+Na
+
] C
12
H
16
F
4
N
5
O
11
P
3
Na 597.9887, found
597.9891.
(α, β), (β, γ) – Bis(CF
2
) CTP 33
O
OH OH
O P
O
O
F
2
C P
F
2
C P
O O
O O
O [Et
3
NH
+
]
4
N
N
NH
2
O

1
H NMR (400 MHz, D
2
O): 1.1 (t, J = 7.2 Hz, 27H, Bu
3
NH
+
), 2.98 (q, J = 7.2 Hz, 18H, Et
3
NH
+
),
4.07-4.11 (m, 2H), 4.15-4.2 (m, 2H), 4.23-4.27 (m, 1H), 5.86 (d, J = 4.8Hz, 1H), 5.89 (d, J = 7.6
Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H) ppm.
19
F NMR (376 MHz, D
2
O): -118.3 (t, J = 75.2 Hz), -
118.8 (t, 76.7 Hz) ppm.
31
P NMR (162 MHz, D
2
O): 2.56-4.42 (m, 2P), 11.87-14.58 (m, 1P)
ppm. HRMS: calculated for [M+Na
+
] C
11
H
15
F
4
N
3
O
11
P
3
573.9775, found 573.9780.

- 20 -

(α, β), (β, γ) – Bis(CF2) UTP 34
O
OH OH
O
NH
O
O N
P
O
O
F
2
C P
F
2
C P
O O
O O
O [Et
3
NH
+
]
4

1
H NMR (400 MHz, D
2
O): 1.12 (t, J = 7.3 Hz, 27H, Et
3
NH
+
), 3.04 (q, J = 7.3 Hz, 18H, Et
3
NH
+

), 4.07 - 4.12 (m,1H), 4.13-4.19 (m, 2H), 4.2 - 4.25 (M, 2H), 5.78 (d, J = 8 Hz, 1H), 5.81 (d, J =
4.8 Hz, 1H), 7.84 (d, J = 8 Hz, 1H) ppm.
19
F NMR (376 MHz, D
2
O): -119.36 (t, J = 72.94 Hz), -
119.37 (t, J = 76.7 Hz) ppm.
31
P NMR (162 MHz, D
2
O): 2.32-3.9 (m, 2P), 9.64-12.05 (m, 1P)
ppm.
(α, β), (β, γ) – Bis(CF
2
) dGTP 35
NH
N
N
O
NH
2
N
O
OH OH
O P
O
O
F
2
C P
F
2
C P
O O
O O
O [Et
3
NH
+
]
4

1
H NMR (400 MHz, D
2
O): 1.1 (t, J = 7.3 Hz, 27H, Et
3
NH
+
), 3.02 (q, J = 7.3 Hz, 18H, Et
3
NH
+
)
4.11 - 4.22 (m, 4H), 4.36 - 4.41 (m, 1H), 5.72-5.81 (m, 1H), 8.11 (s, 1H) ppm.
19
F NMR (376
MHz, D
2
O): -118.9 (t, J = 76.7 Hz) ppm.
31
P NMR (162 MHz, D
2
O): 2.37 - 4.25 (m, 2P), 11.7 -
14.4 (m, 1P) ppm. HRMS: calculated for [M+H
+
] C
12
H
15
F
4
N
5
O
12
P
3
589.9872, found 589.9875.

- 21 -

1.5 Representative NMR spectra and HPLC chromatograms
HPLC traces of ( , ),( ,)-Bis(CF
2
) ATP

DEAE-5PW HPLC 30 min. gradient regime (100% H
2
O/ 0% 1M TEAB to 40% H
2
O/60% 1M
TEAB), retention time 30.4 minutes.

DEAE-5PW HPLC 30 min. gradient regime (80% H
2
O/ 20% 1M TEAB to 50% H
2
O/50% 1M
TEAB), retention time 26.5 minutes.
- 22 -

DEAE-5PW HPLC 30 min. gradient regime (80% H
2
O/ 20% 1M TEAB to 50% H
2
O/50% 1M
TEAB), retention time 26.5 minutes
1
H NMR of ( , ),( , )-Bis(CF
2
) ATP

- 23 -

19
F NMR of ( , ),( , )-Bis(CF
2
) ATP

- 24 -

31
P NMR of ( , ),( , )-Bis(CF
2
) ATP

- 25 -

HPLC traces of ( , ),( ,)-Bis(CF
2
) CTP

DEAE-5PW HPLC 30 min. gradient regime (100% H
2
O/ 0% 1M TEAB to 40% H
2
O/60% 1M
TEAB), retention time 28.3 minutes

DEAE-5PW HPLC 30 min. gradient regime (85% H
2
O/ 15% 1M TEAB to 50% H
2
O/50% 1M
TEAB), retention time 25.7 minutes.

- 26 -

1
H NMR of ( , ),( , )-Bis(CF
2
) CTP

19
F NMR of ( , ),( , )-Bis(CF
2
) CTP

- 27 -

31
P NMR of ( , ),( , )-Bis(CF
2
) CTP

- 28 -

HPLC traces of ( , ),( ,)-Bis(CF
2
) UTP

DEAE-5PW HPLC 30 min. gradient regime (100% H
2
O/ 0% 1M TEAB to 40% H
2
O/60% 1M
TEAB), retention time 29.5 minutes.

- 29 -

DEAE-5PW HPLC 40 min. gradient regime (100% H
2
O/ 0% 1M TEAB to 40% H
2
O/60% 1M
TEAB), retention time 32.2 minutes.
1
H NMR of ( , ),( , )-Bis(CF
2
) UTP

- 30 -

19
F NMR of ( , ),( , )-Bis(CF
2
) UTP

- 31 -

31
P NMR of ( , ),( , )-Bis(CF
2
) UTP

- 32 -

HPLC traces of ( , ),( ,)-Bis(CF
2
) GTP

DEAE-5PW HPLC 30 min. gradient regime (100% H
2
O/ 0% 1M TEAB to 40% H
2
O/60% 1M
TEAB), retention time 33.5 minutes.

DEAE-5PW HPLC 35 min. gradient regime (100% H
2
O/ 0% 1M TEAB to 40% H
2
O/60% 1M
TEAB), retention time 35.52 minutes.

- 33 -

1
H NMR of ( , ),( , )-Bis(CF
2
) GTP

19
F NMR of ( , ),( , )-Bis(CF
2
) GTP

- 34 -

31
P NMR of ( , ),( , )-Bis(CF
2
) GTP

- 35 -

- 36 -

1.6 REFERENCES
1. Pelletier, H.; Sawaya, M. R.; Kumar, A.; Wilson, S. H.; Kraut, J., Science 1994, 264,
1891.
2. Prasad, R.; Batra, V. K.; Yang, X. P.; Krahn, J. M.; Pedersen, L. C.; Beard, W. A.;
Wilson, S. H., DNA Repair 2005, 4, 1347.
3. Pelletier, H.; Sawaya, M. R.; Wolfle, W.; Wilson, S. H.; Kraut, J., Biochemistry 1996, 35,
12742.
4. Beard, W. A.; Wilson, S. H., Chem. Rev. 2006, 106, 361.
5. Sawaya, M. R.; Prasad, R.; Wilson, S. H.; Kraut, J.; Pelletier, H., Biochemistry 1997, 36,
11205.
6. Molecular Anatomy Project (http://maptest.rutgers.edu/drupal/?q=node/83)
7. Prasad, R.; Beard, W. A.; Wilson, S. H., J. Biol. Chem. 1994, 269, 18096.
8. Sawaya, M. R.; Pelletier, H.; Kumar, A.; Wilson, S. H.; Kraut, J., Science 1994, 264,
1930.
9. Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martinek, V.; Xiang, Y.;
Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florian, J.; Warshel, A.;
Goodman, M. F., Biochemistry 2007, 46, 461.
10. Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.;
Wilson, S. H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F., Biochemistry 2008,
47, 870.
11. McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.;
Pedersen, L. C.; Beard, W. A.; Wilson, S. H., J. Am. Chem. Soc. 2007, 129, 15412.
12. Upton, T. G.; Kashemirov, B. A.; McKenna, C. E.; Goodman, M. F.; Prakash, G. K. S.;
Kultyshev, R.; Batra, V. K.; Shock, D. D.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H., Org.
Lett. 2009, 11, 1883.
13. Prakash, G. K. S.; Zibinsky, M.; Upton, T. G.; Kashemirov, B. A.; McKenna, C. E.;
Oertell, K.; Goodman, M. F.; Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Shock, D. D.; Wilson,
S. H.; Olah, G. A., Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 15693.
14. Zhu, X. F.; Scott, A. I., Synth. Commun. 2008, 38, 1346.
15. Cui, Z. Y.; Zhang, L.; Zhang, B. L., Tetrahedron Lett. 2001, 42, 561.
- 37 -

16. Matulic-Adamic, J.; Beigelman, L., Helv. Chim. Acta 1999, 82, 2141.

- 38 -

Chapter 2 Investigations toward bioisosteric replacement
of phosphoric hydroxyl group with difluoromethyl motif
in nucleotide analogues
2.1 INTRODUCTION
Enzymatic phosphorylation of the biologically active compounds is one of the most vital
regulatory processes in the cell cycle of a living system [1, 2]. Therefore, many phosphorus-
containing molecules including nucleosides are valuable drug candidates. Recently a number of
potent nucleosides and nucleotides analogs have been developed as prodrugs demonstrating
activity against neoplastic and viral infections [3-5].
Unfortunately, increased polarity of the phosphorus containing molecules caused by single or
double negative charge of the phosphate group at nearly all physiological pH values impede
them from being effective drugs candidates. Decreased in lipophilicity of polar ionic compounds
is a most common drug-delivery challenge, which inhibits their ability to penetrate cell
membranes leading to low volume of distribution. To overcome these issues, several drug
delivery approaches have been developed that involve alterations of the phosphate functionality.
The increase in the lipophilicity and membrane permeability can be achieved by introduction of
the proper modification in the phosphate group that masks the negative charge [6, 7].
As a next step in our proceeding investigations of the phosphate mimics, we decided to
employ neutral difluoromethyl (CF
2
H) group as isosteric and isopolar replacement of the ionic
phosphate hydroxyl group. Incorporation of such modified phosphate group should improve
lipophilic properties of the poorly adsorbed nucleotides analogs [8].
Incorporation of the fluorine atom into biologicaly active molecules usually leads to drastic
change in physical and chemical properties. Therefore the unique chemical, biological and
structural properties of the fluorinated nucleosides already acquired great deal of attention in the
medicinal chemistry field. Over the past several years a number of efficient and selective
synthetic methodologies toward synthesis of biologically interesting organofluorine compounds
have also been developed [9-11]. Moreover, several fluorinated nucleoside analogues are already
approved for the treatment of viral infections including cancer [10].
- 39 -

Steric considerations revealed that the van der Waals radius of fluorine is (1.35 Å), which is
close to van der Waals radii of oxygen (1.4 Å) and hydrogen (1.2 Å) atoms respectively.
Replacement of hydrogen with fluorine atom causes minimal perturbation in molecular
geometry, retaining complementary relationship between active site of the enzyme and the
compound of interest (Figure 2.1). The size similarities do not allow enzymes and
microorganisms to distinguish between fluorinated and nonfluorinated analogues and permits
them to penetrate into the cells and be involved in the metabolic processes [12].
CF
2
H
isosteric
isopolar
OH
CF
2
isosteric
isopolar
O
(etheral)
C-F
mimic
C-H C-O
1.39 Å 1.1 Å 1.43Å

Figure 2.1.
Difluoromethyl group can be considered as mimic of the hydroxyl group based on the spatial
arrangement of the fluorine atoms in this group that resemble the positioning of the oxygen lone
pairs. Even carbon-fluorine bond sometimes viewed as isopolar and isosteric replacement of a
hydroxyl group because C-O bond length (1.43 Å) is similar to the C-F bond length (1.39 Å).
Indeed due to the high electronegativity of fluorine (4.0 on Pauling scale), its lone pairs
sometimes can act as a hydrogen bond acceptor. On the other hand, the elevated C-H acidity of
the difluoromethyl group allows it to participate in the hydrogen bonding interactions as a
suitable donor (Figure 2.2) [11].
O C
F F

Figure 2.2. The resemblance of the electronic structure and spatial arrangement between oxygen
atom and difluoromethylene group.
Metabolic oxidation reaction involving C-F bond is much less probable because of the high
bond dissociation energy (489 kJ mol
-1
) eliminating the possibility of formation of reactive
radical species during homolitic cleavage processes.
- 40 -

Incorporation of the fluorine atom frequently lead to the stabilization effect on the adjacent
functional groups because it destabilizes any plausible positively charged intermediates or
transition states. In cases when stability of the drug candidate is a crucial factor, such
modification can be used to stabilize essential fragment of the molecule [11, 13]. Biologically
interesting molecules that possess fluorine-containing modifications usually expected to exhibit
increase in hydrophobicity and thus enhancing rate of adsorption of drugs and improved
bioavailability due to increased metabolic stability allowing administration of lower doses of
drugs [13].
Thus we decided to apply well recognized isosteric and isololar concept to the series of
nucleotide analogues that demonstrate poor bioavailability. Substitution of the ionic hydroxyl
group with appropriate mimic function can considerably improve adsorption of the drug
candidate [11, 14].
2.2 RESULTS AND DISCUSSION
Preparation of the difluoromethyl-containing phosphate isosteres
For the preparation of 5'-difluoromethylphosphonates of the various nucleosides, we decided
to employ dichloride 3 as our main electrophilic source of difluoromethylphosphonate function.
Diethyldifluoromethyl phosphonate prepared as described above was treated with the
bromotrimethylsilane for several days. After removing all volatile material and without further
purification, silyl ester 2 was reacted with oxalyl chloride in the presence of catalytic amount of
DMF. The progress of the reaction was monitored by
31
P NMR. When all starting material was
consumed, volatiles were evaporated and difluoromethanephosphonyl dichloride 3 was obtained
in good yield, 87%. Reaction proceeds efficiently with the conversion value close to 100%,
which is extremely important because compound 3 is very moisture sensitive and additional
purification steps significantly decrease the yield (Scheme 2.1).
Scheme 2.1. Preparation of the difluoromethanephosphonyl dichloride 3

O
P CF
2
H
OEt
EtO
TMSBr
O
P CF
2
H
OTMS
TMSO
(COCl)
2
O
P CF
2
H
Cl
Cl
1 2 3

- 41 -

Preparation of nucleotide analogues with the difluoromethyl-modified phosphate moiety
To react difluoromethanephosphonyl dichloride 3 with a number of structurally modified
nucleosides, we implemented the strategy that is widely used for the preparation of different
phosphoramidates [15-18]. The reaction is usually carried out in THF. However, certain
nucleosides exhibit very low solubility in this solvent. To overcome this issue, the reactions were
performed in the solvent mixture THF/pyridine (7:3). After screening several bases, we found
that tert-butyl magnesium chloride is the most efficient base for providing the best conversion
and yield. The nucleosides were reacted with the difluoromethanephosphonyl dichloride 3 in
presence of the tert-butyl magnesium chloride as a base (Scheme 2.2). After reaction achieved
maximum conversion tributylamine was added and followed by quenching reaction mixture with
ice/water mixture. In some cases, the reaction was quenched with 1 M solution of tetrabutyl
ammonium hydroxide in H
2
O/MeOH. 5'-Difluoromethylphosphonates in a free acid form are
rather unstable. Isolation of these nucleotides represents a significant challenge. Carrying out
purification using normal phase (silica gel) chromatography usually leads to decomposition of
much of the material. On the other hand, purification via reverse phase chromatography was a
quite challenging task because desired nucleotide analogues generally eluted from the column
with the dead volume without separation.
Scheme 2.2.
O
P CF
2
H
Cl
Cl + Nucleoside
O
P HF
2
C NUC
Cl
1. Bu
3
N
O
P HF
2
C NUC
O
Bu
3
NH
+
3
t-BuMgCl
THF/Py, rt 2. H
2
O/MeOH
4a-4l

- 42 -

Table 2.1. Chemical structures of nucleotides 5'-difluoromethylphosphonates and their isolated
yields.

Nucleotide 5'-
difluoromethylphosphonate
Yield
%

Nucleotide 5'-
difluoromethylphosphonate
Yield
%
4a
O
OH
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+

35 4f
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
3
NH
+

37
4b
O
N
3
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+

65 4g
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+
OH
F

85
4c
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+

53 4h
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+
OH
Br

21
4d
O
OH
O
N
N
NH
2
O
F
F
P
O
O
HF
2
C
Bu
4
N
+

92 4i
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+
F

43
4e
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+
F OH

79 4j
NH
N
N
O
NH
2
N
O
O P
O
HF
2
C
O
Bu
4
N
+

6

- 43 -

Therefore, in order to increase stability of the compounds and achieve reasonable retention
times on C18 column, nucleotides 5'-difluoromethylphosphonates were transformed in situ into
the tributylammonium or tetrabutylammonium salts. Unfortunately, it was noted that even
though the stability of tributylammonium salts is much greater than the corresponding acid, the
salt still undergoes slow hydrolysis in water/methanol solutions. The yields of the isolated
nucleotide 5‟-difluoromethylphosphonates are represented in Table 2.1.
In the next step in our investigation towards substitution of one of the phosphoric hydroxyl
group with difluoromethyl group, we decided to attempt the synthesis of non-hydrolyzable
bisphosphate and trisphosphate containing nucleotides. To access such modified nucleotide
analogues, we developed the synthetic protocol for the preparation of difluoromethyl-methylene
bisphosphonate structural motif and the corresponding acid which are considered to be the key
substrates in this endeavor (Figure 2.3).
P
F
2
HC
P
10 12
O O
O
O O
[Bu4N
+
]
3
P
F
2
HC
P
O O
OEt
N OEt
P
F
2
HC
P
O O
OH
HO OH
11

Figure 2.3.
Scheme 2.3. Preparation of diethyl[ethoxy(tert-butyldimethylsilyldifluoromethyl)
phosphoryl]phosphonate 9.
TMSBr
O
P CF
2
H
OEt
EtO
LDA, TBDMSCl
1
THF, -78
o
C
O
P
F
2
C
OEt
EtO Si
CH
2
Cl
2
O
P
F
2
C
OTMS
TMSO Si
(COCl)
2
,
DMF (cat.),
CH
2
Cl
2
O
P
F
2
C
Cl
Cl Si
O
P CH
3
OEt
EtO
1. BuLi, THF, -78
o
C
O
P
F
2
C
OEt
H
2
C Si P
O
EtO
OEt
5 6
7 9
2. LiOEt/THF
+

- 44 -

Scheme 2.4. Preparation of [(hydroxydifluoromethylphosphinyl)methyl]- phosphonic acid
tetrabutylammonium salt 10.

O
P
F
2
C
OEt
H
2
C Si P
O
EtO
OEt
9
1. TBAF, THF
P
F
2
HC
P
2. TMSBr/ CH
3
CN
3. Bu
4
N
+
OH
-
/H
2
O
10
O O
O
O O
[Bu4N
+
]
3

The Scheme 2.3 represents the synthetic route towards difluoromethyl-methylene-
bisphosphosphoric acid. In the first step of this protocol, TBDMS group was used to protect
difluoromethyl group, which exhibit rather high acidity and interfere with the strong lithium
bases. Trimethylsilyl protection cannot be utilized instead because it is too labile and readily
removed under acidic conditions used for deprotection of ester groups. Subsequently the general
approach described above to transform phosphorus ethyl ester functional group into phosphonyl
dichloride was utilized. Compound 7 obtained after treatment with oxalyl chloride was coupled
with methyl phosphonate without extra purification. It was noticed that under the described
conditions, only one equivalent of diethyl-methylphosphonate reacts with the substrate 7. In
order to simplify purification steps, reaction mixture was quenched with lithium ethoxide
affording TBDMS-protected difluoromethyl bisphosphonate 9 (Scheme 2.3).
TBDMS group of the compound 9 was deprotected by treating it with the 1M TBAF solution
in THF at room temperature. Finally all ethyl ester groups were removed after reaction with
bromotrimethylsilane and the resulting silyl ester was quenched with water giving access to the
corresponding acid. Without isolation, the acid was reacted with tetrabutylammonium hydroxide
and the obtained salt 10 was purified by reverse phase chromatography.
We also found alternative approach toward desired structural motif. Even though this method
provides similar yield, it can be useful in some cases. The key step in this approach is reaction of
phosphonyl dichloride 7 with 1 equivalent of the lithium diisopropylamide (LDA). LDA usually
reacts with various phosphonyl chlorides with highly regiospecificity, and in this case only one
equivalent of LDA will react with 7 producing compound 11. The latter readily reacts with
methyl phosphonate under general coupling conditions (Scheme 2.5). After performing all
necessary work up procedures, we found that TBDMS protecting group was removed during
isolation and purification steps giving access to 12. Subsequently the compound 12 underwent
- 45 -

the same treatment as described for 9 affording [(hydroxydifluoromethylphosphinyl)methyl]-
phosphonic acid tetrabutylammonium salt, 10.
Scheme 2.5. Alternative synthetic approach toward [(hydroxydifluoromethylphosphinyl)methyl]
-phosphonic acid tetrabutylammonium salt 10.
O
P N
CHF
2
O
P
F
2
C
Cl
Cl Si
O
P CH
3
OEt
EtO
BuLi, THF,
-78
o
C to rt
P
O
OEt
OEt
7 12
1 eq. LDA/THF
O
P
F
2
C
N
Cl Si
P
F
2
HC
P
1. TMSBr/ CH
3
CN
2. Bu
4
N
+
OH
-
/H
2
O
10
O O
O
O O
[Bu4N
+
]
3
11

2.3 CONCLUSIONS
The approach toward synthesis of nucleotides-5'-difluoromethylphosphonate was developed.
A number of diverse nucleotide monophosphate analogues (4a-4l) was synthesized, purified and
fully characterized showing the versatility of this method. Additionally, the synthetic route to
[(hydroxydifluoromethylphosphinyl)methyl]-phosphonic acid 11 was discovered. The acid was
synthesized, purified and characterized in the form of tetrabutylammonium salt 10. This
structural fragment allows the access of more complex hydrolyzable and non-hydrolyzable
nucleotide diphosphates and trisphosphates analogues. Such nucleotide analogues are not only
valuable probes for studying function and structure of different enzymes, but they will also
permit further exploration of the ability of difluoromethyl motif to mimic non-bridging
phosphate hydroxyl group.

- 46 -

2.4 EXPERIMENTAL PART
Preparation of difluoromethanephosphonyl dichloride 3
O
P CF
2
H
Cl
Cl

Solution of 5 g (26.5 mmol) of diethyl (difluoromethyl)phosphonate in 20 mL CH
2
Cl
2
was
placed in a flask filled under nitrogen gas. Next, 10.5 mL of bromotrimethylsilane (12.2 g,
80 mmol) was added to the stirred solution and the reaction mixture was allowed to stir at room
temperature for four days. All volatiles were removed using a rotory evaporator with 10 torr max
vacuum applied. The flask was again kept under N
2
atmosphere and 20 mL of anhydrous CH
2
Cl
2

was added to dissolve the residue. The flask was then connected to the nitrogen line and 6.8 mL
(10.07 g, 80 mmol) of oxalyl chloride was introduced followed by several drops of DMF.
Evolution of large amount of gas was observed. The reaction mixture was then allowed to stir for
three days at room temperature under N
2
. After that, all volatiles were removed using vacuum
(50 torr) and the residue was separated to obtain compound 3 (3.89 g, 87 %) which, was used
without further purification.
19
F NMR (376 MHz, CDCl
3
) δ -129.20 (dd, J = 115.3, 49.2 Hz).
31
P NMR (162 MHz, CDCl
3
) δ
25.78 (t, J = 115.3 Hz).
General procedure for preparation of nucleosides-5'-difluoromethylphosphonate 4a-4l
Appropriate nucleoside (1 mmol) was placed in the round bottom flask filled with argon
atmosphere and 15 ml of freshly distilled THF. Subsequently tert-butyl magnesium chloride (2.4
mmol of 1 M solution in THF) was added dropwise to the suspension and the mixture was stirred
at room temperature for 30 minutes. Solution of difluoromethanephosphonyl dichloride
(3.6 mmol) dissolved in dry THF (3 mL) was added to the reaction mixture and the stirring was
continued for 24 hours. After that, tributylamine or tetrabutylammonium hydroxide was added to
the stirred solution and the reaction was quenched with the addition of ice cold water. All
volatiles were removed by evaporation and the mixture was subjected to column chromatography
on C18 column with water/methanol solution as the eluent.

- 47 -

Preparation of diethyl (tert-butyldimethylsilyl)difluoromethylphosphonate 5
O
P
F
2
C
OEt
EtO Si

Under inert atmosphere (N
2
), solution of 5 g (26.5 mmol) of the diethyl
(difluoromethyl)phosphonate 1 in 60 mL of freshly distilled THF was cooled to -78 °C in dry
ice/acetone bath. 14 mL of 2 M (28 mmol) solution of lithium diisopropylamide (LDA) was
added dropwise in to the flask. The reaction was stirred for 30 minutes under this temperature
when solution TBDMSCl 3.99 g (26.5 mmol) in 10 mL of dry THF was introduced. The cooling
bath was then removed and reaction mixture was allowed to warm up slowly to room
temperature within an hour. All volatiles were removed under reduced pressure and the residue
was distributed between water and hexane. Organic fractions were washed 3 times with water
and dried over MgSO
4
. Hexane was evaporated leaving 6.74 g (84 %) of the desired compound 5
as an yellowish oil, which was used without further purification.
1
H NMR (400 MHz, CDCl
3
) δ 4.06 – 3.96 (m, 4H), 1.13 (t, J = 7.1 Hz, 6H), 0.78 (s, 9H), 0.00
(s, 6H).
13
C NMR (100 MHz, CDCl
3
) δ 128.26 (td, J = 272.7, 163.1 Hz), 63.69 (d, J = 7.1 Hz),
26.5, 20.9, 16.31 (d, J = 5.5 Hz), -7.8.
31
P NMR (162 MHz, CDCl
3
) δ 8.82 (t, J = 92.8 Hz).
19
F NMR (376 MHz, CDCl
3
) δ -126.03 (d, J = 92.8 Hz).
Preparation of (tert-butyldimethylsilyl)difluoromethanephosphonyl dichloride 7
O
P
F
2
C
Cl
Cl Si

tert-Butyldimethylsilyl)difluoromethylphosphonate 5 (5 g, 16.5 mmol) was placed under
argon atmosphere and dissolved in 40 mL of anhydrous CH
2
Cl
2
. Then, 5.2 mL (39.6 mmol)
bromotrimethylsilane was slowly added to the stirred solution of 5. The reaction mixture was
stirred at room temperature for three days. All volatiles were removed under reduced pressure
and the residue was redissolved in dry CH
2
Cl
2
. 8.5 mL of oxalyl chloride (99 mmol) was added
dropwise to the stirred solution followed by several drops of DMF. The reaction mixture was left
under stirring at this temperature for three days under N
2
. All volatiles were evaporated leaving
- 48 -

dark brown oil containing more than 95 % of the target material 4.53 g (97 %), which was used
without further purification.
1
H NMR (400 MHz, CDCl
3
) δ 0.69 (s, 9H), 0.00 (s, Hz, 6H).
13
C NMR (100 MHz, CDCl
3
) δ
127.89 (td, J = 294.0, 94.5 Hz), 26.52, 18.03, -7.0.
19
F NMR (376 MHz, CDCl
3
) δ -115.94 (d, J
= 122.5 Hz, 2F).
31
P NMR (162 MHz, CDCl
3
) δ 36.55 (t, J = 122.5 Hz, 1P).
Diethyl[ethoxy(tert-butyldimethylsilyldifluoromethyl) phosphoryl]phosphonate 9
O
P
F
2
C
OEt
H
2
C Si P
O
EtO
OEt

Under argon atmosphere, a 250 mL round bottom flask was charged with 2.28 g (15 mmol)
diethyl methylphosphonate and 60 mL of freshly distilled THF. The stirred solution was cooled
to -78 °C and 6.6 mL (16.5 mmol) of 2.5 M solution of n-butyllithium in hexanes was added
dropwise. After 30 minutes stirring at this temperature, the solution of the 7 (2.84 g, 10 mmol) in
5 mL of THF was introduced. The resulting mixture was kept at -78 °C for two more hours when
0.78 g (15 mmol) of lithium ethoxide in 5 mL of THF was added and the reaction mixture was
allowed to warm slowly to room temperature. All volatiles were removed under reduced pressure
and the residue was distributed between water and hexane. Organic fraction was collected and
washed 3 times with distilled water and dried over MgSO
4
. The drying agent was filtered off, the
solution was evaporated and purified by flash chromatography on silica gel (80 % EtOAc in
Hexane). Compound 9 (1.3 g, 32 %) was isolated pure as a colorless oil.
1
H NMR (400 MHz, CDCl
3
) δ 4.26 – 3.83 (m, 6H), 2.47 – 2.04 (m, 2H), 1.18 – 1.05 (m, 9H),
0.77 (s, 9H), 0.04 (s, 3H), -0.00 (s, 3H).
13
C NMR (100 MHz, CDCl
3
) δ 127.25 (dt, J = 330.5,
243.8 Hz), 64.48 – 61.96 (m), 60.38, 26.48, 23.08 (dd, J = 134.5, 76.9 Hz), 21, 17.5, 16.13, -7.3.
19
F NMR (376 MHz, CDCl
3
) δ -123.54 (dd, J = 404.4, 80.6 Hz, 1F), -126.13 (dd, J = 404.4, 99.0
Hz, 1F).
31
P NMR (162 MHz, CDCl
3
) δ 33.95 – 32.41 (m), 19.56 (d, J = 19.6 Hz).
Phosphonic acid, [[(diisopropylamino)(difluoromethyl)phosphinyl]methyl]- diethyl ester 12
O
P N
CHF
2
P
O
OEt
OEt

- 49 -

Under an argon atmosphere, the solution of 1.67 g (11 mmol) diethyl methylphosphonate in
anhydrous 60 mL THF was cooled to -78 °C. 4.5 mL (11 mmol) n-butyllithium (2.5 M in
hexanes) was added dropwise to the stirred solution and the mixture was stirred at -78 °C for 30
minutes. In a separate flask kept under N
2,
the solution of compound 7 (2.84 g, 10 mmol) in 5
mL of anhydrous THF was cooled to -40 °C. 5 mL (10 mmol) of a solution of lithium
diisopropyl amide (LDA, 2 M in heptane) was added dropwise to the solution of 7. After stirring
this mixture for 30 minutes at -40 °C, the temperature was raised to ambient temperature and the
stirring was continued for another hour. Subsequently, the second solution was added to the
mixture containing diethyl methylphosphonate cooled to-78 °C. The reaction mixture was stirred
at -78 °C for two additional hours and then cooling bath was removed and the temperature was
allowed to slowly rise to room temperature. All volatiles were removed in vacuum and the
residue was distributed between water and ethyl acetate. Organic fraction was collected and
washed 3 times with distilled water and dried over MgSO
4
. The drying agent was filtered off and
after solvent was evaporated, the reaction mixture was purified by flash chromatography on
silica gel (85 % EtOAc in Hexane). Pure 12 was isolated as a colorless oil.
1
H NMR (400 MHz, CDCl
3
) δ 6.03 (td, J = 49.5, 25.7 Hz, 1H), 4.25 – 4.09 (m, 4H), 3.46 – 3.30
(m, 2H), 2.67 – 2.50 (m, 1H), 2.46 – 2.26 (m, 1H), 1.34 – 1.20 (m, 18H).
13
C NMR
(101 MHz, CDCl
3
) δ 115.46 (dt, J = 263.4, 131.7 Hz), 62.80 (dd, J = 40.4, 6.4 Hz), 47.02 (d, J =
4.4 Hz), 25.50 (dd, J = 132.4, 76.4 Hz), 22.96 (d, J = 53.3 Hz), 16.31 (dd, J = 6.4, 2.4 Hz).
19
F NMR (376 MHz, CDCl
3
) δ -131.21 (ddd, J = 124, 70.1, 49.5 Hz, 1F), -137.28 (ddd, J =
124.0, 75.4, 49.5 Hz, 1F).
31
P NMR (162 MHz, CDCl
3
) δ 25.13 – 23.91 (m, 1P), 18.94 (d, J =
6.6 Hz, 1P).
[(hydroxydifluoromethylphosphinyl)methyl]-phosphonic acid tetrabutylammonium salt 10
P
F
2
HC
P
O O
O
O O
[Bu4N
+
]
3

Solution of 1g (2.45 mmol) of compound 9 in anhydrous acetonitrile was placed in a flask
equipped with a magnetic stirrer under argon atmosphere. 2.6 mL (19.6 mmol) of
bromotrimethylsilane was added and the reaction mixture was left at room temperature for five
- 50 -

days under stirring. After that, the mixture was concentrated in vacuum and distributed between
water and Et
2
O. The water layer was then extracted three times with Et
2
O. The solution of the
tetrabutyl ammonium hydroxide (1 M in water) was added dropwise to the water solution of the
resulted acid 11 until pH reached 8. The water was removed by evaporation and the salt 10 was
purified by flash chromatography on C18 with water/methanol as eluent.
Alternatively, 1 g (2.86 mmol) of compound 12, in anhydrous acetonitrile was placed in a
flask equipped with a magnetic stirrer under argon atmosphere. 3 mL (22.9 mmol, 8 eq.) of
bromotrimethylsilane was added and the reaction mixture was left at room temperature for five
days under stirring.
1
H NMR (400 MHz, CD
3
OD) δ 6.75 (td, J = 50.3, 25.1 Hz, 1H), 3.85 – 3.78 (m, 24H), 2.74 –
2.63 (m, 2H), 2.30 – 2.18 (m, 24H), 2.06 – 1.92 (m, 24H), 1.60 (t, J = 7.4 Hz, 36H).
13
C NMR
(100 MHz, CD
3
OD) δ 114.69 (td, J = 257.6, 136.5 Hz), 58.03, 28.28 (dd, J = 116.5, 81.6 Hz),
23.36, 19.29, 12.54.
19
F NMR (376 MHz, CD
3
OD) δ -140.21 (dd, J = 73.2, 50.3 Hz).
31
P NMR
(162 MHz, CD
3
OD) δ 19.38 (td, J = 73.2, 3.3 Hz), 12.77 (d, J = 3.3 Hz).
Thymidine-5'-difluoromethylphosphonate 4a
O
OH
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+

1
H NMR (400 MHz, cd
3
od) δ 7.79 (d, J = 1.2 Hz, 1H), 6.35 (dd, J = 8.2, 6.0 Hz, 1H), 5.88 (td, J
= 49.4, 23.2 Hz, 1H), 4.52 – 4.42 (m, 1H), 4.18 (dddd, J = 11.5, 7.7, 5.4, 2.8 Hz, 2H), 4.06 –
3.97 (m, 1H), 3.29 – 3.16 (m, 8H), 2.32 – 2.13 (m, 2H), 1.91 (d, J = 1.2 Hz, 3H), 1.72-1.61 (m,
8H), 1.48 – 1.35 (m, 8H), 1.09 – 0.97 (m, 12H).
13
C NMR (100 MHz, cd
3
od) δ 165.04, 151.00,
136.52, δ 114.32 (td, J = 256.1, 196.1 Hz) 110.58, 86.15 (d, J = 6.8 Hz), 84.61, 71.44, 65.54 (d, J
= 6.1 Hz), 58.03, 39.51, 23.35, 19.29, 12.54, 11.03.
19
F NMR (376 MHz, cd
3
od) δ -134.90 (dd, J
= 78.0, 49.4 Hz, 2F).
31
P NMR (162 MHz, cd
3
od) δ 2.08 (t, J = 78.0 Hz). HRMS: ESI, Negative
mode. Calculated for [M-H]
-
C11H14F2N2O7P 355.05122, found 355.05139 (100%).

- 51 -

3’-Azido-3’-deoxythymidine-5'-difluoromethylphosphonate 4b
O
N
3
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+

1
H NMR (399 MHz, cd
3
od) δ 7.76 (d, J = 1.2 Hz, 1H), 6.29 – 6.19 (m, 1H), 5.89 (td, J = 49.4,
23.3 Hz, 1H), 4.48 (dt, J = 6.7, 3.4 Hz, 1H), 4.28 – 4.16 (m, 2H), 4.10 – 4.00 (m, 1H), 3.27 –
3.18 (m, 8H), 2.49 – 2.29 (m, 2H), 1.91 (d, J = 1.2 Hz, 3H), 1.74 – 1.58 (m, 8H), 1.48 – 1.34 (m,
8H), 1.02 (t, J = 7.4 Hz, 12H).
13
C NMR (100 MHz, cd
3
od) δ 164.98, 150.93, 136.25, 114.99
(dt, J = 255.9, 128.0 Hz), 110.75, 84.31, ), 83.26 (d, J = 6.8 Hz), 65.32 (d, J = 6.0 Hz), 61.24,
58.60 – 57.61 (m), 36.68, 23.35, 19.29, 12.56, 11.06.
19
F NMR (376 MHz, cd
3
od) δ -134.84 (dd,
J = 78.3, 49.4 Hz).
31
P NMR (162 MHz, cd
3
od) δ 2.09 (t, J = 78.3 Hz). HRMS: ESI, Negative
mode. Calculated for [M-H]
-
C11H13F2N5O6P 380.05770, found 380.05757 (100%).
3'-deoxy-2',3'-didehydro-thymidine-5'-difluoromethylphosphonate 4c
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+

1
H NMR (399 MHz, cd
3
od) δ 7.68 (d, J = 1.2 Hz, 1H), 7.04 – 6.89 (m, 1H), 6.49 – 6.38 (m, 1H),
5.93 – 5.89 (m, 1H), 5.81 (td, J = 49.5, 23.0 Hz, 1H), 5.51 (s, 1H), 4.18 (dddd, J = 11.8, 8.2, 6.0,
2.9 Hz, 2H), 3.29 – 3.18 (m, 8H), 1.89 (d, J = 1.2 Hz, 3H), 1.65 (s, 8H), 1.49 – 1.35 (m, 8H),
1.02 (t, J = 7.2 Hz, 12H).
13
C NMR (100 MHz, cd
3
od) δ 165.19, 151.43, 137.28, 133.98, 126.17,
114.22 (dt, J = 255.2, 195.3 Hz) 110.57, 89.38, 85.81 (d, J = 6.8 Hz), 66.22 (d, J = 5.9 Hz),
58.03, 23.38, 19.30, 12.58, 10.93.
19
F NMR (376 MHz, cd
3
od) δ -134.78 (ddd, J = 78.0, 49.5,
7.3 Hz).
31
P NMR (162 MHz, cd
3
od) δ 1.93 (t, J = 78.0 Hz). HRMS: ESI, Negative mode.
Calculated for [M-H]
-
C11H12F2N2O6P 337.04065, found 337.04062 (100%).
- 52 -

2’-Deoxy-2’,2’-difluorocytidine-5'-difluoromethylphosphonate 4d
O
OH
O
N
N
NH
2
O
F
F
P
O
O
HF
2
C
Bu
4
N
+

1
H NMR (399 MHz, cd
3
od) δ 7.81 (dd, J = 7.6, 1.4 Hz, 1H), 6.26 (t, J = 8.6 Hz, 1H), 5.87 (d, J =
7.6 Hz, 1H), 5.78 (tdd, J = 49.5, 23.4, 7.9 Hz, 1H), 4.78 (dd, J = 9.0, 6.3 Hz, 1H), 4.34 (dd, J =
10.3, 6.3 Hz, 1H), 4.24 – 4.07 (m, 2H), 3.17 – 3.09 (m, 8H), 1.68 – 1.46 (m, 8H), 1.43 – 1.22 (m,
8H), 0.92 (t, J = 7.4 Hz, 13H).
13
C NMR (100 MHz, cd
3
od) δ 166.31, 156.35, 141.80, 121.18 (t,
J = 260.6 Hz), 114.03 (tdd, J = 255.7, 198.3, 41.0 Hz), 95.17, 83.91 (dd, J = 38.9, 22.2 Hz),
79.90, 73.73 – 71.37 (m), 63.09 (d, J = 5.4 Hz), 58.05, 23.35, 19.28, 12.55.
19
F NMR (376 MHz,
cd
3
od) δ -117.51 (d, J = 242.3 Hz), -119.39 (d, J = 242.3 Hz), -133.93 – -136.11 (m).
31
P NMR
(162 MHz, cd
3
od) δ 1.45 (m, 1H)two triplets. HRMS: ESI, Negative mode. Calculated for [M-
H]
-
C10H11F4N3O6P 376.03271, found 376.03269 (100%).
2’-Deoxy-2’-fluorouridine-5'-difluoromethylphosphonate 4e
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
3
N
+
F OH

1
H NMR (399 MHz, d
2
o) δ 7.62 (d, J = 8.1 Hz, 1H), 6.06 – 5.76 (m, 1H), 5.89 – 5.83 (m, 1H),
5.75 (d, J = 8.1 Hz, 1H), 5.39 – 5.17 (m, 1H), 4.94 – 4.76 (m, 1H), 4.37 – 4.24 (m, 1H), 3.97 –
3.74 (m, 2H), 3.05 – 2.95 (m, 6H), 1.64 – 1.46 (m, 6H), 1.33 – 1.16 (m, 6H), 0.80 (t, J = 7.4 Hz,
9H).
13
C NMR (100 MHz, d
2
o) δ 165.24, 150.14, 142.44, 112.44 (td, J = 254.9, 198.5 Hz),
101.47, 90.48 (d, J = 189.6 Hz), 89.55 (d, J = 36.5 Hz), 79.39 (d, J = 6.6 Hz), 72.27 (dd, J =
15.2, 5.6 Hz), 71.11 (dd, J = 15.3, 5.6 Hz).51.77, 24.33, 18.43, 11.94.
19
F NMR (376 MHz, d
2
o)
δ -129.56 – -130.05 (m, 2F), -187.45 – -187.74 (m, 1F, minor diasteriomer pick), -187.87 – -
- 53 -

188.25 (m, 1F, major diasteriomer pick).
31
P NMR (162 MHz, d
2
o) δ 2.83 (t, J = 82.7 Hz).
HRMS: ESI, Negative mode. Calculated for [M-H-H
2
O]
-
C10H9F3N2O6P 341.01558, found
341.01578 (100%).
2’,3’-Dideoxythymidine-5'-difluoromethylphosphonate 4f
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
3
N
+

1
H NMR (399 MHz, d
2
o) δ 7.56 (q, J = 1.1 Hz, 1H), 6.01 – 5.96 (m, 1H), 5.79 (dt, J = 48.8, 24.4
Hz, 1H), 4.24 – 4.16 (m, 1H), 4.15 – 3.95 (m, 2H), 3.03 – 2.91 (m, 6H), 2.36 – 2.21 (m, 1H),
2.07 (s, 3H), 2.04 – 1.83 (m, 3H), 1.57 – 1.45 (m, 6H), 1.28 – 1.16 (m, 6H), 0.77 (t, J = 7.4 Hz,
9H).
13
C NMR (100 MHz, d
2
o) δ 166.53, 151.63, 137.44, 113.54 (td, J = 254.9, 196.5 Hz),
111.05, 85.87, 79.95 (d, J = 6.8 Hz), 67.73, 66.65 (d, J = 6.0 Hz), 52.55, 30.87, 25.09, 24.91,
19.18, 12.67, 11.45.
19
F NMR (376 MHz, d
2
o) δ -134.37 (dd, J = 81.0, 48.8 Hz).
31
P NMR (162
MHz, d
2
o) δ 3.54 (t, J = 81.0 Hz). HRMS: ESI, Negative mode. Calculated for [M-H]
-

C11H14F2N2O6P 339.05630, found 339.05629 (100%).
2’-Deoxy-5-fluorouridine-5'-difluoromethylphosphonate 4j
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
3
N
+
OH
F

1
H NMR (399 MHz, d
2
o) δ 7.82 (d, J = 6.3 Hz, 1H), 6.23 – 6.12 (m, 1H), 5.84 (td, J = 48.9, 24.6
Hz, 1H), 4.91 – 4.80 (m, 1H), 4.32 – 4.21 (m, 1H), 3.82 – 3.70 (m, 2H), 3.02 – 2.94 (m, 6H),
2.51 – 2.40 (m, 1H), 2.39 – 2.27 (m, 1H), 1.57 – 1.47 (m, 6H), 1.28 – 1.17 (m, 6H), 0.79 (t, J =
7.4 Hz, 9H).
13
C NMR (100 MHz, d
2
o) δ 159.20 (d, J = 26.0 Hz), 149.80, 141.78, 139.45,
125.50 (d, J = 34.3 Hz), 113.33 (td, J = 254.9, 197.5 Hz), 85.31, 84.39 (d, J = 5.2 Hz), 76.47 (d,
- 54 -

J = 5.8 Hz). 75.69 (d, J = 5.8 Hz), 52.55, 43.75, 25.10, 19.20, 12.71.
19
F NMR (376 MHz, d
2
o) δ
-134.74 (dd, J = 81.8, 48.9 Hz, 2F), -165.52 (d, J = 6.3 Hz, 1F).
31
P NMR (162 MHz, d
2
o) δ 2.86
(t, J = 81.8 Hz). HRMS: ESI, Negative mode. Calculated for [M-H-H
2
O]
-
C10H9F3N2O6P
341.01558, found 341.01564 (100%).
5-(E)-(2-Bromovinyl)-2’-deoxyuridine-5'-difluoromethylphosphonate 4h
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+
OH
Br

1
H NMR (399 MHz, cd
3
od) δ 7.99 (s, 1H), 7.40 (d, J = 13.5 Hz, 1H), 6.95 (d, J = 13.5 Hz, 1H),
6.34 (t, J = 7.0 Hz, 1H), 5.88 (td, J = 49.4, 23.2 Hz, 1H), 4.56 – 4.44 (m, 1H), 4.28 – 4.12 (m,
2H), 4.05 (m, 1H), 3.27 – 3.18 (m, 8H), 2.34 – 2.21 (m, 2H), 1.75 – 1.55 (m, 8H), 1.46 – 1.36
(m, 8H), 1.02 (t, J = 7.3 Hz, 12H).
13
C NMR (100 MHz, cd
3
od) δ 162.33, 149.82, 138.67,
129.17, 114.33 (td, J = 255.8, 195.6 Hz), 111.05, 107.67, 86.43 (d, J = 6.5 Hz), 85.15, 71.45,
65.56 (d, J = 5.8 Hz), 58.03, 39.8, 23.34, 19.329, 12.53.
19
F NMR (376 MHz, cd
3
od) δ -134.86
(dd, J = 77.5, 49.4 Hz).
31
P NMR (162 MHz, cd
3
od) δ 2.95 (t, J = 77.5 Hz). HRMS: ESI,
Negative mode. Calculated for [M-H]
-
C12H13BrF2N2O7P 444.96173, found 444.96181
(100%).
2’,3’-Dideoxy-3’-fluorothymidine-5'-difluoromethylphosphonate 4i
O
O
N
NH
O
O
P
O
HF
2
C
O
Bu
4
N
+
F

1
H NMR (399 MHz, cd
3
od) δ 7.75 (d, J = 1.2 Hz, 1H), 6.23 (dd, J = 9.3, 5.5 Hz, 1H), 5.64 (m,
1H), 5.19 (m, 1H), 4.14 (m, , 1H), 3.70 – 3.67 (m, 2H), 3.19 – 3.07 (m, 8H), 2.48 – 2.10 (m,
2H), 1.78 (d, J = 1.2 Hz, 3H), 1.64 – 1.50 (m, 8H), 1.38 – 1.24 (m, 8H), 0.93 (t, J = 7.4 Hz,
- 55 -

12H).
19
F NMR (376 MHz, cd
3
od) δ -112.17 (d, J = 98.5 Hz), -134.86 (dd, J = 78.1, 49.8 Hz).
31
P NMR (162 MHz, cd
3
od) δ 2.49 (t, J = 74.4 Hz).
2.5 REPRESENTATIVE NMR SPECTRA
19
F NMR difluoromethanephosphonyl dichloride (3)

31
P NMR difluoromethanephosphonyl dichloride (3)

- 56 -

1
H NMR diethyl (tert-butyldimethylsilyl)difluoromethylphosphonate (5)

13
C NMR diethyl (tert-butyldimethylsilyl)difluoromethylphosphonate (5)

- 57 -

19
F NMR diethyl (tert-butyldimethylsilyl)difluoromethylphosphonate (5)

31
P NMR diethyl (tert-butyldimethylsilyl)difluoromethylphosphonate (5)

- 58 -

1
H NMR (tert-butyldimethylsilyl)difluoromethanephosphonyl dichloride (7)

13
C NMR (tert-butyldimethylsilyl)difluoromethanephosphonyl dichloride (7)

- 59 -

19
F NMR (tert-butyldimethylsilyl)difluoromethanephosphonyl dichloride (7)

31
P NMR (tert-butyldimethylsilyl)difluoromethanephosphonyl dichloride (7)

- 60 -

1
H NMR Diethyl[ethoxy(tert-butyldimethylsilyldifluoromethyl) phosphoryl]phosphonate
(9)

13
C NMR Diethyl[ethoxy(tert-butyldimethylsilyldifluoromethyl) phosphoryl]phosphonate
(9)

- 61 -

19
F NMR Diethyl[ethoxy(tert-butyldimethylsilyldifluoromethyl) phosphoryl]phosphonate
(9)

31
P NMR Diethyl[ethoxy(tert-butyldimethylsilyldifluoromethyl) phosphoryl]phosphonate
(9)

- 62 -

1
H NMR Phosphonic acid, [[(diisopropylamino)(difluoromethyl)phosphinyl]methyl]-,
diethyl ester (12)

13
C NMR Phosphonic acid, [[(diisopropylamino)(difluoromethyl)phosphinyl]methyl]-,
diethyl ester (12)

- 63 -

19
F NMR Phosphonic acid, [[(diisopropylamino)(difluoromethyl)phosphinyl]methyl]-,
diethyl ester (12)

31
P NMR Phosphonic acid, [[(diisopropylamino)(difluoromethyl)phosphinyl]methyl]-,
diethyl ester (12)

- 64 -

1
H NMR [(hydroxydifluoromethylphosphinyl)methyl]-phosphonic acid
tetrabutylammonium salt (10)

13
C NMR [(hydroxydifluoromethylphosphinyl)methyl]-phosphonic acid
tetrabutylammonium salt (10)

- 65 -

19
F NMR [(hydroxydifluoromethylphosphinyl)methyl]-phosphonic acid
tetrabutylammonium salt (10)

31
P NMR [(hydroxydifluoromethylphosphinyl)methyl]-phosphonic acid
tetrabutylammonium salt (10)

- 66 -

1
H NMR Thymidine-5'-difluoromethylphosphonate (4a)

13
C NMR Thymidine-5'-difluoromethylphosphonate (4a)

- 67 -

19
F NMR Thymidine-5'-difluoromethylphosphonate (4a)

31
P NMR Thymidine-5'-difluoromethylphosphonate (4a)

- 68 -

1
H NMR 3’-Azido-3’-deoxythymidine-5'-difluoromethylphosphonate (4b)

13
C NMR 3’-Azido-3’-deoxythymidine-5'-difluoromethylphosphonate (4b)

- 69 -

19
F NMR 3’-Azido-3’-deoxythymidine-5'-difluoromethylphosphonate (4b)

31
P NMR 3’-Azido-3’-deoxythymidine-5'-difluoromethylphosphonate (4b)

- 70 -

1
H NMR 2’-Deoxy-2’-fluorouridine-5'-difluoromethylphosphonate (4e)

13
C NMR 2’-Deoxy-2’-fluorouridine-5'-difluoromethylphosphonate (4e)

- 71 -

19
F NMR 2’-Deoxy-2’-fluorouridine-5'-difluoromethylphosphonate (4e)

31
P NMR 2’-Deoxy-2’-fluorouridine-5'-difluoromethylphosphonate (4e)

- 72 -

1
H NMR 5-(E)-(2-Bromovinyl)-2’-deoxyuridine-5'-difluoromethylphosphonate (4h)

13
C NMR 5-(E)-(2-Bromovinyl)-2’-deoxyuridine-5'-difluoromethylphosphonate (4h)

- 73 -

19
F NMR 5-(E)-(2-Bromovinyl)-2’-deoxyuridine-5'-difluoromethylphosphonate (4h)

31
P NMR 5-(E)-(2-Bromovinyl)-2’-deoxyuridine-5'-difluoromethylphosphonate (4h)

- 74 -

2.6 REFERENCES
1. Voet, D.; Voet, J. G., Biochemistry. Vol. 2
nd
. John Wiley and Sons; New York: 1995.
2. Gumina, G; Chong, Y; Choo, H; Song, G; Chu, C. K., Curr. Topics Med. Chem. 2002, 2,
1065.
3. De Clercq, E.; Field, H. J., Br. J. Pharmacol. 2006, 147, 1.
4. Orr, D. C.; Figueiredo, H. T.; Mo, C. L.; Penn, C. R.; Cameron, J. M., J. Biol. Chem.
1992, 267, 4177.
5. Anderson, K. S., Biochim. Biophys. Acta-Mol. Basis Dis. 2002, 1587, 296.
6. Krise, J. P.; Stella, V. J., Adv. Drug Deliv. Rev. 1996, 19, 287.
7. Zemlicka, J., Biochim. Biophys. Acta-Mol. Basis Dis. 2002, 1587, 276.
8. Berkowitz, D. B.; Bose, M., J. Fluor. Chem. 2001, 112, 13.
9. Tozer, M. J.; Herpin, T. F., Tetrahedron 1996, 52, 8619.
10. Liu, P.; Sharon, A.; Chu, C. K., J. Fluor. Chem. 2008, 129, 743.
11. (a) Welch, J. T., Tetrahedron 1987, 43, 3123. (b) Purrington, S. T.; Kagen, B. S.; Patrick,
T. B., Chem. Rev. 1986, 86, 997. (c) Olah, G. A.; Chambers, R. D.; Prakash, G. K. S., Synthetic
Fluorine Chemistry. John Wiley and Sons; New York: 1992.
12. Bondi, A., J. Phys. Chem. 1964, 68, 441.
13. (a) Lin, G-Q.; You, Q-D.; Cheng, J-F., Chiral Drugs: Chemistry and Biology action.
John Wiley and Sons, 2011. (b) Ojima, I., Fluorine in Medicinal Chemistry and Chemical
Biology. John Wiley & Sons, 2009.
14. Tatlow, J. C.; Smart, B. E.; Banks, R. E., Organofluorine Chemistry: Principles and
Commercial Application, Phenum Press, N.Y and London, 1994, 80.
15. McGuigan, C.; Madela, K.; Aljarah, M.; Gilles, A.; Brancale, A.; Zonta, N.;
Chamberlain, S.; Vernachio, J.; Hutchins, J.; Hall, A.; Ames, B.; Gorovits, E.; Ganguly, B.;
Kolykhalov, A.; Wang, J.; Muhammad, J.; Patti, J. M.; Henson, G., Bioorg. Med. Chem. Lett.
2010, 20, 4850.
16. McGuigan, C.; Madela, K.; Aljarah, M.; Gilles, A.; Battina, S. K.; Ramamurty, C. V. S.;
Rao, C. S.; Vernachio, J.; Hutchins, J.; Hall, A.; Kolykhalov, A.; Henson, G.; Chamberlain, S.,
Bioorg. Med. Chem. Lett. 2011, 21, 6007.
- 75 -

17. Zhang, H.-W.; Zhou, L.; Coats, S. J.; McBrayer, T. R.; Tharnish, P. M.; Bondada, L.;
Detorio, M.; Amichai, S. A.; Johns, M. D.; Whitaker, T.; Schinazi, R. F., Bioorg. Med. Chem.
Lett., 2011, 21, 6788.
18. Mehellou, Y.; Valente, R.; Mottram, H.; Walsby, E.; Mills, K. I.; Balzarini, J.;
McGuigan, C., Bioorg. Med. Chem. Lett. 2010, 18, 2439.

- 76 -

Chapter 3 Synthesis of monofluoroalkenes
via Julia-Kosinski reaction
3.1 INTRODUCTION
Fluorinated bioactive molecules have had substantial influence on drug development in recent
years [1]. Even though the number of fluorine-containing natural products is relatively small,
currently, almost a quarter of marketed drugs contain at least one fluorine atom. It has been
demonstrated that incorporation of the fluorine atom into the biologically interesting molecules
may have tremendous impact on it pharmacokinetic and pharmacodynamic properties and
protein–ligand interactions [2, 3]. This has raised great interest for the development of synthetic
pathways to access a wides variety of fluorinated compounds [4]. A number of efficient
fluorinating reagents for stereoselective introduction of fluorine atom has been introduced [5].
Particularly, the systems, whith the fluorine atom adjacent to the carbon-carbon double bond,
attracted special attention of medicinal chemists [6]. The major reason for this is the fact that
fluoroolefinic fragment demonstrates the closest structural resemblance to the peptide bond
(Figure 3.1). Charge distribution in the alkene moiety induced by the presence of electronegative
fluorine atom mimics the dipolar nature of the peptide function [7, 8].
Peptides demonstrate great utility in the investigation the nature structural behavior and
properties of biological systems [6]. However, they exhibit considerable lack in stability when
exposed to hydrolytic enzymes such as peptidases. Because of their poor bioavailability and
short physiological lifetimes, they cannot be used as potential therapeutic agents. Therefore,
structural modifications that can prevent rapid degradation, improve stability and bioavailability
while retaining the properties of peptide bond are the subject of intense research [9].
One of the most commonly recognized idea is to employ monofluoroalkenes as stable, non-
isomerable and non-hydrolyzable peptide bioisostere [6]. Amide bond possesses a partial double
bond character with a shorter bond length and greater rotation energy compared to a C-N single
bond. Incorporation of the electronegative fluorine atom into double bond creates polarizing
pattern that mimics electrostatic charge distribution of the natural peptide motif (Figure 3.1) [7].
- 77 -

Figure 3.1. Monofluoroalkenes as stable mimic of the peptide bond [7].
Even though monofluoroalkenes attracted considerable interest of organic chemists as a
potential peptidomimetics [7] currently, only a few versatile methods for the preparation of
terminal monofluoroalkenes are known. Among them, preparation of vinyl fluorides using Wittig
reaction [10-11], nucleophilic alkylation/hydroxylation of fluoromethyl
sulfones/sulfoximines/sulfoxides followed by removal of sulfonate/sulfinamide/sulfinate [13-16],
electrophilic fluorination of alkenes and reduction of terminal gem-difluoroolefins are the major
ones [17]. Recently, sulfone-based chemistry demonstrates substantial advances for the
preparation of fluorine-containing unsaturated structural motifs [18-23].
Herein, we will discuss the development of the novel simple and efficient synthetic approach
for the preparation of monofluoroalkenes via Julia-Kosinski reaction.
3.2 RESULTS AND DISCUSSION
Preparation of monofluoroalkenes via Julia-Kocienski olefination reaction
In this chapter, the synthesis of novel and efficient fluoromethylating reagent based on
monofluoromethylarylsulfone 1 is described. Furthermore, development of a new one step
method for the preparation of a wide variety of monofluoroalkenes via Julia-Kocienski reaction
also discussed.
- 78 -

Prepartion of monofluoromethyl 3,5-bis(trifluoromethyl)phenyl sulfone
Scheme 3.1. Synthesis of sulfone 1.
CF
3
F
3
C SH
2
Na
Et
2
O
CF
3
F
3
C SNa
CH
2
FCl S CF
3
CF
3
F
S CF
3
CF
3
F
H
2
O
2
/AcOH
O O
1
CH
3
CN

Sulfone 1 was prepared according to the Scheme 3.1 with the three step procedure as shown.
Initially, 3,5-bis(trifluoromethyl)benzenethiol 2 was treated with sodium metal in diethylether
followed by reaction with fluorochloromethane gas dissolved in acetonitrile. In situ oxidation of
the intermediate monofluoromethyl 3,5-bis(trifluoromethyl)phenyl sulfide using hydrogen
peroxide/acetic acid afforded sulfone 1 in 61% overall yield.
In order to find the best conditions for the fluoromethylation reaction, monofluoromethyl
3,5-bis(trifluoromethyl)phenyl sulfone was exposed to several reaction conditions usually
employed for similar analogues [23] (Table 3.1). After screening of several solvents and bases, it
was determined that the best results were obtained when KOH or CsF were used as a base and
reaction was carried out in DMSO.
The KOH / DMSO system provides good yields for the aldehyde and ketones with electron
donating substituents. However, these conditions were not efficient when electron deficient
carbonyl compounds were utilized. When CsF was used instead of KOH, though the reaction
proceeds slower, the desired monofluoroalkenes with both electron rich and electron
withdrawing substituents can be obtained in excellent yields. Unfortunately, due to the side
condensation reactions and significant volatility, the yields of enolizable carbonyl compounds
did not exceeded 20%.
A number of aldehyde and ketones represented in Table 3.2 were converted into
monofluoroolefins via Julia-Kosinski reaction using either KOH/DMSO or CsF/DMSO
conditions. Most of the monofluoroalkenes prepared by this method appeared as mixture of E
and Z isomers in different ratios. The yields of the products as well as ration of E/Z isomers were
determined by
19
F and
1
H NMR spectroscopy (Table 3.2).

- 79 -

Table 3.1. Optimization of reaction conditions for monofluoroolifination of carbonyl compounds
using 1.
a

a
All reactions were performed at room temperature except entry 3 where the reaction mixture was firstly cooled -
78 C and after addition of all components the temperature was slowly raised to ambient.
b
Entry 11 have
demonstrated much better yields compared to other screened solvent/base systems when carbonyl compounds with
electron donating substituents were used.

Solvent
THF

DMF

DMSO
MeCN
Base
KOH/n-Bu
4
N
+
Br
-
CsOH/n-Bu
4
N
+
Br
-
LDA
CsF
Py
NaH (2 eq.)
CsF
NaH (2 eq.)
NaH (5 eq.)
NaOH
KOH
CsOH
Py
Et
3
N
DBU
n-Bu
4
N
+
OH
-
KF
CsF
NaH
KOH/n-Bu
4
N
+
Br
-
CsF
Yield of 4e, %

30
36

3



15
5

26
b
25


31


95
5
20
2
Entry

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
CHO
Cl
+
CF
3
CF
3
S
O O
F
Cl
F
3e 1 4e
Time

12 h
12 h
5 h
12 h
12 h
12 h
12 h
12 h
12 h
12 h
2 h
2 h
12 h
12 h
72 h
5 min
12 h
48 h
12 h
12 h
12 h
- 80 -

Table 3.2. Preparation of terminal alkenes via Julia-Kosinski olifination reaction.

All compounds were purified using silica gel chromatography and fully characterized with
1
H,
19
F, and
13
C NMR. The molecular weights of all alkenes 4a-4t were confirmed by GC-MS
analysis. Additionally compounds 4f, 4g, 4i, 4j, 4k, 4l, 4m, 4p were subjected to high resolution
mass spectral analysis; HRMS analysis of the other monofluoroolefins (4a-4e, 4h, 4n, 4o, 4q-4t)
could not be carried out because of their low stability and tendency to polymerize [24, 25].
However we were able to separate (E)- and (Z)-isomers for compounds 4m, 4r-t (Table 3.2) and
individually characterize them.

- 81 -

3.3 CONCLUSIONS
In summary, a novel reagent based on 3,5-bis(trifluoromethyl)phenyl sulfone 1 for
monofluoromethylation of variety of carbonyl compounds was introduced. Additionally, a new
facile and efficient methodology that employs mild and robust reaction conditions for the
preparation of terminal monofluoroalkenes was also developed. The products containing
mixtures of E/Z isomers can be obtained in good to excellent yields.
3.4 EXPERIMENTAL PART
General
Unless otherwise mentioned, all reagents were purchased from commercial sources.
1
H,
13
C
and
19
F NMR spectra were recorded on a Varian Inova 400 MHz NMR spectrometer.
1
H NMR
chemical shifts were determined relative to internal (CH
3
)
4
Si (TMS) standard at 0.00 ppm.
13
C
NMR chemical shifts were determined relative to the
13
C signal of the residual CHCl
3
in CDCl
3

(77.16 ppm). CFCl
3
was used as internal standard for
19
F NMR. High resolution mass spectra
were recorded in EI or FAB mode on a high resolution mass spectrometer at the Mass
Spectrometry facility, University of Arizona.
Preparation of 3,5-bis(trifluoromethyl)phenyl monofluoromethyl sulfone
Sodium metal (1.0 g, 43.48 mmol) in 40 mL of diethyl ether was taken in a three-necked flask
equipped with a dropping funnel, reflux condenser, and a stirr bar. The solution of 10 g (40.62
mmol) of 3,5-bis(trifluoromethyl)benzenethiol in 50 mL of Et
2
O was added to sodium in Et
2
O
dropwise; the stirred mixture was heated to reflux and was kept under reflux overnight. The
solution was cooled and the remaining sodium was carefully quenched after filtering. Most of
Et
2
O was removed under reduced pressure, and large amount of hexane (~100 ml) was added to
the remaining concentrated ethereal solution. The precipitated white solid was filtered off and air
dried. Sodium salt of 3,5-bis(trifluoromethyl)thiophenol (10.02 g,92%) was obtained and used
for the next step without additional purification.
Sodium 3,5-bis(trifluoromethyl)thiophenolate (5 g, 18.65 mmol), was placed in a pressure
tube, dissolved in 60 mL acetonitrile, and the solution was rapidly cooled down to -78
o
C.
Previously cooled CH
2
ClF (6 mL, 68.94 mmol)was added to the thiophenolate solution at -78
o
C.
The reaction mixture was allowed to warm up to 40
o
C and was stirred overnight. Subsequently,
- 82 -

the reaction mixture was brought to room temperature and all the remaining volatile products
were vented out. Water (100 mL) was added to the residual solution. The mixture was extracted
with chloroform (4 x 25 mL) and the combined organic phase was dried over anhydrous
magnesium sulfate. Solvents were evaporated under reduced pressure, the residue (crude 3,5-
bis(trifluoromethyl)phenyl monofluoromethyl sulfide) was dissolved in 60 mL glacial acetic
acid, and 8.46 g of 30% hydrogen peroxide was added to the solution. The reaction mixture was
heated to 80
o
C and stirred overnight at this temperature. The resulting solution was poured onto
ice and the precipitated white crystals of 3,5-bis(trifluoromethyl)phenyl monofluoromethyl
sulfone were collected by suction filtration, washed with water and air dried. The yield of
3,5-bis(trifluoromethyl)phenyl monofluoromethyl sulfone was 3.7 g (64%).
1
H NMR (400 MHz, CDCl
3
): δ 5.23 (d, J

= 47 Hz), 8.24 (s), 8.43 (s) ppm.
19
F NMR (376 MHz,
CDCl
3
): -63.4 (s), -210.9 (t, J

= 47 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
): 91.8 (d, J

= 220 Hz),
122.3 (q, J

= 272 Hz), 128.7 (t, J

= 4 Hz), 129.6, 133.7 (q, J

= 34 Hz), 138.8 ppm.
Typical procedure for the monofluoroolefination of carbonyl compounds with
3,5-bis(trifluoromethyl)phenyl monofluoromethyl sulfone in the presence of KOH and TBAF
Aldehyde or ketone (1.5 mmol) and 1.5 mmol (1 eq.) of 3,5-bis(trifluoromethl)phenyl
monofluoromethyl sulfone are placed in a 50 mL round bottom flask and 15 mL of DMSO was
added. A solution of 9 eq of KOH in 10 mL DMSO was added dropwise to the reaction mixture
over a period of two hours. The reaction mixture was left at room temperature and monitored by
19
F NMR until completion (1 – 12 hours). After completion of the reaction, the solution was
poured into cold saturated solution of ammonium chloride, extracted with ethyl acetate (4 x 25
ml), and the organic layer was separated, dried over anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure and the resulting crude product was purified by column
chromatography (EtOAc/Hex = 1/10).
Typical procedure of monofluoroolefination of carbonyl compounds with
3,5-bis(trifluoromethyl)phenyl monofluoromethyl sulfone in the presence of CsF
Under inert atmosphere 9 eq of CsF was placed in a 50 mL round bottom flask and 20 mL of
DMSO was introduced. Subsequently, the mixture of 1.5 mmol of aldehyde or ketone and 1.5
mmol of 3,5-bis(trifluoromethl)phenylmonofluoromethyl sulfone dissolved in 10 mL of DMSO,
- 83 -

and was added to the solution of CsF. The reaction mixture was stirred overnight, poured into
cold saturated solution of ammonium chloride, extracted with ethyl acetate (4 x 15 mL), and the
organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated under
reduced pressure and the resulting crude product was purified by column chromatography
(EtOAc/Hex = 1/10).
3.5 SPECTRAL DATA OF PRODUCTS
1-(2-Fluorovinyl)benzene 4a
1
H NMR (400 MHz, CDCl
3
) δ 5.63(dd, J
HF
=45 Hz, J
HH
=5.2Hz, 0.9H), 6.42 (dd, J
HF
=19 Hz,
J
HH
=12 Hz, 1H), 6.67 (dd, J
HF
=83 Hz, J
HH
=5.2 Hz, 0.9H), 7,19 (dd, J
HF
=83 Hz, J
HH
=12 Hz, 1H),
7,25-7.38 (m, 5.7), 7.17 (d, J
HH
=9.8 Hz, 2H), 7.54 (d, J
HH
=9.2 Hz, 1.8H) ppm.
19
F NMR (376
MHz, CDCl
3
) δ -122.7 (dd, J
HF
=83 Hz, J
HF
=45 Hz), -130.5 (dd, J
HF
=83 Hz, J
HF
=19 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 40.47, 40.57, 110.69, 112.28, 112.74, 113.68 (d, J
CF
=16 Hz),
120.59 (d, J
CF
=12 Hz), 121.1, 127.9 (d, J
CF
=3 Hz), 129.93 (d, J
CF
=7 Hz), 146.15 (d, J
CF
=264
Hz), 148.13 (d, J
CF
=252 Hz), 149.77 (d, J
CF
=3 Hz), 150.05 (d, J
CF
=1 Hz) ppm.
1-(2-Fluorovinyl)-4-methoxybenzene 4b
1
H NMR (400 MHz, CDCl
3
) δ3.84 (s, 3H), 3.85 (s, 2.5), 5.6 (dd, J
HF
=46 Hz, J
HH
=5.2 Hz, 0.8H),
6.4 (dd, J
HF
=20 Hz, J
HH
=11.2 Hz, 1H), 6.64 (dd, J
HF
=84 Hz, J
HH
=5.2 Hz, 0.8H), 6.88-6.94 (m,
3.6H) 7,15 (dd, J
HF
=84 Hz, J
HH
=11.2 Hz, 1H), 7.22 (d, J
HH
=9.2 Hz, 2H), 7.51 (d, J
HH
=8.4 Hz,
1.6H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -125.8 (dd, J
HF
=84 Hz, J
HF
=46 Hz), -133.15 (dd,
J
HF
=84 Hz, J
HF
=20 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 55.27, 55.31, 110.30, 113.37 (d,
J
CF
=16 Hz), 113.98, 114.32 125.10 (d, J
CF
=11 Hz), 125.44 (d, J
CF
=2 Hz), 127.37 (d, J
CF
=3 Hz),
130.22 (d, J
CF
=7 Hz), 148.09 (d, J
CF
=266 Hz), 149.08 (d, J
CF
=256 Hz), 158.94 (d, J
CF
=3 Hz),
159.16 (d, J
CF
=1 Hz) ppm.
4-(2-Fluorovinyl)-N,N-dimethylbenzenamine 4c
1
H NMR (400 MHz, CDCl
3
) δ 2.94 (s, 6H), 3.00 (s, 5.4H), 5.54(dd, J
HF
=46 Hz, J
HH
=5.2 Hz,
0.9H), 6.37 (dd, J
HF
=20 Hz, J
HH
=11.2 Hz, 1H), 6.59 (dd, J
HF
=84 Hz, J
HH
=5.2 Hz, 0.9H), 6.69-
6.74 (m, 3.8H), 7,11 (dd, J
HF
=84 Hz, J
HH
=11.2 Hz, 1H), 7.17 (d, J
HH
=9.8 Hz, 2H), 7.46 (d,
J
HH
=10 Hz, 1.8) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -127.7 (dd, J
HF
=84 Hz, J
HF
=46 Hz), -135.9
(dd, J
HF
=84 Hz, J
HF
=20 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 40.47, 40.57, 110.69, 112.28,
- 84 -

112.74, 113.68 (d, J
CF
=16 Hz), 120.59 (d, J
CF
=12 Hz), 121.1, 127.9 (d, J
CF
=3 Hz), 129.93 (d,
J
CF
=7 Hz), 146.15 (d, J
CF
=264 Hz), 148.13 (d, J
CF
=252 Hz), 149.77 (d, J
CF
=3 Hz), 150.05 (d,
J
CF
=1 Hz) ppm.
1-Chloro-4-(2-fluorovinyl)benzene 4d
1
H NMR (400 MHz, CDCl
3
) δ 5.58 (dd, J
HF
=44 Hz, J
HH
=5.4 Hz, 1H), 6.36 (dd, J
HF
=18.8 Hz,
J
HH
=11.2 Hz, 1H), 6.66 (dd, J
HF
=82 Hz, J
HH
=5.4 Hz, 1H), 7.14 (dd, J
HF
=82 Hz, J
HH
=11.2 Hz,
1H), 7.17 (d, J
HH
=11.2 Hz, 2H), 7.26-7.33 (m, 4H), 7.44 (d, J
HH
=8.4 Hz, 2H) ppm.
19
F NMR
(376 MHz, CDCl
3
) δ -121.9 (dd, J
HF
=82 Hz, J
HF
=44 Hz), -129 (dd, J
HF
=82 Hz, J
HF
=18.8 Hz)
ppm.
13
C NMR (100 MHz, CDCl
3
) δ109.90, 113.6 (d, J
CF
=17 Hz), 127.45 (d, J
CF
=3 Hz), 128.77
129.05, 130.16 (d, J
CF
=7 Hz), 131.08 (d, J
CF
=13 Hz), 131.24 (d, J
CF
=12 Hz), 133.25, 133.27,
148.68 (d, J
CF
=270 Hz), 150.5 (d, J
CF
=259 Hz) ppm.
1-Fluoro-4-(2-fluorovinyl)benzene 4e
1
H NMR (400 MHz, CDCl
3
) δ 5.58 (dd, J
HF
=44 Hz, J
HH
=5.4 Hz, 0.9H), 6.36 (dd, J
HF
=19.2 Hz,
J
HH
=11.6 Hz, 1H), 6.63 (dd, J
HF
=83 Hz, J
HH
=5.4 Hz, 0.9H), 6.98-7.05 (m, 4.8H), 7.19-7.26 (m,
4.8H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -114.2 (m), -114.9 (m), -124 (dd, J
HF
=83 Hz, J
HF
=44
Hz), -130.6 (dd, J
HF
=83 Hz, J
HF
=19.2 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ109.88, 113.5 (d,
J
CF
=17 Hz), 115.7, 116.0, 127.8 (d, J
CF
=3 Hz), 127.9 (d, J
CF
=3 Hz) 130.6 (d, J
CF
=7 Hz), 130.7
(d, J
CF
=7 Hz), 131.24 (d, J
CF
=12 Hz), 148.0 (d, J
CF
=267 Hz), 150.0 (d, J
CF
=258 Hz), 161.1,
163.53 ppm.
4-(2-Fluorovinyl)benzonitrile 4f
1
H NMR (400 MHz, CDCl
3
) δ 5.66 (dd, J
HF
=43 Hz, J
HH
=5.4 Hz, 1H), 6.4 (dd, J
HF
=18.4 Hz,
J
HH
=11.2 Hz, 1H), 6.75 (dd, J
HF
=82 Hz, J
HH
=5.4 Hz, 1H), 7.25 (dd, J
HF
=82 Hz, J
HH
=11.2 Hz,
1H), 7.32-7.35 (m, 2H), 7.57-763 (m, 6H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -117.2 (dd,
J
HF
=82 Hz, J
HF
=44 Hz), -124.0 (dd, J
HF
=82 Hz, J
HF
=18.4 Hz) ppm.
13
C NMR (100 MHz,
CDCl
3
) δ, 109.8, 110.9, 111.05, 113.1 (d, J
CF
=17 Hz), 118.8, 118.9, 126.7 (d, J
CF
=3 Hz), 128.7
(d, J
CF
=4 Hz), 132.4, 132.7, 137.2, 137.7 (d, J
CF
=13 Hz), 150.5 (d, J
CF
=275 Hz), 152.3 (d,
J
CF
=264 Hz) ppm. HRMS: Calculated: 147.0484, found: 147.0490.
Methyl 4-(2-fluorovinyl)benzoate 4g
- 85 -

1
H NMR (400 MHz, CDCl
3
) δ 3.87 (s, 3H), 3.88 (s, ), 5.64 (dd, J
HF
=44 Hz, J
HH
=5.4 Hz, 0.8H),
6.38 (dd, J
HF
=18.8 Hz, J
HH
=11.2 Hz, 1H), 6.68 (dd, J
HF
=82 Hz, J
HH
=5.4 Hz, 0.8H), 7.22 (dd,
J
HF
=82 Hz, J
HH
=11.2 Hz, 1H), 7.13-7.28 (d, J
HH
=8.4 Hz, 2H), 7.53 (d, J
HH
=8.8 Hz, 1.6H), 7.93-
798 (m, 4H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -118.8 (dd, J
HF
=82 Hz, J
HF
=44 Hz), -126.2
(dd, J
HF
=82 Hz, J
HF
=18.8 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 52.2, 110.3, 113.5 (d,
J
CF
=16.8 Hz), 126.1 (d, J
CF
=3 Hz), 128.8 (d, J
CF
=7 Hz), 129.8, 130.2, 137.2 (d, J
CF
=2 Hz), 137.5
(d, J
CF
=12 Hz), 149.8 (d, J
CF
=273 Hz), 151.6 (d, J
CF
=262 Hz), 166.8, 166.9 ppm. HRMS:
Calculated: 180.0587, found: 180.0589.
1-((E)-2-Fluorovinyl)-2,4,5-trimethoxybenzene 4h
1
H NMR (400 MHz, CDCl
3
) δ 3.80 (s, 3H), 3.81 (s, 3H), 3.86 (s, 3H), 6.4 (dd, J
HF
=22 Hz,
J
HH
=11.2 Hz, 1H), 6.48 (s, 1H), 6.67 (s, 1H), 7.29 (dd, J
HF
=86 Hz, J
HH
=11.2 Hz, 1H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -128.4 (dd, J
HF
=86 Hz, J
HF
=22 Hz) ppm.
13
C NMR (100 MHz,
CDCl
3
) δ 56.3, 56.5, 56.8, 97.8, 110.2 (d, J
CF
=18 Hz), 112.0, 113.2 (d, J
CF
=11 Hz), 143.3, 149.4,
150.4 (d, J
CF
=231 Hz), 151.9.
1-((Z)-2-Fluorovinyl)-2,4,5-trimethoxybenzene 4h
1
H NMR (400 MHz, CDCl
3
) δ 3.78 (s, 3H), 3.82 (s, 3H), 3.87 (s, 3H), 5.95 (dd, J
HF
=47 Hz,
J
HH
=5.4 Hz, 1H), 6.58 (dd, J
HF
=84 Hz, J
HH
=5.4 Hz, 1H), 7.23 (s, 1H), 7.36 (s, 1H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -126.8 (dd, J
HF
=84 Hz, J
HF
=47 Hz) ppm.
13
C NMR (100 MHz,
CDCl
3
) δ 56.2, 56.6, 56.9, 97.4, 110.1 (d, J
CF
=2.3 Hz), 113.6 (d, J
CF
=12 Hz), 143.1, 147.2 (d,
J
CF
=266 Hz), 149.4, 151.1 ppm.
2-Fluoro-1,1-diphenylethene 4l
1
H NMR (400 MHz, CDCl
3
) δ 7.00 (d, J
HF
=82.5 Hz, 1H), 7.27-7.29 (m, 2H), 7.35-7.38 (m, 4H),
7.39-7.41(m, 4H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -128.5 (d, J
HF
=82.5 Hz) ppm.
13
C NMR
(100 MHz, CDCl
3
) δ 126.35 (d, J
CF
=6 Hz), 127.89, 127.92, 128.34, 128.64, 128.79 (d, J
CF
=3
Hz), 129.88 (d, J
CF
=5 Hz), 137.08, 137.15, 145.90 (d, J
CF
=267 Hz) ppm. HRMS: Calculated:
198.0845, found: 198.0852.
1-(2-Fluoro-1-phenylvinyl)-4-methyl)-4-methylbenzene 4j
- 86 -

1
H NMR (400 MHz, CDCl
3
) δ 2.44 (s, 3H), 2.45 (s, 2.55H), 7.00 (d, J
HF
=84 Hz, 0.85H), 7.02 (d,
J
HF
=84 Hz, 1H) 7.25-7.27 (m, 3.65H), 7.31-7.36 (m, 4.5H), 7.38-7.42 (m, 4.5H), 743-7.45(m,
4H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -128.8 (d, J
HF
=84 Hz), δ -129.25 (d, J
HF
=84 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 21.25, 21.36, 126.18 (t, J
CF
=5 Hz), 127.80, 127.84, 128.28,
128.57, 128.67 (d, J
CF
=3 Hz), 128.80 (d, J
CF
=3 Hz), 129.05, 129.34, 129.76 (d, J
CF
=4 Hz),
129.87 (d, J
CF
=4 Hz), 134.13, 134.21, 137.25, 137.33, 137.68, 137.73, 145.55 (d, J
CF
=266 Hz),
145.66 (d, J
CF
=266 Hz) ppm. HRMS: Calculated: 196.0888, found: 196.0896.
1-(2-Fluoro-1-phenylvinyl)-4-methoxybenzene 4k
1
H NMR (400 MHz, CDCl
3
) δ 3.84 (s, 2.7H), 3.85 (s, 3H), 6.9-6.95 (m, 3.8H), 6.94 (d, J
HF
=84
Hz, 1H), 6.96 (d, J
HF
=84 Hz, 0.9H) 7.19-7.22 (m), 7.26-7.30 (m, 1.8H), 7.33-7.4(m, 11.5H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -129.6 (d, J
HF
=84 Hz), δ -129.9 (d, J
HF
=84 Hz) ppm.
13
C NMR
(100 MHz, CDCl
3
) δ 55.35, 55.30, 113.71, 114.03, 125.79 (d, J
CF
=5 Hz), 125.80 (d, J
CF
=5 Hz),
127.80, 127.85, 128.28, 128.57, 128.86 (d, J
CF
=3 Hz), 129.83 (d, J
CF
=5 Hz), 129.90 (d, J
CF
=3
Hz), 131.58 (d, J
CF
=5 Hz), 137.31, 137.39, 145.19 (d, J
CF
=266 Hz), 145.33 (d, J
CF
=265 Hz),
159.13, 159.48 ppm. HRMS: Calculated: 228.0950, found: 228.0953.
4-(2-Fluoro-1-phenylvinyl)-N,N-dimethylbenzeneamine 4l
1
H NMR (400 MHz, CDCl
3
) δ 3.01 (s, 4.8H), 3.02 (s, 6H), 6.72-6.77(m, 3.6H), 6.91 (d, J
HF
=84
Hz, 1H), 6.98 (d, J
HF
=84 Hz, 0.8H) 7.14-7.18 (m, 1.6H), 7.3-7.35 (m, 4H), 7.36-7.45 (m, 6H)
ppm.
19
F NMR (376 MHz, CDCl
3
) δ -130.8 (d, J
HF
=84 Hz), δ -131.9 (d, J
HF
=84 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 40.46, 40.55, 111.91, 112.38, 124.73, 124.80, 126.23 (d, J
CF
=5
Hz), 126.08 (d, J
CF
=5 Hz), 127.60, 127.67, 128.19, 128.44, 129.09 (d, J
CF
=3 Hz), 129.45 (d,
J
CF
=3 Hz), 129.92 (d, J
CF
=4 Hz), 130.68 (d, J
CF
=3 Hz), 137.71, 137.80, 144.60 (d, J
CF
=264 Hz),
144.74 (d, J
CF
=265 Hz), 149.92, 150.26 ppm. HRMS: Calculated: 241.1267, found: 241.1265.
4-(2-Fluoro-1-phenyl)benzonitrile 4m-a
1
H NMR (400 MHz, CDCl
3
) δ 7.05 (d, J
HF
=82 Hz, 1H), 7.35-7.42 (m, 4H), 7.63-7.65 (m, 5H)
ppm.
19
F NMR (376 MHz, CDCl
3
) δ -127.8 (d, J
HF
=82 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ
111.6, 118.8, 125.7 (d, J
CF
=7.6 Hz), 128.5, 128.6, 129.2 (d, J
CF
=3 Hz), 129.8 (d, J
CF
=4 Hz),
132.5, 133.9, 142.8 (d, J
CF
=8 Hz), 147.1 (d, J
CF
=270 Hz) ppm. HRMS: Calculated: 223.0797,
found: 223.0789.
- 87 -

4-(2-Fluoro-1-phenyl)benzonitrile 4m-b
1
H NMR (400 MHz, CDCl
3
) δ 6.975 (d, J
HF
=82 Hz, 1H), 7.15-7.18 (m, 2H), 7.33-7.37 (m, 3H),
7.42-7.49 (m, 2H), 7.6-7.64 (m, 2H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -128.1 (d, J
HF
=82 Hz)
ppm.
13
C NMR (100 MHz, CDCl
3
) δ 111.5, 118.9, 125.1 (d, J
CF
=4 Hz), 128.5, 128.9 (d, J
CF
=3
Hz), 130.5(d, J
CF
=4 Hz), 120.5, 132.2, 135.7 (d, J
CF
=8 Hz), 140.0, 147.2 (d, J
CF
=272 Hz) ppm.
1-Chloro-4-(2-fluoro-1-phenylvinyl)benzene 4n
1
H NMR (400 MHz, CDCl
3
) δ 6.96 (d, J
HF
=83 Hz, 1H), 6.97 (d, J
HF
=83 Hz, 0.8H) 7.17-7.20 (m,
2H), 7.21-7.26 (m, 2H), 7.28-7.42 (m, 12.2H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -127.4 (d,
J
HF
=83 Hz), δ -127.5 (d, J
HF
=83 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 125.35 (d, J
CF
=5 Hz),
125.50 (d, J
CF
=7 Hz), 128.11, 128.15, 128.44, 128.56, 128.75, 128.79, 128.85, 129.78 (d, J
CF
=4
Hz), 130.03 (t, J
CF
=3 Hz), 131.16, 131.21, 135.57, 135.65, 136.54, 136.62, 145.95 (d, J
CF
=268
Hz), 146.16 (d, J
CF
=268 Hz) ppm.
1,1-Bis(4-chlorophenyl)-2-fluoroethene 4o
1
H NMR (400 MHz, CDCl
3
) δ 6.9 (d, J
HF
=83 Hz, 1H), 7.14-7.16 (m, 2H), 7.24-7.27 (m, 2H),
7.31-7.35(m, 4H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -126.7 (d, J
HF
=83 Hz) ppm.
13
C NMR
(100 MHz, CDCl
3
) δ 124.56 (d, J
CF
=6 Hz), 128.72, 129.018, 130.04, (d, J
CF
=3 Hz), 131,12 (d,
J
CF
=4 Hz), 134.00, 134.22, 135.08, 135.16, 146.25 (d, J
CF
=269 Hz) ppm.
2-Fluoro-1,1-dip-tolylethene 4p
1
H NMR (400 MHz, CDCl
3
) δ 2.43 (s, 3H), 2.44 (s, 3H) 6.98 (d, J
HF
=84 Hz, 1H), 7.20-7.25 (m,
4H), 7.32-7.34 (m, 4H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -129.7 (d, J
HF
=84 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 21.26, 21.37, 126.02 (d, J
CF
=4 Hz), 128.69 (d, J
CF
=3 Hz),
129.02, 129.30, 129.75 (d, J
CF
=5 Hz), 134.30, 134.38, 137.60, 137.65, 145.33 (d, J
CF
=266 Hz)
ppm. HRMS: Calculated: 226.1158, found: 226.1160.
1-Fluoroundec-1-ene 4q
1
H NMR (400 MHz, CDCl
3
) δ 0.88 (t, J
HH
=7.2Hz, 4.35H), 1.26-1.36 (m, 20.3H), 1.86-1.9(m,
2H), 2.07-2.13 (m, 0.9H), 4.64-4.79 (m, 0.45H), 5.28-5.39 (m, 1H) 6.43(ddt, J
HF
=88 Hz, J
HH
=4
Hz, J
HH
=1.6 Hz 0.45H), 6.48(ddt, J
HF
=88 Hz, J
HH
=12 Hz, J
HH
=1.2 Hz 1H) ppm.
19
F NMR (376
MHz, CDCl
3
) δ -131.64 (dd, J
HF
=86.8 Hz, J
HF
=19 Hz), -132.02 (dd, J
HF
=86 Hz, J
HF
=43 Hz)
- 88 -

ppm.
13
C NMR (100 MHz, CDCl
3
) δ 14.2, 22.7, 22.8, 25.0, 25.1, 25.2, 29.1, 29.2, 29.3, 29.5,
29.6, 29.7, 29.8, 32.0, 11.3 (d, J
CF
=5.3 Hz), 111.8 (d, J
CF
=8.4 Hz), 147.6 (d, J
CF
=254 Hz), 148.6
(d, J
CF
=252 Hz) ppm.
2-((E)-2-Fluorovinyl)pyridine 4r
1
H NMR (400 MHz, CDCl
3
) δ 6.36(dd, J
HF
=17.6 Hz, J
HH
= 11.2Hz, 1H), 7.06-7.1 (m, 2H), 7.53-
7.58 (m, 1H), 7.58 (dd, J
HF
=83 Hz, J
HH
=11.2 Hz, 1H), 8.43-8.45 (m, 1H) ppm.
19
F NMR (376
MHz, CDCl
3
) δ -127.92 (dd, J
HF
=83 Hz, J
HF
=17.6 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ
113.9 (d, J
CF
=16 Hz), 121.8 (d, J
CF
=5 Hz), 122.3 (d, J
CF
=3 Hz), 136.8, 149.5, 152.7 (d, J
CF
=4
Hz), 154.4 (d, J
CF
=264 Hz) ppm.
2-((Z)-2-Fluorovinyl)pyridine 4r
1
H NMR (400 MHz, CDCl
3
) δ 5.86 (dd, J
HF
=44 Hz, J
HH
=5.4Hz, 1H), 6.75 (dd, J
HF
=82 Hz,
J
HH
=5.4 Hz, 1H), 7.07-7.1(m, 1H), 7.59-7.63 (m, 1H), 7.7 (d, J
HH
=8 Hz, 1H), 8.49-8.51 (m, 1H)
ppm.
19
F NMR (376 MHz, CDCl
3
) δ -118.31 (dd, J
HF
=82 Hz, J
HF
=44 Hz) ppm.
13
C NMR (100
MHz, CDCl
3
) δ 112.3 (d, J
CF
=2 Hz), 122.1 (d, J
CF
=2 Hz), 124.5 (d, J
CF
=2 Hz), 136.5, 149.2,
150.6 (d, J
CF
=262 Hz), 152.2 (d, J
CF
=2 Hz) ppm.
2-Bromo-5-((E)-2-fluorovinyl)thiophene 4s
1
H NMR (400 MHz, CDCl
3
) δ 6.44 (dd, J
HF
=17.2 Hz, J
HH
=11.6Hz, 1H), 6.79 (dd, J
HF
=88 Hz,
J
HH
=4 Hz, 1H), 6.96 (d, J
HH
=11.4 Hz, 1H), 7.16 (d, J
HH
=11.4 Hz, 1H) ppm.
19
F NMR (376
MHz, CDCl
3
) δ -128.3 (dd, J
HF
=88 Hz, J
HF
=17.2 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ
107.84 (d, J
CF
=20.6 Hz), 110.5 (d, J
CF
=4 Hz), 126.3 (d, J
CF
=6 Hz), 130.3, 136.6 (d, J
CF
=12.2
Hz), 149.9 (d, J
CF
=262 Hz) ppm.
2-Bromo-5-((Z)-2-fluorovinyl)thiophene 4s
1
H NMR (400 MHz, CDCl
3
) δ 5.89 (dd, J
HF
=44 Hz, J
HH
=4.8Hz, 1H), 6.64 (dd, J
HF
=82 Hz,
J
HH
=5.2 Hz, 1H), 6.8 (d, J
HH
=4 Hz, 1H), 7.16 (d, J
HH
=6 Hz, 1H) ppm.
19
F NMR (376 MHz,
CDCl
3
) δ -120.4 (dd, J
HF
=82 Hz, J
HF
=44 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 105.2 (d,
J
CF
=3 Hz), 113.4 (d, J
CF
=10 Hz), 127.1 (d, J
CF
=3 Hz), 129.5, 136.2 (d, J
CF
=2 Hz), 147.2 (d,
J
CF
=268 Hz) ppm.
(Z)-4-Fluoro-1,1-diphenylbuta-1,3-diene 4t
- 89 -

1
H NMR (400 MHz, CDCl
3
) δ 5.51 (ddd, J
HF
=41.8 Hz, J
HH
=4.8Hz, J
HH
=11.2Hz 1H), 6.45 (ddd,
J
HF
=88 Hz, J
HH
=4.8Hz, J
HH
=1.2Hz 1H), 6.94 (d, J
HH
11.2 Hz, 1H), 7.19-7.28(m, 7H), 7.32-
7.39(m, 3H) ppm.
19
F NMR (376 MHz, CDCl
3
) δ -125.6 (dd, J
HF
=88.6 Hz, J
HF
=41.8 Hz) ppm.
13
C NMR (100 MHz, CDCl
3
) δ 109.6, 117.5 (d, J
CF
=5 Hz), 127.7, 127.8, 128.3, 128.4, 130.4,
139.4, 141.9, 143.2 (d, J
CF
=5 Hz), 149.3 (d, J
CF
=268 Hz) ppm.
(E)-4-Fluoro-1,1-diphenylbuta-1,3-diene 4t
1
H NMR (400 MHz, CDCl
3
) δ 6.11 (ddd, J
HF
=17.6 Hz, J
HH
=11.6Hz, J
HH
=11.2Hz 1H), 6.49 (d,
J
HH
11.6 Hz, 1H), 6.95 (dd, J
HF
=83.6 Hz, J
HH
=10.8 Hz, 1H) 7.17-7.28(m, 7H), 7.32-7.40(m, 3H)
ppm.
19
F NMR (376 MHz, CDCl
3
) δ -124.51 (dd, J
HF
=83.6 Hz, J
HF
=17.6 Hz) ppm.
13
C NMR
(100 MHz, CDCl
3
) δ 112.7 (d, J
CF
=17Hz), 119.7 (d, J
CF
=11 Hz), 127.5, 127.6, 127.7, 128.3,
128.5, 130.32, 139.3, 142.1, 143.2 (d, J
CF
=13 Hz), 153.3 (d, J
CF
=262 Hz) ppm.

- 90 -

3.6 REFERENCES
1. Filler, R.; Saha, R., Future Medicinal Chemistry 2009, 1, 777.
2. Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V., Chem. Soc. Rev. 2008, 37, 320.
3. Cahard, D.; Xu, X. H.; Couve-Bonnaire, S.; Pannecoucke, X., Chem. Soc. Rev. 2010, 39,
558.
4. Kirsch, P., Modern fluoroorganic chemistry: synthesis, reactivity, applications. Wiley-
VCH, 2004.
5. Hara, S., Top. Curr.Chem. 2012, 327, 59.
6. Van der Veken, P.; Senten, K.; Kertesz, I.; De Meester, I.; Lambeir, A. M.; Maes, M. B.;
Scharpe, S.; Haemers, A.; Augustyns, K., J. Med. Chem. 2005, 48, 1768.
7. Jacobsen, C. B.; Nielsen, M.; Worgull, D.; Zweifel, T.; Fisker, E.; Jorgensen, K. A., J.
Am. Chem. Soc. 2011, 133, 7398.
8. Muller, K.; Faeh, C.; Diederich, F., Science 2007, 317, 1881.
9. McKinney, B. E.; Urban, J. J., J. Phys. Chem. A 2010, 114, 1123.
10. Schlosser, M.; Zimmermann, M., Synthesis, 1969, 75.
11. Cox, D. G.; Gurusamy, N.; Burton, D. J., J. Am. Chem. Soc. 1985, 107, 2811.
12. Kataoka, K.; Tsuboi S., Synthesis 1999, 452.
13. Reutrakul, V.; Rukachaisirikul, V., Tetrahedron Lett. 1983, 24, 725.
14. Inbasekaran, M.; Peet, N. P.; McCarthy, J. R.; Letourneau, M. E., J. Chem. Soc.-Chem.
Commun. 1985, 678.
15. Boys, M. L.; Collington, E. W.; Finch, H.; Swanson, S.; Whitehead, J. F., Tetrahedron
Lett. 1988, 29, 3365.
16. McCarthy, J. R.; Matthews, D. P.; Barney, C. L., Tetrahedron Lett. 1990, 31, 973.
17. Tanaka, K.; Nakai, T.; Ishikawa, N., Chem. Lett. 1979, 175.
18. Lee, S. H.; Schwartz, J., J. Am. Chem. Soc. 1986, 108, 2445.
19. Petasis, N. A.; Yudin, A. K.; Zavialov, I. A.; Prakash, G. K. S.; Olah, G. A., Synlett 1997,
606.
20. Greedy, B.; Gouverneur, V., Chem. Commun. 2001, 233.
21. Zhao, Y. C.; Huang, W. Z.; Zhu, L. G.; Hu, J. B., Org. Lett., 2010, 12, 1444.
22. Ghosh, A. K.; Zajc, B., J. Org. Chem. 2009, 74, 8531.
- 91 -

23. Alonso, D. A.; Fuensanta, M.; Najera, C.; Varea, M., J. Org. Chem. 2005, 70, 6404.
24. Vinson, W. A.; Prickett, K. S.; Spahic, B.; Demontellano, P. R. O., J. Org. Chem. 1983,
48, 4661.
25. Bergmann, F.; Kalmus, A.; Breuer, E., J. Am. Chem. Soc. 1958, 80, 4540.

- 92 -

Chapter 4. Bioisosteric replacement of α,- β,- and γ- phosphate of
trisphosphoric acid with the squaryl moiety. Synthesis of
corresponding nucleotide analogues and evaluation of their chemical
properties.
4.1 INTRODUCTION
Since the first synthesis of cyclobutendione accomplished by the pioneering work of Cohen
[1, 2] and West [3], 3,4-dihydroxy-3-cyclobutene-1,2-dione (squaric acid) has attracted attention
of the researchers form many diverse research areas [4]. Even though various applications as
well as theoretical and synthetic studies were reported to date, only recently the unusual physical
and chemical properties of squaric acid motif were fully examined [5]. In the last decade,
immense amount of biological and pharmaceutical applications of cyclobutenediones have been
discovered [4, 6].
Squaric acid is a unique diacid system that possess two hydroxyl groups and demonstrates
unusual high double acidity with the pK
a1
= 0.54 and pK
a2
= 3.58 (Figure 4.1). The reason for
such high acidity stands in resonance stabilization and increase in aromaticity of dianion in
comparison with the protonated form. Deprotonated form of squaric acid is a unique structure
that efficiently distributes two negative charges through the zwitter ionic aromatic system
(Figure 4.2) [2, 5].
OH HO
O O
pK
a
= 0.54
O HO
O O
pK
a
= 3.58
O O
O O
O O
O O
O O
O O
O O
O O
O O
O O

Figure 4.1. Acidity and charge delocalization of squaric acid [5].

OH HO
O O
O O
O O
2+
+ 2H
+

Figure 4.2. Aromaticity increases upon deprotonation of squaric acid [5].
- 93 -

However, observing vibrational spectra of carbanions with the general structure C
n
O
n
2-
West
et. al. [3] established that significant stabilization energy effect cannot be described only by
delocalization of electron density (Figure 4.3) [4, 5]. Thus it was established that oxocarbon
dianions with the general formula C
n
O
n
2-
associated to previously unrecognized class of aromatic
compounds [7].
O O
O O
sqarate
O O
O
deltate
O
O O
O O
O
O
O
O
O
O
croconate rhodizonate

Figure 4.3. Oxocarbon dianions with the general formula C
n
O
n
2-
[7].
Further examination of geometric (bond length equalization, bond order indices) and
energetic parameters (aromatic stabilization energies) of squarate dianion indicate that it is truly
an aromatic system. This fact was also supported by the work of Schleyer et al. [8], who
introduced nucleus independent chemical shift (NICS) magnetic parameter as additional
characteristic of aromaticity. Systems with more aromatic character tend to have more negative
NICS value [8, 9]. In order to decrease the effect of local shielding of nearby σ-bonds which
complicates the analysis of small rings, the NICS parameters were determined by calculating
chemical shift at 0.6 Å above ring centers (Table 4.1).
Table 4.1. Relative aromaticity of oxocarbon dianions in terms of NICS values and chemical
shift.
Benzene (reference) NICS (0.6) = -10.1 ppm
C
3
O
3
2-
NICS (0.6) = -21.3 ppm highly aromatic
C
4
O
4
2-
NICS (0.6) = -7.1 ppm
moderate
C
5
O
5
2-
NICS (0.6) = -0.5 ppm
less aromatic
C
6
O
6
2-
NICS (0.6) = -0.6 ppm less aromatic

- 94 -

Preparation of cyclobutenediones
Wide-ranging studies of Liebeskind [10, 11], Moore [12, 13], Paquette [14] and others have
shown that squaric acid template can be used as a versatile synthon for the synthesis of highly
functionalized carbocyclic and heterocyclic compounds. To date a number of general and
practical methods to access cyclobutenediones have been developed [4, 15].
All major methods for the synthesis of cyclobutenediones could be divided into three groups:
thermal or photochemical cycloadditions reactions, reactions involving transition metal
complexes and cyclopropene ring expansion reactions. More complex cyclobutenediones can be
accessed by derivatization of more simple cyclobutenediones. Examples of these preparation
methods are briefly discussed below[15].
Cycloaddition reactions. Thermal cyclodimerization of tetrahaloalkenes as well as
cycloaddition involving alkynes and dichloroketenes have demonstrated to be the best methods
for preparation of simple squaric acid derivatives. Chlorovinylene carbonate can undergo
cyclodimerization upon irradiation in acetone providing squaric acid after dehydration with SO
2
(Scheme 4.1) [16-20].
Scheme 4.1. Preparation of cyclobutanones via cycloaddition reactions.
R
2
R
1
C O
Cl
Cl
+
Cl
Cl
O
R
2
R
1
H
3
O
O
R
2
R
1
O
F
F F
Cl
2
delta
F
Cl
F
F F
F
Cl
F
Zn
F
F F
F
F F
ROH
F
F F
F
RO OR
H
2
SO
4
RO OR
O O
delta
O O
Cl Cl
O
2
hv
O
O O
O
O O
Cl Cl
Cl Cl
H
3
O
OH
OH
OH
OH HO
OH
OH
HO
SO
2
HO OH
O O

Reactions involving transition metal complexes. Thermal [2+2] reactions involving
chromium, tungsten [21], nickel, iron [22] carbonyl complexes with electron rich double or triple
bonds afford cyclobutendiones with the good yields under mild conditions [23-25]. Below is an
example of novel cycloaddition reaction with alkyne involving nickel complex. Although those
reactions usually provide good yield and could be carried out under mild conditions, there are
difficulties associated with the use of toxic transition metal carbonyl complexes. Additionally,
- 95 -

these methods are uncommon because they allow the access of only a limited diversity in
substituents (Scheme 4.2).
Scheme 4.2. Preparation of cyclobutanones via formation of transition metal complexes
(bpy)Ni(CO)
2
(bpy)Ni(R R)
(bpy)Ni(COD)
R R
Ni(bpy)
R
R
O
O
R R
CO
CO
R
R
O
O
O
O O
or CO

Cyclopropene ring expansion reactions. Cyclobutendiones can be prepared through ring
expansion of cyclopropene. However, it is not very practical method because of problems
associated with preparation of the starting material (Scheme 4.3) [26].
Scheme 4.3. Preparation of cyclobutanones through ring expansion reactions
O
R
2
R
1
+
CCl
3
R
1
R
2
O CCl
3
- Cl
Cl
Cl
O R
2
R
1
H
2
SO
4
O R
2
R
1
O

Derivatisation of simple cyclobutenediones. Preparation of cyclobutenediones via
cycloaddition reactions or transition metal complex is limited to either squaric acid or simple aryl
or alkyl derivatives. However, multifunctionalized cyclobutendiones could be effectively
synthesized through derivatization of simple cyclobutadienes. Extensive studies [27, 28] have
shown that simple diones could be functionalized by reacting with different carbon and
heteroatom nucleophiles. The reactions usually proceed through two major pathways.
Nucleophile either attacks vinyl carbon of cyclobutendione or targets carbonyl group of of dione,
the latter usually requires acid hydrolysis of resulting alcohol. First method is usually employed
when nucleophiles are O or N or neutral C –H C-Zn, C-Cd, C-Sn, whereas the second pathway
is followed mostly in case of carbon nucleophiles generated using Li or Mg bases (Scheme 4.4)
[29, 30].

- 96 -

Scheme 4.4. Preparation of cyclobutedione derivatives via reactions with nucleophiles
R
R
O
O
Nu
R = OH,Cl, Br, OR
Nu
R
O
O
Nu
R
R
O
O
Nu
R
R
O
O
R
O
O
Nu
H
3
O
Nu
R
R
O
OH
2
Nu
H
2
O

Application to organic synthesis and catalysis
In the last few years, the areas of asymmetric hydrogen-bonding and bifunctional catalysis
have been of great interest for the synthetic community and significant progress in those fields
has been achieved [31, 32]. Traditional asymmetric organocatalysis employs chiral urea or
thiourea catalysts which can activate electrophilic groups such as carbonyl through hydrogen
bonding interactions and catalyze reactions with nucleophiles [33]. Recently, amide derivatives
of squaric acid (squaramides) have gained considerable attention in the area of organocatalysis
[5]. For example, Jorgensen et. al. have developed asymmetric addition of 1,3-dicarbonyls to
acyl phosphonate derivatives using a squaramide based catalyst [34].
Squaramides can be easy obtained from squaric acid or esters. Lately, wide varieties of
different squaramides were synthesized and used as efficient ligands in organocatalysis. Easily
accessible squaramide skeleton permits screening large amount of analogues and enables fine
tuning of the pKa‟s of their NH‟s as well as efficient optimization of catalyst‟s chiral
environment. Moreover, structurally well-defined chiral environment provided by rigid squaric
acid template can be used for asymmetric induction [5].
Rawal et. al. [35] examined a number of squaramide catalysts in order to compare them with
the conventional urea and thiourea analogues (Figure 4.4). It was established that the rigid
structure of scquaramide motif creates a well defined chiral pocket that significantly improves
the enantioselectivities of the reactions.

- 97 -

O
O
Ph
N
NH
Ph
Ph
HO
NH N
H
O O
CF
3
F
3
C
N
H
H
N
O O
N
H
N
H
N
CF
3
F
3
C

Figure 4.4. Examples of organocatalysts based on squaramide structural motif.
Application to medicinal chemistry
Squaric acid as metal chelators. Trying to develop new chemosensors for recognition of
metal ion in solution Brucker et. al. [36] was attracted by the squaric acid functionality. Squaric
acid possesses two vinylogous acid moieties cross-conjugated with the two carbonyl groups
forming a resonance stabilized system. Several squaric acids based moieties, such as squaric
acid, hydroxamic-ester and hydroxamic-amide were examined on their chelating abilities. It was
found that early representations about η
2
cyclobutendione coordination to the first and second
row of transition metals were not correct. Chelation cannot happen because bite angle of squaric
acid ring is too large (3.3 Å) for the effective binding and it requires 2.6 Å. Therefore only η
1

coordination mode can be present in the solution. Interestingly investigation showed that
hydroxamate ester did not show any sight of chelation and only hydroxamate amide were able to
demonstrate significant binding to the Fe(III). Authors suggested that stabilization of existing
limiting resonance structure that reveals amide character of the carbonyl group is responsible for
binding to the metal. Thus N-hydroxyl group is cross-conjugated with the opposite carbonyl
group, but it does not have significant effect on the adjacent carbonyl group. In a similar manner,
carbonyl group that chelates to the metal is in conjugation with the substituent across the ring,
therefore its electron donating ability correlates to that of the fourth substituent. Thus amide
substituent possesses much higher donating ability and much better stabilizing resonance
structure responsible for metal chelation than the ester substituent (Figure 4.5) [36].

- 98 -

2+
O
O O
O
M
O
O O
O
M
M
2+
O
OR N
O
O
R
M
O
N
H
N
O
O
R
M
R

O
R N
O
R
M
O
R N
O
R
M
O O
R' N
O
R
M
O O
R' N
O
R
M
Strong chelation
Chelating ability of O
-
depends
on electronic conjugation effect
of R'

Figure 4.5. Chelation of squaric acid with different metals.
Hydroxamic acids (Figure 4.6) are considered to be one of the best Zn chelation agents, used
by medicinal chemists for long time for targeting metalloproteases containing Zn in the active
site [36]. Unfortunately, hydroxamic acids are susceptible to metabolic degradation and
glucuronidation and generally adsorbed insufficiently. Therefore, there is an extensive interest in
discovering a proper substitute that can be incorporated into the structures of metalloprotease
inhibitors. Thus, squaramide analogues of hydroxamic acid have been examined as a potential
new Zn chelating group for the inhibition of matrix metalloprotease (MMP) enzymes. Hanessian
et. al. [37], who investigate squaric acid analogues of hydroxamic acid, were able to syntheses
compounds with less than 100 μM activity, which is considered to be promising for further
optimizations.
O O
R' N
O
R
Zn
2+
O O
R' N
O
R
Zn
2+ O
R N
O
R
Zn
2+

O O
N
H
N
H
HO
OBu
ADAMTS-5 IC50 = 2.6 mM

Figure 4.6. Zn
2+
chelation of hydroxamic and squaric acids.
- 99 -

Squaric acid as isosteric replacement of carboxylic acids and amino acids
Kim and Misco in their work proposed that squaric acid moiety can be used as isoelectronic
replacement of carboxylic acid in biological systems [38]. They reported the synthesis of squaric
acid analogues of phosphonoacetic acid (PAA) and phosphonoformic acid (PFA), which were
approved as inhibitors for the treatment of cytomegalovirous in patients with AIDS.
Interestingly, despite the conformational rigidity of squaric acid motif, such compounds
exhibited binding activity very similar to that of PFA (Figure 4.7).
O
P HO
OH
O
OH
PFA
O
OH
P
O
HO
HO
PAA
P
O
HO
HO
O
O
HO

Figure 4.7. Squaric acid mimics carboxylic acid function.
This promoted the interest of many research groups to investigate the possibility for
incorporation squaryl moiety into design of biomolecules, showing that it can be sufficient
bioisostere for carboxylic acid function. These concepts were expanded in the works of Shinada
et al. [39] who tried to incorporate squaric acid into the side chain of amino acid of natural toxins
(Figure 4.8). Several analogues of nephilatoxin-8 (NPTX-8) were synthesized with replacement
in tyrosine or asparagine linker (Figure 4.8). Preliminary tests using crickets showed that
replacement with glutamine based squaryl amino acid functionality exhibit ten times more
paralytic activity than the natural toxin NPTX-8. This example demonstrates that that squaric
acid can function as an effective isostere for hydrophilic amino acid side chain.
H
N
N
H
N
H
N
H
H
N NH
2
HN
O
O
CONH
2
O
H
N
N
H
N
H
N
H
H
N NH
2
HN
O
O O
R
O
O
R=OH
R=NH2

Figure 4.8. Nephilatoxin-8 analogues with squaric acid moiety replacing carbamide function of
- 100 -

tyrosine or asparagine linker.
Proposing that novel squaric acid bioisostere has sufficient electronic similarities to the amino
acid, it was attempted to introduce squaramide function into N-methyl-D-aspartic acid (NMDA)
[40]. These investigations result in the design of EAA-90 (Perzinfotel), a potent, selective,
competitive NMDA receptor antagonist. Structure-activity relationship studies (SARs) have
indicated that cyclic molecule provides more possibility for the formation of hydrogen bonds
with the receptor, eliminating intramolecular hydrogen bonding (Figure 4.9).
OH P
O
HO
HO
NH
2
O
IC50 = 390 nM
N
H
P
O
HO
HO
H
2
N
O
O
IC50 = 470nM
P
O
OH
HO
N
NH
O
O
IC50 = 49nM
N P
O
HO
HO
NH
O
OH
IC50 = 110 nM
EAA-90 (Perzinfotel)

Figure 4.9. Squaric acid mimics the amino acid structural motif.
Sqauric acid as natural isosteres of guanidines and cyanoguanidines
Development of effective treatment for peptidic ulcer disease based on guanidine stimulated
the interest for the development of improved H2 antagonists. Thus, cyanoguanidine group was
substituted with wide range of structural mimetics, including squaramide. Although this
replacement wasn‟t as potent as starting cimetidine, it retains some activity demonstrating that
squaramide is a considerable isostere (Figure 4.10) [41].
N
NC
N
H
N
H
S
N
NH
N
H
S
N
NH
HN
O
O

Figure 4.10. Replacement of guanidine moiety by squaric acid.
In the effort to find inhibitor for HIV-1 Tat-TAR in 2005, Rana et. al. [42] screened a library
of peptidomimetic compounds which revealed that compound with R = guanidine exhibits anti-
HIV-1 activity (Figure 4.11). Further structure-activity relationship (SAR) study showed that
elevated basicity of guanidine substituent lead to poor pharmacokinetic properties. First efforts to
- 101 -

reduce basicity by attaching electron-withdrawing groups to guanidine moiety lead to decrease in
activity. However, significant drop in basicity without losing any activity was achieved by
substitution of guanidine with squaramido function (Figure 4.11) [42].
N
N
H
N
H
N
NC
N
H
N
H
O O
Cl
Cl
O
N O
H
N
O
NH
2
O
O
NH
2
NH
2
H
2
N
Peptidomimetic inhibitors of HIV-1 Tat-TAR
interactions
Lee et al.
R
NH
NH
2
O
O
NH
NH
2
N
O
2
N
NH
NH
2
HN

Figure 4.11. Replacement of guanidine moiety by squaramide.
Traditional and novel phosphate mimic
Ionic character of the phosphate functionality efficiently retains phosphorylated intermediates
inside the cells and significantly reduces the degradation via hydrolysis. However, substantial
charge leads to the lack in membrane permeability and do not allow phosphate containing
molecules to be suitable biological probes [43-44].
Moreover, phosphate esters and anhydride functionalities are very labile toward enzymatic
hydrolysis. Thus, discovery of the appropriate phosphate mimics are very important for finding
enzyme inhibitors. The challenge is to design functional groups that mimic both the electronic
properties and the spatial arrangement of the phosphate group while improving cell permeability
and stability [45].
The phosphate anion plays a major role in enzymatic binding and recognition events.
Therefore phosphate isostere with reduced charge may not be able to bind the target protein.
Moreover, the unique capability of tetrahedral phosphate moiety to form anhydride linkage while
keeping a negative charge makes this endeavor very difficult [46]. Thus, recent progress in
finding right phosphate mimics focuses on their interactions with target protein that resemble
natural phosphate interactions with improved stability in similar physiologic environment and
better bioavailability.
- 102 -

Below four major categories of phosphate isostere replacements will be described: three
“traditional” replacement motifs (phosphorus-containing, sulfur-containing, and carboxylate
linkages) and the more unique, cyclic mimics (Figure 4.12) [45].
P
O
O
O
P
O
OR
OR
P
O
O
O
F
F
R
O
O
O O
S
R R
1
S
R R
1
S
R R
1
S
RO N
H
O
O O O
O
O
R
1
O
HO
O
O
OH
OH
HN
S
O
HN
S
O
S
Z
Y
F
OR
F
X
N
NH
S
R
O
R
1
O
O
N
NH
S
O
O
O
R
1
R
O
OH HO
R R
3
R
2
R
1
hydroxytropolone reverse
sulfhydantoin
sulfhydantoin perfluorylarl
X,Y=OH or
F, Z=N or C-
R, where R
is EW groop
tetronic acid squaric acid thiazolidinone rhodanine
dimethylene
sulfide
sulfoxide sulfone acetylsulfamate
dicarboxylate
phosphonate phosphonate
ester
fluorophosphonate
Novel cyclic phosphate mimics
Traditional phosphate mimics

Figure 4.12. Traditional and novel phosphate mimics.
Phosphonates, sulfones, and dicarboxylates. Although phosphonates, sulfones, and
dicarboxylates are traditional and dominant phosphate mimics in medicinal chemistry, these
compounds usually have poor cell membrane permeability and not always display acceptable
biological activity. Under physiological conditions, phosphonates do not possess di-ionic
character because of their high ionization constant. Moreover, replacement of oxygen with
- 103 -

methylene group eliminates the possibility of hydrogen bonding, which usually reduces the
binding affinities significantly. Nevertheless extensive studies have been conducted on the
development of inhibitors of glycosyltransferases, fucosyltransferases and squalene synthetase
(Figure 4.13) [47-49].
Incorporation of one or two fluorine atom drops the pKa of the phosphonate, which better
resemble pKa of the natural phosphate (Figure 4.13). Incorporatioin of Pmp or F
2
Pmp by
hexameric peptide sequences inhibit protein tyrosine phosphatase 1B [50].
O
P
O
O
O
N
H
HN
O
P
O
O
OH
N
H
HN
O
C
F
2
P
O
O
O
N
H
HN
O
pTyr Pmp
F
2
Pmp

Figure 4.13. Phosphotyrosyl (pTyr), nonhydrolyzable phosphonomethyl phenylalanine (Pmp
IC
50
= 200 μM) and phosphonodifluoromethyl phenylalanine (F2Pmp IC
50
= 0.1 μM) mimetics.
Sulfur-containing mimics. Multiple efforts have been made to replace phosphorous containing
linkages with sulfur-containing bioisosteres. Introduction of the oxidized forms of sulfur can
increase stability and the membrane permeability of phosphates in vivo, however, sulfones
providing isosteric replacements for a phosphate, have a negative charge deficiency (Figure 4.14)
[51].
O
O
RO
A
P
O
OR
O
T
O
O
natural DNA
O
RO
A
O
OR
T
sulfone DNA
S
O
O

Figure 4.14. Backbone distortion in the DNA analogues with sulfone linkage, making suitable as
inhibitors of restriction enzymes.
Dicarboxylates. Dicarboxylate structural motif represents another class of compounds that can
potentially mimic the anionic monophosphate or diphosphate moieties. It was proposed that
compounds containing dicarboxylates are able to chelate to the metal ions in a similar manner as
- 104 -

phosphate containing natural substrates (Figure 4.15). Additional computational study indicated
that, inspite of large size of the dicarboxylate, the distance between anionic oxygen atoms of
these two moieties varies only by 0.1 Å [52; 53].
O
P
O O
P
O
O O
O
O O
O
O
O
O
O
H
+
-H
+
farnesyl pyrophosphate natural substrate
Chaetomellic acid A

Figure 4.15. Chaetomellic acid A, an example of novel dicarboxylate-containing mimic of
farnesyl pyrophosphate, which inhibits the enzyme FTPase
Cyclic Phosphate Mimics. Cyclic structural motifs serving as isosteric replacement of
phosphate such as tetronic acid-, squaric acid-, thiazolidinone-, rhodanine-, perfluorylaryland
sulfhydantoin-based derivatives were introduced relatively recently and therefore there is limited
amount of data that exist about their actual ability to mimic phosphate function. Phosphate
mimic usually tends to decrease charge on the phosphate and improve bioavailability. Large
amount of the enzyme active sites are filled with neutral amino acids and do not contain
positively charged metal ion, which enhance the potentials of using neutral compounds as
sufficient inhibitors [45].
Tetronic acid. Molecular modeling simulations predicted that tetronic acid anion can exhibit
hydrogen bonding properties similar to phosphate. Furthermore, novel and effective dual
specificity phosphatase (DSP) inhibitor based on 3-acyltetronic acid was recently discovered [54,
55].
Perfluoroaryl compounds. Calculations also showed that general spatial and electronic
distribution of perfluoroaryl compounds are analogous to those of di- and tri-phosphate motif
and potentially can mimic guanosine 5‟-triphosphate or guanosine 5‟-diphosphate [56, 57].
Hydroxytropolones. Hydroxytropolones were discovered as potent inhibitors of IMPase
mainly because of their ability to chelate magnesium ions in the active sight. Additional analysis
- 105 -

indicates that binding of hydroxytropolones to the active site mimics the binding of the natural
phosphate ester [58].
Thiazolidinines and rhodanines. Computational studies suggested that thiazolidinines bearing
carboxylic acid substituent at R1 position can potentially mimic interactions of the
diphosphonate with the lysine moiety. Moreover, it was suggested that thiazolidinine ring can
position the side chains inside the active site and can fill the space designated for uridine and
glucosamine moieties [59].
Derivatives of thiazolidinones and rhodanines are already considered to be a novel class
of anticancer agents and are very useful for medicinal chemistry applications. Based on their
ability to mimic natural substrates bearing the phosphate group several potent inhibitors have
been synthesized. Thus, compounds a-c (Figure 4.16) represent replacement motif for
lysophosphatidic acid (LPA) with the increase of stability and enhanced activity against prostate
cancer cell lines [59].
O
P O
O
O
O
OH
(CH
2
)
n
CH
3
O
Lysophosohatidic acid
N
S
O
O
HN
C
18
H
37
a
N
S
O
O
HN
C
18
H
37
b
O
N
S
O
O
HN
C
18
H
37
O
O
c

Figure 4.16. Compounds a-c containing thiazolidinones as phosphate bioisostare exhibit activity
against various cancer cell lines (a:IC
50
7.1-12.6 μM; b: IC
50
5.4-11.2 μM; c: IC
50
5.5-15.5 μM)
Squaric acid-structural motifs as phosphate mimic
For many years, squaric acid has been a target of great interest in the field of medicinal
chemistry, for example, providing valuable derivatives that can serve as enzyme inhibitors [45,
60, 61].

Figure 4.17. Calculations of electrostatic charge distribution of squaric acid moiety compared to
that of phosphate ester [62].
- 106 -

One of the most remarkable examples to use squaric acid as phosphate mimic has been
demonstrated by Sato et al. (Figure 4.18) [62, 63]. At first, ab initio calculations showed that
electrostatic charge distributions in dimethyl phosphate and N –isopropyl–N –
methylsquaryldiamide are very similar (Figure 4.18). Based on these calculations, researchers
attempted the synthesis of modified thymidine dimer that contained squaric acid moiety instead
of a phosphate (Figure 4.18). Subsequently, modified dimer TsqT was incorporated into
oligodeoxynucleotides chain which was further subjected to enzymatic hydrolysis of squaryl
modified oligodeoxynucleotides. After hydrolysis the TsqT structural motif was isolated along
with other deoxynuleotides, demonstrating enzymatic stability of squarimide linkage.
O
NH
O
N
NH
O
O
NH
O
O
O
O
N
NH
O
O
O
HN
HO
N
NH
O
O
NH
O
O
O
O
N
NH
O
O

Figure 4.18. Oligonucleotide analogues containing squaril group incorporated instead of
phosphate group
Another example of successful employment of squaric acid derivatives as a non-hydrolyzable
phosphotyrosine mimic is the discovery of potent inhibitors of Yersinia PTP, which show
decrease in overall charge and increase in membrane permeability [64] (Figure 4.19).
OH O
O
OH O
O
NO
2
O
O
O
NH
3
P
O
O
O
IC50 = 47 uM
IC50 = 56 uM phosphotyrosine

Figure 4.19. Squaric acid derivatives that are used as non-hydrolysable phosphotyrosine
mimicetics.

- 107 -

Design squaric acid based nucleotide analogues
As was described before, modeling studies of electrostatic charge distributions of squaric acid
relative to the natural phosphate moiety indicate that the squaric acid based phosphate isostere
frequently have decreased overall charge due to its distribution through the multiple bonds.
Nevertheless, polarizing patterns of these two moieties are very similar [62] (Figure 4.20).
Although a large amount of the computational study was performed to demonstrate structural
and electrostatic correlations between cyclic phosphate substitutes and their parent phosphates, in
fact, only limited information exists about their actual ability to mimic the phosphate group.
Additionally, similar to phosphate functionality, the squaric acid motif contains carbonyls that
can function as hydrogen-bonding partners and binding sites for divalent metal ions.
HO OH
O O
pK
1
= 0.54,
pK
2
= 3.48
-2H
+
O O
O O
O O
O O
2+
HO OH
O O
2+
RO OR
O O
2+ P
OR
O
RO
O
=

Figure 4.20. Compared polarizing patterns between squaric acid and phosphate
In our initial design, we decided to incorporate squaric acid motif instead of the β-phosphate
of triphosphoric acid. Additionally, in order to prevent enzymatic hydrolysis we intended to
replace bridging oxygen atoms in α, β or β, γ positions of triphosphoric acid with the isopolar and
isosteric CF
2
group. This modification was recently applied to achieve non-hydrolizable
properties of nucleotide analogues [65]. Towards this endeavor, the synthesis of new non-
hydrolyzable squaric acid analogue of triphosphoric acid has been carried out. Subsequently,
synthesis of squaric acid based non-hydrolysable deoxynucleotide analogues that can be applied
to study structure and function of DNA polymerase β has been attempted (Figure 4.21).
- 108 -

X X
O O
P P
O
OH
O
HO
OH HO
OR RO
O O
OR RO
O O
2+
O
P
OR
OR O
O C
F F
O
OH
O P
X X
P O
O O
O O
O O
B
X = CH
2
, CF
2
, NH
O
P
O
P
O
O
P
OH
O
HO
HO OH
OH
Non-hydrolizable properties
Phosphate isostere

Figure 4.21. Design of nucleotide analogues with the squaryl moiety incorporated instead of the
β-phosphate
2.2 RESULTS AND DISCUSSION
Studies toward synthesis of squaric acid-based dNTP Analogues
Below, we describe synthetic approach towards squaric acid analogues of triphosphoric acid
(Scheme 4.5). Our initial attempts to afford compound 1 using coupling reaction have failed.
Likewise, reactions of squaric acid dichloride [66] with various diethyl difluoromethylene
phosphonate derivatives in presence of various bases also did not provide access to desired
squaryl analogues (Scheme 4.5) [67-79].
Scheme 4.5. Initial attempts to prepare squaryl analogue of triphosphoric acid.
C
F
2
CF
2
P P
O O
OEt
OEt EtO
EtO
O O
Cl Cl
O O
O
P OEt
OEt
BrZnF
2
C
O
P OEt
OEt
BrCuF
2
C
O
P OEt
OEt
BrCdF
2
C
O
P OEt
OEt
F
2
C
O
P OEt
OEt
HF
2
C
O
P OEt
OEt
Bu
3
SnF
2
C
Cl Cl
O O
Cl Cl
O O
Cl Cl
O O
Cl Cl
O O
Cl Cl
O O
a
b
c
d
e
f
S

a: i).THF, rt; ii), DMA, rt; iii) THF/DMA, CuCN/CuBr/CuCl, 2LiCl, 0 to rt; b: i) THF, rt; ii) DMA, rt, 2LiCl; c i)
DMA/DMF/Triglyme/HMPA, rt,ii) DMF, CuCl, rt; d:i) TBAT/CsF/TBAF/KF/K
2
CO
3
, DMF/CH
3
CN/THF, rt; e: i)
LDA/LTMPA/NaH/t-BuOK/CsF/K
2
CO
3
/DBU/Et
3
N/Lutidine/2,6-Di-t-BuPy, THF/DMF, -78°C to rt, f: i).
Pd(PPh
3
)
4
, ClCH
2
CH
2
Cl,ii) PdCl
2
(PPh
3
)
2
, CuI, DMF, rt.
- 109 -

Since direct coupling of different phosphonates with squaric acid dichloride or ethyl ester did
not give any positive results, we decided to attempt the synthetic approach developed by
Liebeskind et. al. [80] This method involves stepwise addition of phosphonate to the squaric acid
ring. We choose squaric acid diethyl ester and diethyl difluoromethyl phosphonate as starting
materials for our synthesis. Both the starting materials can be accessed in large scale from
commercially available compounds. Squaric acid diethyl ester can be easily obtained by treating
squaric acid with triethylorthoformate under reflux, followed by chromatographic separation
[81], whereas diethyldifluoromethyl phosphonate is easily accessible in large scale through the
reaction of sodium salt of diethylphosphite with difluorochloromethane (Freon 22) [82] (Scheme
4.6). Thus, we started our synthesis with the coupling of squaric acid diethyl ester 1 with one
equivalent of diethyl difluoromethyl phosphonate 2.
Scheme 4.6. Preparation of difluoromethylphosphonate derivatives of squaric acid.
OEt EtO
O O
OH HO
O O
P EtO
OEt
O
H
P EtO
OEt
O
CF
2
H
OH
F
2
C P
O
OEt
OEt
O
OEt EtO
O
F
2
C P
O
OEt
OEt
O
OEt EtO
Si
TMSOTf
OTMS TMSO
O
OEt
O
O
F
2
C P
O
OEt
OEt
TMSCl / Et
3
N
O
OEt
F
2
C P
O
OEt
OEt
+
O
CHOEt
3
\ EtOH
+
CHF
2
Cl \ Na
N
Li
1
2
3
4
5 6

After several adjustments of the reaction conditions and exploring different bases we found
that employment of the sterically hindered base lithium 2,2,6,6-tetramethylpiperidine amide
(LTMPA) provide access to desired alcohol 3 in good yield, 75 %. Interestingly, hydrolysis of
the alcohol 3 in presence of the TFA, acetic acid or 12 M hydrochloric acid did not give the 3-
substituted squaric acid derivative, but led to decomposition products only. It is known [83-86]
that such alcohols bearing electron donating substituents readily undergo hydrolysis. Apparently,
- 110 -

difluoromethylene substituent highly destabilizes the positively charged cationic transition state
formed during typical hydrolysis, thus preventing the reaction.
Therefore, in order to proceed with our synthesis, we decided to employ transformation
developed by Liebeskind [80], now frequently used to access 3,4-substituted squaric acid
derivatives. In this reaction, trace amount of trimethylsilyltrifluoromethane sulfonate (TMSOTf)
is used to initiate the elimination of alcohol moiety, whereas 1,2-bis(trimethylsiloxy)ethane is
employed to protect forming carbonyl group across the ring and recover TMSOTf catalyst.
Scheme 4.7 represents the mechanism of this reaction.
Scheme 4.7. Mechanism of the reaction that convert 4 into 5
OEt EtO
O
O
F
2
C P
O
OEt
OEt
Si
OEt
O
O
O
TMSOTf
OEt EtO
O F
2
C P
O
OEt
OEt
Si O
Si
O S
O
CF
3
OEt O
O
O S
O
CF
3
O
O
TMSO OTMS
OEt
O
EtO
O
TMSO S
O
CF
3
O
+ + EtOTMS
Et
F
2
C P
O
OEt
OEt
F
2
C P
O
OEt
OEt
O S
O
CF
3
O
Si +
OTMS
F
2
C P
O
OEt
OEt
4
5
4a 4b 4a

Alcohol 3 following this approach obtained was converted into trimethylsilyl derivative by
treating it with the chlorotrimethylsilane (TMSCl) and Et
3
N in anhydrous diethyl ether for
several days. The resulting compound 4 was then converted to the cyclic ketal 5 as described
above. After screening different reaction conditions, we realized that in order to achieve
maximum conversation we had to use large amount of catalyst and run the reaction for a
prolonged time. However, excess of the TMSOTf in the reaction mixture led to the deprotection
of the desired monoketone 5 generating diketone 6. Unfortunately, diketone 6 had no use for the
synthetic pathway we were following, because nucleophilic attack always occur on activated C-1
carbonyl group generating the 2,4-substituted squaric acid derivatives. To the best of our
knowledge, it has not been previously reported that such transformation occurs with squaric acid
bearing strong electron withdrawing groups. In fact, this not only decreases the yield
significantly, but also slows down the reaction due to destabilization of cationic intermediate 4a.
- 111 -

After numerous attempts to adjust reaction conditions we found that in order to achieve
maximum yield of 5 one gram of the starting material 4 had to be treated with 1 equivalent of
1,2-bis(trimethylsiloxy)ethane, 0.4 equivalents of TMSOTf catalyst in acetonitrile at room
temperature for 12 hours. Under these conditions, convertion to 4 had increased to 85 % with 35-
45 % yield of the product 5.
Scheme 4.8. Preparation of bis[difluoromethylphosphonate] derivatives of squaric acid.
THF/-78
o
C
O
OEt
O
O
F
2
C P
O
OEt
OEt
P EtO
OEt
O
CF
2
H
OEt
O
O
OR
F
2
C P
O
EtO
EtO
F
2
C P
O
OEt
OEt
TMSOTf
OTMS TMSO
CH
3
CN
O
O O
O
C
F
2
CF
2
P P
O O
OEt
OEt EtO
EtO
C
F
2
CF
2
P P
O O
OH
OH HO
HO
O O
H
+
R = H
R= TMS
TMSCl / Et
3
N
H
2
O/TMSCl
LTMPA
5
7
8
9

With the monoketone 5 in hands that has one carbonyl group protected and another one
available for the next nucleophilic attack, we attempted the next step in our synthetic pathway.
The conversion of 5 into 7 was carried out by employing the same condition as we used to access
compound 3 (Scheme 4.8). Compound 8 can be afforded by using TMSCl/Et
3
N conditions.
However, at this step we quenched the reaction mixture with TMSCl generating compound 8 in
situ. Even though addition of chlorotrimethylsilane did not convert all lithium salt into TMS-
protected alcohol, this simple step in the purification protocol turned out to be a very critical
factor. During the work up procedures the TMS-ether 8 was extracted into hexane and following
treatment with water allowed the isolation and characterization of pure 8. In contrast, alcohol 7
was not soluble in hexane and its purification required column chromatography. Purification of
the compound 7on silica gel represents significant challenge because of its high polarity and low
UV-absorption properties. Moreover, reaction cannot be scaled up and thus, we had to work with
a very small amount of the product. Generation of squaric acid derivative 9 from 8 was
accomplished with 100% conversion using the conditions developed for preparation of 5.
Unfortunately, we did not succeed in our attempts to deprotect carbonyl groups of compound
9. It turned out that huge electron withdrawing effect of two difluoromethylenephosphonate
groups turn carbonyl groups into hydrated form and thus, deprotection of such groups is not
driven by formation of the carbon-oxygen double bond. It was reported [87] that this reaction can
- 112 -

only happen under extreme conditions and carbonyl group predominantly exists in equilibrium
with hydrated form. In this case, the resonance stabilization of the aromatic ring is significantly
decreased and squaryl bioisostere could be less likely serves as a phosphate mimic. However, in
spite of our best efforts, squaric acid derivative 9 could not sustain harsh deprotection conditions
and decomposed upon treatment with acids under elevated temperatures. To overcome this issue
we decided to decrease electron withdrawing effect on the squaric acid rings by substituting one
of the difluoromethylene groups with a methylene group.
Scheme 4.9. Attempts to prepare non-hydrolysable squaryl analogue of triphosphoric acid.
THF/-78
o
C
O
OEt
O
O
F
2
C P
O
OEt
OEt
P EtO
OEt
O
CH
3
OEt
O
O
OR
H
2
C P
O
EtO
EtO
F
2
C P
O
OEt
OEt
TMSOTf
OTMS TMSO
CH
3
CN
O
O O
O
C
F
2
CH
2
P P
O O
OEt
OEt EtO
EtO
C
F
2
CH
2
P P
O O
OH
OH HO
HO
O O
H
+
R = H
R= TMS
TMSCl / Et
3
N
H
2
O/TMSCl
BuLi
9
10
11
12

Thus, we proceeded with already described material 5 using the same synthetic approach
employed earlier to access compound 9 (Scheme 4.9). In order to introduce methylene group we
reacted monoketone 5 with lithiated diethyl methylphosphonate by prior treatment with n-
butyllithium. The reaction mixture was quenched with TMSCl and squaric acid derivative 11
was isolated according to protocol described for 8. Compound 12 was obtained via already
established procedure involving bis(trimethylsiloxy)ethane and TMSOTf as a catalyst. It is worth
to mention that the substitution of CF
2
group by CH
2
positively affected overall yields of all
reactions with significant increase (2-3 times) in the course of the reactions. Nevertheless,
compound 12 as well as squaryl analogue 9 could not be deprotected without decomposition.
Consequently, we came to the conclusion that both difluoromethylene groups have to be
replaced with the methylene groups in order to reduce even more electron withdrawing effect on
the aromatic ring. Therefore, we followed already established synthetic approach by using twice
the amount of diethyl methylphosphonate instead of diethyl difluoromethylphosphonate (Scheme
4.10). Interestingly, the yields throughout the synthetic scheme were significantly improved,
compounds became more stable and the isolation of the products became much easier.

- 113 -

Scheme 4.10. Attempts to prepare non-hydrolysable squaryl analogue of triphosphoric acid
OEt EtO
O O
O
H
2
C P
O
OEt
OEt
O
OEt EtO
TMSOTf
OTMS TMSO
O
OEt
O
O
H
2
C P
O
OEt
OEt
THF/-78
o
C
H
3
C P
O
OEt
OEt
+
1. BuLi,
2. TMSCl
Si
1. BuLi,THF -78
o
CH
3
C P
O
OEt
OEt
P
O
OEt
OEt
P
O
EtO
EtO
P
O
OEt
OEt
P
O
EtO
EtO
O
HO
OH
P
O
OEt
OEt
P
O
EtO
EtO
OH
OH
O
1. TMSBr
2. Bu
4
N
+
OH
-
P
O
O
O
P
O
O
O
O
HO
OH
[ Bu
4
N
+
]
4
OEt
O
O
P
O
OEt
OEt
O
2. TMSCl
P
O
EtO
EtO
CH
3
COOH
CH
3
OTMS / TMSOTf
O O
Si
13 14 15
16 17 19
1

To our delight, bismethelenephosphonate squaryl ester 17 (
3,4
BM-β-SQTPE) can be easily
deprotected by treating it with acetic acid for several days. However, it appeared that right after
deprotection, it is almost completely hydrated to one carbonyl group (Scheme 4.10). And the
hydrated form exists in equilibrium with the non-hydrated form. Fortunately, we discovered that
the hydrated form can be almost exclusively converted into non-hydrated form by treating it with
excess of TMSOTf in acetonitrile (Scheme 4.11). On the other hand, final purifications and
probing of nucleotide analogues have to be performed in aqueous solutions and as soon as we
exposed the acid or the ester to water, the carbonyl group reacts and become hydrated.
Subsequently
3,4
BM-β-SQTPE 17 analogue can undergo deprotection of the phosphate ester
groups by treating it with the bromotrimethylsilane according to the method established by
McKenna and co-workers [88]. The acid 18 (
3,4
BM-β-SQTPA) was quantitatively converted into
tetrabutylammonium salt by treating it with tetrabutyl ammonium hydroxide and drying the
material under high vacuum.

- 114 -

Scheme 4.11.
31
P NMR spectra of the mixture containing hydrated and non-hydrated forms of
the
3,4
BM-β-SQTPE ester 17 and corresponding acid (
3,4
BM-β-SQTPA) 18 obtained after
TMSBr hydrolysis
CH
3
CN
2 weeks
TMSOTf
O
OH
HO
P P
O
OEt
OEt
O
EtO
OEt
O
P P
O
OEt
OEt
O
EtO
OEt
O
2. H
2
O
1. TMSBr
P P
O
OH
OH
O
HO
OH
O
Hydrated form (17)
Non-hydrated form (17)
18
O
P P
O
OH
OH
O
HO
OH
O
OH
OH

Preparation of DNTP analogues based on
3,4
BM-β-SQTPA
The synthesis of
3,4
BM-β-SQTPA based deoxynucleotide analogues was attempted adopting
general preparation methods involving tosylate or iodide substitution on 5‟-position of the
nucleoside. As starting materials for that reaction four benzoyl protected 5‟-tosylates of dA, Th,
dC, dG and 5‟-thymidine iodide were prepared and were subjected to this substitution reaction.
However, after screening of various coupling reaction conditions, we could not access desired
deoxynucleotide analogues (Scheme 4.12). It turned out that no substitution reaction occurs at
- 115 -

room temperature and
3,4
BM-β-SQTPA (Bu
4
N
+
)
4
salt rapidly decomposes upon heating, without
producing any trace of deoxynucleotide analogues.
Scheme 4.12. Unsuccessful attempts to prepare nucleotide analogues using salt 19
O
OBz
O
BaseBz
Ts
+
O O
P P
O O
O
O
O
O
4
Bu
4
N
O
OBz
O
BaseBz
P
O
O O
P
O
O
O O
3
Bu
4
N
20-23 19 24-27

Redesign of squaric acid based triphosphoric acid analogues
We attempted a redesign of squaryl analogues of triphosphoric acid which sustain hydration
of carbonyl group by decreasing even more electron withdrawing effect of the phosphates
(Figure 4.22).
Replacement of the β-phosphate. At first, incorporation of the squaric acid motif into the β-
position of the triphophosphoric acid can be accomplished via the 2,4-substituted derivative.
Furthermore, incorporation of another bridging atom such as nitrogen between the phosphate and
the squaric acid moiety will decrease phosphate effect on the aromatic ring and also provide
additional flexibility to tetrahedral phosphate. In contrast to the phosphonate group, squaramide
motif contributes to the resonance stabilization by donating electrons to the aromatic system.

Figure 4.22. Attempts to redesign squaryl analogues of triphosphoric acid
- 116 -

Replacement of the γ-phosphate and α-phosphate. In the case of γ-incorporation, the influence
of single phosphate substituent would enhance the aromaticity of the squaryl motif, increasing
isosteric properties of phosphate replacement. The similar effect can be expected in the case of
replacement the α-phosphate with squaric acid, which can be achieved via 3,4-substituted
squaramide analogue.
The investigation toward the synthesis of 2,4-substituted BMPA- β-SQ
Scheme 4.13 describes the synthetic approach we endeavored to access
2,4
BMPE-β-SQ
analogue 31. Solution of acetic acid in acetonitrile quantitatively hydrolyzes protected squaryl
derivative 15 resulting in the formation of squaric acid moiety. Obtained squaric acid analogue
(MPESQA) 28 was converted into squaric acid chloride derivative 29 by facile reaction with
oxalyl chloride in chloroform in presence of catalytic amount of DMF. Finally, the preparation of
the target
2,4
BMPE-β-SQ analogue was attempted via reaction of methylenephosphonate squaric
acid chloride 29 with lithium salt of methyl phosphonate followed by acid treatment promoting
elimination of water.
Scheme 4.13. Synthetic route toward
2,4
BMPE-β-SQ 31
H
2
C
O O
P
O
OEt
OEt
HO
(COCl)
2
CHCl
3
/DMF H
2
C
O O
P
O
OEt
OEt
Cl
H
2
C
O
P
O
OEt
OEt
EtO
O
O
CH
3
COOH
15 28 29
O
OH
P
P
O
OEt
OEt
O
EtO
EtO
THF/-78
o
C 1. BuLi,
2. H
2
O
C
H
2
O
P
O
OEt
OEt
HO
OH
H
2
C P
O
EtO
EtO
1M HCl
30 31
H
3
C P OEt
OEt
O

As it was expected, nucleophilic attack on 29 proceeds exclusively on activated carbonyl
group across the ring from the methylenephosphonate. After quenching the reaction with the
water the resulting alcohol was purified by C-18 chromatography. Treatment with 1 M
- 117 -

hydrochloric acid solution promotes elimination of water giving access to 31. The desired
diphosphate ester 31 was isolated using reverse phase chromatography (Scheme 4.13).
Scheme 4.14. Synthesis of analogues of triphosphoric acid with squaramide functional fragment.
O O
N
H
P
O
OEt
OEt
P
O
EtO
EtO
O O
HN N
H
P
O
EtO
EtO
P
O
OEt
OEt
O O
P
O
OEt
OEt
Cl
O
P
NH
2
EtO
OEt
+
DMF, 60
o
C
DMF, RT
2
O O
Cl Cl
O
P
NH
2
EtO
OEt
+ 4
29 32 33
1c 32 34

As shown on the scheme 4.14, squaramide derivatives 33 and 34 can be prepared through
reaction of corresponding acid chloride derivatives with diethyl aminomethylphosphonate in
DMF under various temperatures.
Study toward incorporation of squaryl moiety into γ-position of triphosphoric acid
Pβ–C
XY
–Pγ nucleotide analogs have been extensively utilized as probes of molecular
interactions with base excision repair (BER) enzymes that are overexpressed in some cancer
cells [89]. Thus, we also began our work on the synthesis of nucleotide analogues, where Pβ–O–
Pγ function was replaced by SQA–CH
2
–Pγ motif. Squaric acid derivative that possess only one
substituent on the four-membered ring more efficiently distributes electron density through the
aromatic system. Therefore, squaryl modification at the γ–phosphate would most probably well
resemble electrostatic charge distributions of natural phosphate. Additionally, introduction of this
group could potentially reveal new interaction with the active site not observed with the natural
nucleotide triphosphate.
Our initial idea was to explore preparation of triphosphoric acid analogue in which terminal
phosphate was replaced by squaric acid function with the bridging oxygen atom. Thus, squaric
acid dichloride 1c was treated with tetrabutylammonium salt of methylene bisphosphonate 36b.
Yet less than 10% conversion was observed, even after applying various reaction conditions. On
the other hand, reactions proceeded very smoothly when squaric acid dichloride 1c was reacted
- 118 -

with the tetrabutylammonium salt of difluoromethylene bisphosphonate 36c (Scheme 4.15).
After quenching the reaction mixture with water the desired analogue was isolated using C18
flash chromatography and characterized by NMR spectroscopy. Compound 36d was found to be
very unstable and despite our best effort, it decomposed within short period of time. Therefore,
we decided to utilize more stable linkage between squaryl moiety and phosphate and focused on
the nature of the bridging methylene group.
Scheme 4.15. Preparation of γ-squaryl analogues of triphosphoric acid with oxygen bridging
atom.
O O
Cl Cl
H
2
C P
O
O
OH
[Bu
4
N
+
]
3
P
O
O
O
+
O O
Cl Cl
F
2
C P
O
O
OH
[Bu
4
N
+
]
3
P
O
O
O
+
F
2
C P
O
O
OH
[Bu
4
N
+
]
3
P
O
O
O
O
O
OH
1c
1c
36c
36b
36d
H
2
C P
O
O
OH
[Bu
4
N
+
]
3
P
O
O
O
O
O
OH
36e

Scheme 4.16. Preparation of analogues of nucleotide diphosphates in which β-phosphate
substituted by squaryl function
H
2
C
O O
P
O
OEt
OEt
HO H
2
C
O O
P
O
OH
OH
HO
28 35
1. TMSBr
2. H2O
H
2
C
O O
P
O
O
OH
O
Bu
4
N
+
OH
-
[Bu
4
N
+
]
2
36

O
OBz
O P
O
O
O
OBz
O
O
O O
P
O
O
OH
[Bu
4
N]
2
+
+
BBz
n
BBz
n
B =
N
N
N
N
NH
2
NH
N
O
O
[Et
3
NH
+
]
2
Ts
DMF, RT
O
O
O NH
3
/MeOH
O
OH
O P
O
O
B
[Et
3
NH
+
]
2
O
O
O
36 37 B = dA
38 B = Th
39 B = dA
40 B = Th
20 B = dA
21 B = Th

- 119 -

Consequently, we attempted the incorporation of squaric acid instead of γ-phosphate having
stable CH
2
linkage. Although, we decided initially to explore the preparation of the squaryl
analogues of diphosphonate in which terminal phosphate was substituted with squaric acid motif.
This was undertaken in order to predict the behavior of the γ-analogue based on strong
similarities between these two substances.
MPESQA 28 prepared from 15 as described above was reacted with bromotrimethylsilane
and the afforded silyl ester was hydrolyzed with water. The resulting acid 35 was quantitatively
converted into tetrabutylamonium salt 36. The final coupling reaction was conducted by treating
MPSQA [Bu
4
N
+
]
2
salt 36 with benzoyl protected 5‟tosyl deoxynucleosides (Scheme 4.16). After
stirring the reaction mixture for 3 days at room temperature in DMF deprotection of the formed
deoxynucleotide analogues was carried out without isolation by treating with the mixture
NH
3
/H
2
O/MeOH 7:1:1. Deoxynucleotide analogues were purified using weak anion exchange
column PW-DEAE using water/1 M triethylamonium carbonate sol. (TEAB) as eluent system.
Furthermore, we were able to prepare the MPSQA [Ph
4
P
+
]
2
salt, which was purified using
C18 chromatography and crystals suitable for X-ray analysis were obtained. Acquired X-ray
crystallography data showed that none of the carbonyl groups exhibit any sign of hydration
(Figure 4.23). Particularly, bond length of C52-O4 is approximately 2.4 Å similar to that of C50-
O6, whereas bond lengths of C50-C51 and C51-C52 are very close and 1.43 Å, indicating that
these bonds are involved in resonance stabilization.
Indeed,
13
C NMR spectrum of the compound MPSQA [Bu
4
N
+
]
2
36 (Figure 4.24) also
supports these findings. Thus, in the high field part of carbon NMR of the salt 36 appeared only
as one peak at 201.9 ppm, which corresponds to the both C50 and C52 carbons. This in fact
indicates that C1 hydroxyl group and C3 carbonyl group are inter-exchangeable.
Inspired by these results, we undertook the synthesis of γ-substituted squaric acid
deoxynucleotide analogues. To our delight, we were able to adopt the general approach for the
preparation of α,β-hydrolyzable nucleotide analogues, where α,β-phosphoranhydride bond is
formed via nucleophilic substitution of morpholidate in the nucleotide monophosphate (Scheme
4.17). Corresponding morpholidate derivatives were synthesized according to a well-established
protocol [89]. After most of the starting material was consumed (apr. 7 days), the reaction
mixture was loaded on flash SAX anion exchange column and the target nucleotide analogues
were isolated using water/1 M TEAB mixture. Subsequently, these analogues were finally
- 120 -

purified using weak PW-DEAE anion exchange HPLC system, which allowed preparation of the
compounds with the good overall yield.

Figure 4.23. X-ray structure of MPSQA [Ph
4
P
+
]
2
.
3H
2
O.

Figure 4.24. Down field part of the
13
C NMR spectrum of MPSQA [Bu
4
N
+
]
2
salt 36.

- 121 -

Scheme 4.17. Synthesis of the analogues of nucleotide triphosphates in which γ-phosphate
substituted by squaric acid function.
O
OH
O P
O
O
O
P
O
H
2
C
O
O
O
O O
OH
O P
O
N
OH
O
B
B
B =
N
N
N
N
NH
2
NH
N
O
O
[Et
3
NH
+
]
3
DMSO \ RT
7 days
O
O O
P
O
O
OH
[Bu
3
NH
+
]
2
41
+
44 B = dA
45 B = Th
42 B = dA
43 B = Th

Study toward incorporation of squaryl moiety into α-position of triphosphoric acid
To extend our studies of employing squaric acid as a phosphate mimic, we attempted the
synthesis of dNTP analogues in which α-phosphate was substituted by squaric acid functionality
and α, β–oxygen linkage replaced by methylene group. This change will make nucleotides stable
toward enzymatic hydrolysis and provide the possibility for structure, function and fidelity
studies of DNA polymerase β. This approach may provide access to potential interactions of
squaric acid functionality inside the enzyme active site.
In order to access α-squaryl nucleotide analogues, we decided to attempt coupling of squaric
acid moiety with the nucleoside via squaramide bond formation. The route for of this pathway
was based on our observations that squaric acid site of MPSQA [Bu
4
N
+
]
2
salt 36 acts as a very
weak nucleophile. Bearing an electron withdrawing substituent enhances this effect.
Nucleophilic attack involving squaric acid (OH) oxygen atoms is very improbable because the
resonance stabilization of the ring pulling electrons from hydroxyl group equally distribute
electron throughout the aromatic system. Therefore, it seemed reasonable to turn squaric acid
into an electrophile as acid chloride (MPSQCl) 29 and perform nucleophilic attack on it from the
nucleoside site.

- 122 -

Scheme 4.18. Synthesis 5‟aminonucleosides 54 and 55.
O
OBz
O
BBz
n
S
O
O
H
3
C
O
OBz
N
3
BBz
n
O
OH
H
2
N
B
NaN
3
DMF
MsCl
pyridine
H
2
, Pd/C
48 B = dA
49 B = Th
50 B = dA
51 B = Th
46 B = dA
47 B = Th
O
OBz
HO
BBz
n
O
OH
N
3
B
NH
4
OH/MeOH
54 B = dA
55 B = Th
52 B = dA
53 B = Th

Consequently, as starting materials for this coupling reaction 5‟-amino nucleosides had to be
obtained [90]. Initially benzoyl protected nucleoside was reacted with methanesulfonyl chloride
giving access to 5‟-sulfonyl derivatives 48-49 (Scheme 4.18). Following reaction with sodium
azide was carried out in DMF at 80 °C for 3 days. 5‟-Azidodeoxynucleosides 50, 51 produced in
the reaction were subjected to deprotection with the mixture NH
4
OH/MeOH without further
purification. Deprotected azide derivative 52-53 were found to be very polar and were isolated
using normal phase chromatography with 100% methanol as an eluent. Finally, functional azide
was reduced with hydrogen using palladium poisoned with carbon as a catalyst. 5‟-
Azidodeoxynucleoside was dissolved in methanol, placed in the steel reactor containing
palladium catalyst and the reactor was pressurized with 8 atmospheres of hydrogen. This reaction
required a significant amount of time to achieve maximum conversion. After two weeks, reaction
was stopped and 5‟-aminonucleoside was purified on silica gel with the 100% methanol as an
eluent.

- 123 -

Scheme 4.19. Synthesis of analogues of nucleotide diphosphates in which α-phosphate
substituted by squaryl function
O
OH
N
H
C
H
2
P
O
EtO
EtO
O O
H
2
C Cl
O O
P
O
OEt
OEt
+
DMF, rt
O
OH
H
2
N
N
N
NH
2
O
N
N
NH
2
O
29 55 56

Initially 5‟-aminothymidine was firstly coupled with MPSQCl 29 and the corresponding
nucleotide analogues were isolated using reverse phase column in very good yield 78 %.
However, all our attempts to remove phosphate ester groups without hydrolyzing squaramide
bond were unsuccessful. Therefore, we decided to remove ester groups prior to the coupling
reaction (Scheme 4.20).
Scheme 4.20. Preparation of MPSQCl 29 for coupling with 5‟-amino nucleosides.
H
2
C Cl
O O
P
O
OTMS
OTMS
H
2
C
O O
P
O
OEt
OEt
HO
(COCl)
2
CHCl
3
\ DMF H
2
C
O O
P
O
OEt
OEt
Cl
TMSBr
28 29 57

In fact, it turned out to be challenging to find the right base for this reaction, because squaric
acid chloride primarily reacts with any type of base forming the ylide. Therefore, we decided to
utilize two equivalents of the 5‟-aminonucleoside, where one equivalent would form squaramide
bond, whereas another one would act as a base (Scheme 4.21). After the reaction was carried out,
it was quenched with 1 M solution of triethylammonium carbonate and the reaction mixture was
initially run through SAX anion exchange column followed by more precise HPLC purification.
Subsequent conversion of these dNDP analogues into dNTP analogues could be accomplished
by enzymatic γ–phosphorylation [91] that in addition would provide the successful example of
squaric acid mimick of the phosphate moiety.

- 124 -

Scheme 4.21. Synthesis of nucleotide triphosphate analogues in which α-phosphate were
substituted by squaryl function.
O
OH
N
H
C
H
2
P
O
O
OH
O O
P
O
HO
OH
O
OH
N
H
C
H
2
P
O
O
O
O O
O
OH
H
2
N
enzymatic
phosphorylation
[Et
3
NH
+
]
2
H
2
C Cl
O O
P
O
OTMS
OTMS
+ B B
B =
N
N
N
N
NH
2
NH
N
O
O
DMF, RT
B
57 58 B = dA
59 B = Th
60 B = dA
61 B = Th
54 B = dA
55 B = Th
2

Computational Investigations
Squaric acid is often viewed as phosphate isostere because of its similar polarizing patterns [63,].
Thus distribution of electron density and aromaticity of the squaryl structural motif are the most
important factors that control its ability to serve as a phosphate mimic. To evaluate the
aromaticity of the synthesized squaryl analogues and the factors that affect the aromaticity a
computational study was initiated. Using the NICS approach developed by Schleyer and co-
workers [8]the aromaticiy values for a number of squaryl analogues of trsphosphoric acid were
obtained and are presened in Table 4.2.

- 125 -

Table 4.2. NICS (0.6) Values for squaric acid derivatives calculated at the B3LYP/aug-cc-
pVTZ//B3LYP/cc-pVTZ level of theory using Gaussian 09
[a,b]
[92]
Cl Cl
O O
C
H
2
P
O
OEt
OEt
O O
C
F
2
CF
2
P P
O O
OEt
OEt EtO
EtO
O O
Cl C
H
2
CH
2
P P
O O
OEt
OEt EtO
EtO
O O
C
F
2
P
O
OEt
OEt
O O
HO C
H
2
P
O
OEt
OEt
O O
EtO C
H
2
P
O
OEt
OH
O O
EtO C
H
2
P
O
OH
OH
O O
EtO
C
H
2
P
O
OEt
OEt
O O
HO
O
OH
P
P
O
OEt
OEt
O
EtO
EtO
C
H
2
P
O
OEt
OEt
O O
NH
H
2
C P
O
EtO
OEt
OH HO
O O
O O
O O
OEt EtO
O O
C
H
2
P
O
OEt
OEt
O O
NH Th C
H
2
P
O
O
OH
O O
EtO
N
H
O O
NH
H
2
C P
O
EtO
OEt
H
2
C P
O
OEt
OEt
N
H
NH P P
O O
OEt
OEt EtO
EtO
O O
O
P
C
F
2
O
O
O
HO
P
O
OH
O O
O
P
C
H
2
O
O
O
O
P
O
OEt
O O
O
P
C
H
2
O
O
O
HO
P
O
OH
O O
1
9 17
6a 28
31
C
H
2
P
O
O
OH
O O
O
36
29
6 6b 35
36i
1a
1b
1c
33
34 34b 36f
36e 36d
O
P
O C
H
2
O
O
HO
P
O
OH
O O
44b
56
-1.08 -1.48 -1.61 -0.81
Not Aromatic
ModeratelyAromatic
Aromatic
-2.83 -3.24 -3.39 -3.20 -3.45
-3.56
-3.49
-5.82
-6.92
-5.80
-5.12
-5.22
-6.05 -5.31 -6.43
-6.24 -6.24
-4.22
-4.99

[a] For reference, benzene is -9.93 under the same conditions [b] To simplify computational investigations all
simulations were performed with phosphate ethyl esters substituted by methyl ester groups additionally for 12
Thymidine was replaced with Me. These substitutions do not cause to any significant changes in NICS values
The computational investigations showed that all compounds can be devided into three groups
according to their aromaticity values: Not Aromatic, Moderately Aromatic and Aromatic. As can
- 126 -

be seen the substituents on squaryl ring significantely impact the aromaticity. Compounds with
the electron donating substituents tend to have higher NICS values wereas compounds with
electron withdrawing substituent have overall decreased NICS values. It is interresting to note
that the changes on the ionization state of adjasent phosphate functionality (compounds 35 and
28 compare to 36 and 36i) have significant affect on aromaticity values placing 36 and 36i
among the highly aromatic compounds.
4.3 CONCLUSIONS
In this work the possibility for incorporation of squaryl moiety instead of the phosphate
functionality into the α-, β-, and γ- positions of the nucleotide trisphosphate was investigated.
The synthetic approach towards corresponding analogues of trisphoshoric acid was found and
methodologies for their preparation were developed. All prepared compounds were characterized
by
1
H,
13
C,
31
P NMR spectroscopy and high resolution mass spectrometry. Performed
computational study using NICS approach demonstrated the dependency of substituent effects on
aromaticity of the squaryl ring. This synthetic work together with computational investigations
can help to predict the properties and find the access towards novel biologically important
molecules based on squaryl moiety.
4.4 EXPERIMENTAL PART
Preparation of compound 3
OEt EtO
O O
O
P EtO
OEt
CF
2
H +
THF, -78oC,
OEt EtO
O
OH
F
2
C P
O
OEt
OEt
LTMPA

Absolute THF (200 mL) was placed in 500 mL round bottom flask equipped with magnetic
stirrer under inert atmosphere. 2,2,6,6-Tetramethylpiperidine (8.7 g, 10.48 mL, 61.7 mmol, 1.05
eq.) was added and solution was cooled down to 0 °C in ice-water bath. After that, 2.5 M n-BuLi
solution in hexanes (24.8 mL, 61.7 mmol, 1.05 eq.) was added dropwise to the amine solution
and the resulting reaction mixture was maintained at 0 °C for 30 minutes. Then reaction mixture
was cooled to -78 °C and THF solution of diethyldifluoromethylphosphonate 2 (11.05 g, 58.8
mmol) was slowly introduced. After stirring the reaction mixture at this temperature for another
40 minutes, a solution of 3,4-diethoxy-3-cyclobutene-1,2-dione 1 (10 g, 58.8 mmol) in 25 mL of
- 127 -

THF was slowly added to the reaction mixture. After two hours of stirring at -78
o
C the reaction
mixture was quenched with saturated solution of ammonium chloride and allowed to warm to
room temperature. THF was removed under reduced pressure; the crude mixture was purred into
water and extracted three times with EtOAc. Organic layers were combined, washed with water,
brine and dried over MgSO
4
. EtOAc was removed and crude reaction mixture was subjected to
column chromatography on silicagel with EtOAc/ Hexane as eluent (gradient conditions: from
0/100 to 60/40) to afford 3 (11.8 g 56 %) as a pale yellow oil.
1
H NMR (400 MHz, CDCl
3
) δ: 1.24 (t, J = 6.8 Hz, 3H), 1.3 (t, J = 6.8 Hz, 6H), 1.35 (t, J = 7.2
Hz, 3H), 4.2 (m, 6H), 4.4 (m, 2H).
13
C NMR (100 MHz, CDCl
3
) δ: 14.9, 15.2, 16.1, 16.2, 65.2
(d, J=6.8 Hz), 65.4 (d, J=6.7 Hz), 67.1, 69.8, 86.8 (dt, J=17.3 Hz, J=25 Hz), 117.2 (dt, J=205.8
Hz, J=267.4 Hz), 135.7, 160.6, 177.9.
19
F NMR (376 MHz, CDCl
3
) δ: -115 (dd, J=312.4 Hz,
J=101.4 Hz), -117.3 (dd, J=312Hz, J=101.4 Hz).
31
P NMR (162 MHz, CDCl
3
) δ: 4.89 (t,
J=101.4 Hz).
Preparation of compound 4
OEt EtO
O
OH
F
2
C P
O
OEt
OEt
Et
3
N, TMSCl
Et
2
O
OEt EtO
O
O
F
2
C P
O
OEt
OEt
Si

To a stirring solution of compound 3 (0.5 g, 1.4 mmol) in anhydrous diethyl ether (10 mL)
under inert atmosphere was added TMSCl (0.227 mg, 2.1 mmol, 1.5 eq.). The mixture was
cooled to 0 °C and solution of triehtylamine (0.425 mg, 4.21 mmol, 3 eq.) in anhydrous ether
(5 mL) was added drop wise. The reaction mixture was allowed to warm to room temperature
and stirring was continued for two days. After that, the reaction mixture was diluted with ether
and poured into ice-water. Ethereal solution was washed with water and brine, and then dried
over MgSO
4
. Volatiles were removed under reduced pressure and crude reaction mixture was
purified with flash chromatography on silica with EtOAc/ Hexane as the eluent (gradient
conditions: from 0/100 to 40/60) affording the pure product as colorless oil 505 mg 84 %.
1
H NMR (400 MHz, CDCl
3
) δ: 0.00 (s, 9H), 1.13 (t, J=7.2 Hz, 3H), 1.14-1.19 (m, 6H), 1.24 (t,
J=7.2 Hz, 3H), 3.94-4.19 (m, 6H), 4.22-4.36 (m, 2H).
13
C NMR (100 MHz, CDCl
3
) δ: 0.00,
14.1, 14.6, 15.35, 15.4, 63.7 (d, J=6.7 Hz), 66.2, 68.8, 87.2 (dt, J=18 Hz, J=25.2 Hz), 117.1 (dt,
- 128 -

J=208.2 Hz, J=267.5 Hz), 135.1, 160.8, 177.4.
19
F NMR (376 MHz, CDCl
3
) δ: -114.03 (dd,
J=319.6 Hz, J=103 Hz), -114.98 (dd, J=308Hz, J=103 Hz).
31
P NMR (162 MHz, CDCl
3
) δ: 4.73
(t, J=103 Hz)
Preparation of compound 5
OEt EtO
O
O
F
2
C P
O
OEt
OEt
Si
TMSO OTMS
OEt
O
O
O
S
O
O
O
F
F
F
Si
F
2
C P
O
OEt
OEt

Solution of compound 4 (1 g, 2.3 mmol) in anhydrous acetonitrile (15 mL) was placed in the
flask purged with inert gas. Then ethylenedioxybis(trimethylsilane) (0.484 g, 2.3 mmol ) was
added to reaction mixture followed by TMSOTf (0.258 g, 0.94 mmol, 0.4 eq). The solution was
stirred at room temperature for 12 hours. Acetonitrile was removed in vacuum; reaction mixture
was poured into the water and extracted three times with ether. Organic layers were combined,
dried over MgSO
4
, reaction mixture was concentrated in vacuum and subjected for column
chromatography on silica with EtOAc/ Hexane as eluent (gradient conditions: from 0/100 to
80/20) affording the pure product as pale yellow oil 262 mg 32 %.
1
H NMR (400 MHz, CDCl
3
) δ: 1.28-1.34 (m, 9H), 3.99-4.07 (m, 4H), 4.17-4.32 (m, 4H), 4.47
(q, J=7.2 Hz, 2H).
13
C NMR (100 MHz, CDCl
3
) δ: 15.2, 16.1, 16.2, 65.1 (d, J=6.6 Hz), 66.2,
69.2, 114.8 (dt, J=218 Hz, J=258.3 Hz), 116.2, 142.3 (dt, J=13.1 Hz, J=25.2Hz), 163.5 (m),
190.7.
19
F NMR (376 MHz, CDCl
3
) δ: -109.6 (d, J=107 Hz).
31
P NMR (162 MHz, CDCl
3
) δ:
3.87 (t, J=107 Hz).
Compound 6
OEt
O F
2
C P
O
OEt
OEt
O

1
H NMR (400 MHz, CDCl
3
) δ: 1.33 (t, J=7.2 Hz, 6H), 1.47 (t, J=7.2 Hz, 3H), 4.25-4.32 (m,
4H), 4.82 (q, J=7.2 Hz, 2H).
13
C NMR (100 MHz, CDCl
3
) δ: 15.4, 16.2, 16.3, 65.9 (d, J=6.7
Hz), 73.2, 114.1 (dt, J=215.8 Hz, J=260.6 Hz), 166.5 (dt, J=13.5Hz, J=26.1 Hz), 186.3, 192.6,
- 129 -

196.1.
19
F NMR (376 MHz, CDCl
3
) δ: -112.9 (d, J=105 Hz).
31
P NMR (162 MHz, CDCl
3
) δ:
3.12 (t, J=105 Hz).
Preparation of compound 11
OEt
O
O
F
2
C P
O
OEt
OEt
O
Si
P
O
EtO
EtO

Diethyl methyl phosphonate 13 (427 mg 2.8 mmol) was placed in a flask filled with inert gas,
dissolved in 30 mL of freshly distilled THF and cooled to -78 °C. A solution of n-butyllithium
(1.23 ml of 2.5 M in hexanes) was slowly added to the stirred solution. The reaction was kept at
-78 °C for a 30 min when solution of 5 (1 g, 2.8 mmol) in anhydrous THF was added to reaction
mixture and it was stirred at -78 °C for two more hours. The reaction mixture was quenched with
addition of 332 mg (3.08 mmol) of TMSCl and the temperature was raised to ambient
temperature. All volatiles were removed under reduced pressure and the residue was distributed
between hexanes and water. The organic fractions were washed with distilled water, brine and
then dried over MgSO
4
. Hexane was evaporated to afford 552 mg (34 %) of the product ,which
was used in the next step without further purifications.
1
H NMR (400 MHz, CDCl
3
) δ: 0.00 (s, 9H), 1.09-1.24 (m, 15H), 2.06 (m, 1H), 2.59 (m, 1H),
3.63-4.28 (m, 14H).
13
C NMR (100 MHz, CDCl
3
) δ: 0.00, 13.3, 14.2, 14.3, 14.4, 14.5, 31.5 (d,
J=137.9 Hz), 58.3 (d, J=6.2 Hz), 59.4 (d, J=6 Hz), 62.6 (d, J=6.7 Hz), 62.7 (d, J=6.9 Hz), 63.7,
63.8, 64.6, 81.4, 108.7, 110.7, 113.2, 152.3.
19
F NMR (376 MHz, CDCl
3
) δ: -99.5 (dd, J=117.5
Hz, J=327 Hz), -112 (dd, J=114.7Hz, J=324Hz).
31
P NMR (162 MHz, CDCl
3
) δ: 4.9 (dd, J=114.7 Hz, J=117.5 Hz), 27.3 (d, J=3.6 Hz).

- 130 -

Compound 9
F
2
C P P
O
OEt
OEt
O
EtO
OEt
O
O O
O

The compound 9 was prepared according to the procedure described for 5 with 55% isolated
yield.
1
H NMR (400 MHz, CDCl
3
) δ: 1.29 (t, J=7.2Hz, 6H), 1.35 (t, J=7.2Hz, 6H), 3.02 (d, J=24Hz,
2H), 3.88-3.92 (m, 4H), 4.06-4.14 (m, 8H), 4.24-4.32 (m, 4H).
13
C NMR (100 MHz, CDCl
3
) δ:
15.2, 15.25, 15.3, 15.4, 23.27 (d, J=141.1 Hz), 62.7 (d, J=6.8 Hz), 65.3, 65.5, 65.6, 113.1 (m),
113.5 (m), 114.4 (dt, J=217.7 Hz, J=257.8 Hz), 140.6 (m), 150 (m).
19
F NMR (376 MHz,
CDCl
3
) δ: -109.6 (dd, J=7.2 Hz, J=108 Hz).
31
P NMR (162 MHz, CDCl
3
) δ: 4.51 (dt, J=7.2 Hz,
J=108 Hz), 21,9 (q, J=7.2 Hz).
Preparation of compound 12
F
2
C C
F
2
P P
O
OEt
OEt
O
EtO
EtO
O
O O
O

The compound 12 was prepared in 18% isolated yield according to the procedure described
for 5.
1
H NMR (400 MHz, CDCl
3
) δ: 1.35 (t, J=7.2 Hz, 12H), 3.92-3.95 (m, 4H), 4.07-4.11 (m, 4H),
4.23-4.33 (m, 8H).
19
F NMR (376 MHz, CDCl
3
) δ: -109 (d, J=109 Hz).
31
P NMR (162 MHz,
CDCl
3
) δ: 3.4 (t, J=109 Hz).

- 131 -

Preparation of compound 14
OEt EtO
O O
O
P EtO
OEt
CH
3
+
1. BuLi
2. TMSCl
THF, -78oC,
OEt EtO
O
O
H
2
C P
Si
O
OEt
OEt

To 5 g of diethyl methyl phosphonate 13 (32.9 mmol) in 50 mL of freshly distilled THF, 13.8
mL 2.5 M. sol of n-butyl lithium in hexanes was added at -78 °C under inert atmosphere. After
stirring the solution for 30 minutes, 5.6 g of 3,4-diethoxy-3-cyclobutene-1,2-dione 1 (32.9 mmol)
was introduced and reaction mixture was stirred for additional hour. After that, the reaction was
quenched with the addition of 4 g (4.67 mL) TMSCl and the temperature was raised to ambient
temperature. All volatiles were removed under reduced pressure, the crude product was
redissolved in hexanes and washed with water, brine and dried over MgSO
4
. Solution was
concentrated and the reaction mixture was purified by column chromatography on silica-gel
using EtOAc / Hexane as eluent (gradient conditions: from 0/100 to 35/65) to afford the pure 14
(11.1 g 86 %) as colorless oil.
1
H NMR (400 MHz, CDCl
3
) δ: 0.00 (s, 9H), 1.18 (m, 9H), 1.3 (t, J = 7.2 Hz, 3H), 2.24 (m, 2H),
3.92 (m, 4H), 4.15 (m, 2H), 4.33 (q, J = 7.2 Hz, 2H).
13
C NMR (100 MHz, CDCl
3
) δ: 1.57, 15.5,
16, 16.6, 16.7, 32 (d, J = 138.9 Hz), 62 (d, J = 5.9 Hz), 62.1 (d, J = 6.2Hz), 67, 69.3, 84.4 (d, J =
1.8 Hz), 133.3, 166.9 (d, J = 2.3 Hz), 184.3 (d, J = 3.3 Hz).
31
P NMR (162 MHz, CDCl
3
) δ: 20.2
(s).
Preparation of compound 15
OEt EtO
O
O
H
2
C P
O
OEt
OEt
Si
TMSO OTMS
OEt
O
O
O
S
O
O
O
F
F
F
Si
H
2
C P
O
OEt
OEt

A solution of compound 14 (4 g, 10.1 mmol) in anhydrous acetonitrile (100 mL) was placed
in the flask filled with inert gas. Ethylenedioxybis(trimethylsilane) (2.09 g, 10.1 mmol ) was
added to the reaction mixture followed by catalytic amount of TMSOTf (0.224 g, 1.01 mmol, 0.1
eq). The solution was stirred at room temperature for one hour. Acetonitrile was removed under
- 132 -

vacuum, reaction mixture was poured into water and extracted three times with ether. Organic
layers were combined and dried over MgSO
4
. The organic fractions were concentrated in
vacuum and subjected for column chromatography on silica with EtOAc/ Hexane as eluent
(gradient conditions: from 0/100 to 80/20) affording pure compound 15 as pale yellow oil 2.87 g
(89 %).
1
H NMR (400 MHz, CDCl
3
) δ: 1.07 (t, J=6.4 Hz, 3H), 1.2 (t, J=6.8 Hz, 6H), 2.76 (d, J=21.6 Hz,
2H), 3.49 (q, J= 6.8 Hz, 2H), 4.01 (m, 8H).
13
C NMR (100 MHz, CDCl
3
) δ: 15.7, 15.8, 17.5,
20.58 (d, J=139.9 Hz), 57.3, 63.1 (d, J=6.6 Hz), 66.6, 117.4 (d, J=2.5 Hz), 125.1 (d, J=10.3 Hz),
186.1 (d, J=7 Hz), 193.2 (d, J=3 Hz).
31
P NMR (162 MHz, CDCl
3
) δ: 22.2 (s).
Preparation of compound 16
O
P OEt
OEt
Me
+
1. BuLi
2. TMSCl
OEt
O
O
O
H
2
C P
O
OEt
OEt
THF, -78oC
OEt
O
O
P
O
OEt
OEt
O
Si
P
O
EtO
EtO

Under an argon atmosphere, 1.96 mL (4.9 mmol, 1.6 eq) of 2.5 M solution of n-butyllithium
was added dropwise to the solution of diethyl methyl phosphonate 13 (0.712 mg, 4.69 mmol 1.5
eq) in anhydrous THF cooled to -78 °C. After stirring the solution at -78 °C for 30 minutes, a
solution of compound 15 (1g, 3.125 mmol, 1 eq.) in 5 mL of anhydrous THF was slowly
introduced into reaction mixture and stirred for two more hours. Then, the reaction was quenched
with the addition of 1mL of TMSCl and the temperature was raised to ambient. All volatiles
were removed under reduced pressure, the reaction mixture was dispersed between water and
hexanes, and the organic layer was washed with brine and dried over MgSO
4
. Evaporation under
reduced pressure gave the compound 16 (663 mg, 45 %), which was used in the next step
without further purification.

- 133 -

Preparation of tetraethyl-3,4-bis(methylene)phosphonate-cyclobut-3-ene-1,2-dione (
3,4
BM-β-
SQTPE) 17
CH
3
OTMS
OEt
O
O
P
O
OEt
OEt
O
Si
P
O
EtO
EtO
P P
O
OEt
OEt
O
EtO
OEt
HO
HO
O 1.
TMSOTf
2. AcOH

Methoxytrimethylsilane (185 mg, 0.9 mmol) was added to the solution of crude 16 (663 mg in
anhydrous acetonitrile) at room temperature. Fresh TMSOTf (204 mg, 0.9 mmol) was then added
dropwise and reaction mixture was stirred at room temperature overnight. All volatiles were
removed under reduced pressure and the residue was dissolved in acetic acid (20 mL) and stirred
at room temperature for 24 hours. Reaction mixture was then concentrated and the crude product
was purified by silica gel chromatography using Hex:EtOAc:MeOH as eluent (100:0:0 to
0:100:0 to 0:88:12) to yield the title compound 17 as a brown oil (320 mg, 57 %).
P P
O
OEt
OEt
O
EtO
OEt
HO
HO
O
P P
O
OEt
OEt
O
EtO
OEt
O O
TMSOTf
CH
3
CN

Excess of TMSOTf (4.44 g, 20 mmol) was added to the solution of 17 (800 mg, 2.09 mmol)
in anhydrous acetonitrile under stirring. The progress of the reaction was monitored by
31
P NMR.
When the reaction achieved maximum conversion, all volatiles were removed under reduced
pressure and the residue was subjected to column chromatography on silica gel using
EtOAc/MeOH as the eluent.
Non-hydrated:
1
H NMR (400 MHz, CDCl
3
) δ: 1.15 (t, J=7.2 Hz, 12H), 3.4 (d, J=21.6 Hz, 4H),
3.97 (m, 8H).
13
C NMR (100 MHz, CDCl
3
) δ: 16.1, 25.1 (dd, J= 133Hz, J=2.7 Hz), 63, 194.3,
196.3.
31
P NMR (162 MHz, CDCl
3
) δ: 19.9 (s).
Hydrated:
1
H NMR (400 MHz, CDCl
3
) δ: 1.12 (t, J=7.2 Hz, 6H), 1.12 (t, J=6.8 Hz, 6H), 2.82
(dd, J=22.8 Hz, J=2.4 Hz, 2H), 3.12 (dd, J=24 Hz, J=2.8 Hz, 2H) 3.97 (m, 8H).
13
C NMR (100
MHz, CDCl
3
) δ: 16.2, 21.8 (d, J=134.9 Hz), 24.6 (d, J=135.8 Hz), 120.3, 152.9, 174.7, 194.4.
31
P NMR (162 MHz, CDCl
3
) δ: 21.5 (d, J=14.7 Hz), 22.7 (d, J=14.7 Hz).
- 134 -

Preparation of (2-hydroxy-3,4-dioxocyclobut-1-enyl)methylphosphonic acid diethyl ester
(MPESQA) 28
H
2
C P
O
OEt
OEt
O
O
OH

Solution of compound 15 (4g, 12.5 mmol) in methanol was treated with 50 mL of 70 % acetic
acid at room temperature for three days. After all of the starting material had been consumed, the
volatiles were removed by evaporation and the crude mixture was purified by column
chromatography on silica gel using EtOAc/Hex (30/70) as eluent. The pure product 28 was
obtained as an yellow oil (2.4 g, 77 %).
1
H NMR (400 MHz, CDCl
3
) δ: 1.26 (t, J=6.8 Hz, 6H), 3.24 (d, J=22.8 Hz, 2H), 4.1 (m, 4H).
13
C NMR (100 MHz, CDCl
3
) δ: 16, 16.1, 22.7 (d, J=137.9 Hz), 63.8 (d, J=6.7 Hz), 171 (d,
J=10.4 Hz), 197.8 (d, J=2.4 Hz), 198.5 (d, J=4.6 Hz).
31
P NMR (162 MHz, CDCl
3
) δ: 22.5 (s).
Preparation of (2-hydroxy-3,4-dioxocyclobut-1-enyl)methylphosphonic acid (MPSQA) 36
H
2
C P
O
O
OH
O
O
O
[Bu
4
N
+
]
2

Compound 28 (1.2 g, 4.8 mmol) was placed in a flask filled with argon and dissolved in 20
mL of dry acetonitrile. Subsequently, 3 g (19.2 mmol) of bromotrimethylsilane was added
dropwise, at room temperature with stirring, and the reaction was allowed to continue for 72
hour. After that, all volatiles were removed under reduced pressure from the reaction mixture and
the residue was distributed between water and diethyl ether. Aqueous fraction was further
extracted three times with diethyl ether. Tetrabutylammonium hydroxide was added to the water
solution of the corresponding acid until pH = 8. Water was evaporated and the residue was
purified by flash C18 chromatography with H
2
O / MeOH as eluent. Isolated 2.94 g (91 %) of salt
36.
- 135 -

1
H NMR (400 MHz, CDCl
3
) δ: 2.81 (d, J = 21.7 Hz, 2H).
13
C NMR (100 MHz, CDCl
3
) δ: 12.8,
19, 23, 26.3 (d, J=123.1 Hz), 57.9, 181.9 (d, J=9.9 Hz), 201.9 (d, J=3.3 Hz), 210.2.
31
P NMR
(162 MHz, CDCl
3
) δ: 15.7 (s).
Preparation of (2-chloro-3,4-dioxocyclobut-1-enyl)methylphosphonic acid diethyl ester
(MPESQCl) 29
H
2
C P
O
OEt
OEt
O
O
Cl

A solution of compound 28 (1 g, 4.03 mmol) in 40 mL anhydrous CHCl
3
was placed in the
flask under an argon atmosphere. Subsequently, 1.52 g (12.1 mmol) of oxalyl chloride followed
by catalytic amount of DMF was slowly added with stirring. The reaction mixture was allowed
to stir at room temperature overnight. All volatiles were evaporated and the obtained crude
product was used in the next step without further purifications.
1
H NMR (400 MHz, CDCl
3
) δ: 1.14 (m, 6H), 3.26 (d, J=23.2 Hz, 2H), 3.97 (m, 4H).
13
C NMR
(100 MHz, CDCl
3
) δ: 16.1, 16.2, 25.3 (d, J=131.9 Hz), 63.2 (d, J=6.4 Hz), 188.2 (d, J=8.5 Hz),
191.3 (d, J=3 Hz), 193.7 (d, J=11.3 Hz), 193.9 (d, J=1.9 Hz).
31
P NMR (162 MHz, CDCl
3
) δ:
17.5 (s).
Preparation of compound 30
H
2
C
O
OH
P O EtO
OEt
OH
H
2
C P
O
OEt
OEt

At -78 °C, 5.54 mL of 2.5 M sol of n-butyllithium (3.86 mmol, 1.05 eq) was added dropwise
into a flask containing the solution of diethyl methyl phosphonate 13 (2 g, 13.2 mmol) in
anhydrous THF. After stirring the reaction mixture at -78 °C for 30 minutes, the solution of 29 (3
g, 13.3 mmol) in 5 mL of dry THF was added. After stirring at this temperature for two hours,
the reaction mixture was brought to room temperature. The solvent was evaporated and the
- 136 -

residue was purified using column C18 chromatography (H
2
O/MeOH) affording 3.6 g (68 %) of
the product as a colorless oil.
1
H NMR (400 MHz, CDCl
3
) δ: 1.1-1.23 (m, 12H), 2.28 (m, 2H), 3.22 (dd, J=28 Hz, J=14.4 Hz,
1H), 3.58 (dd, J=25.2 Hz, J=14.4 Hz, 1H), 3.83-4.01 (m, 8H).
13
C NMR (100 MHz, CDCl
3
)
δ:16, 16.1, 16.14, 16.2, 25.9 (d, J=134.4 Hz), 30.3 (d, J=139.2 Hz), 61.7 (d, J=6.7 Hz), 62.5 (d,
J=6.3 Hz), 63.2 (m), 89 (m), 133.8 (d, J=12 Hz), 172.7 (dd, J=10.5 Hz, J=4.5 Hz), 187 (dd,
J=9.7 Hz, J=3.4 Hz).
31
P NMR (162 MHz, CDCl
3
) δ: 22 (s), 24.9 (s).
Preparation of (
2,4
BMPE-β-SQ) 31
O
OH
P
P O
OEt
OEt
O EtO
OEt

To a flask containing 200 mg (0.5 mmol) of compound 30 was added 10 mL of 1 M solution
of HCl. The reaction mixture was stirred at room temperature for 12 hours. All volatiles were
removed on a rotary evaporator and the residue was subjected tor C18 column chromatography.
The desired compound 31 (1.1 g 30 %) was isolated using a gradient regime 0% to 60% MeOH
in H
2
O.
1
H NMR (400 MHz, CD
3
OD) δ: 1.28 (t, J=7.2 Hz, 6H), 1.33 (t, J=7.2 Hz, 6H), 2.6 (d, J=19.2
Hz, 2H), 4.1(m, 4H), 4.2 (m, 4H).
13
C NMR (100 MHz, CD
3
OD) δ: 15.5, 15.6, 15.67, 15.68,
28.9 (d, J=141.3 Hz), 63.4 (d, J=6.8 Hz), 63.6 (d, J=6.5 Hz), 64.2 (d, J=4.3 Hz), 64.3 (d, J=4.3
Hz), 89 (m), 134.5 (d, J=10.6 Hz), 174.4 (dd, J=11.4 Hz, J=4.7 Hz), 190 (dd, J=5 Hz, J=3.1Hz).
31
P NMR (162 MHz, CD
3
OD) δ: 22.6 (s), 25.9 (s).

- 137 -

4.4 REPRESENTATIVE NMR SPECTRA
1
H NMR of compound (3)

13
C NMR of compound (3)

- 138 -

19
F NMR of compound (3)

31
P NMR of compound (3)

- 139 -

1
H NMR of compound (4)

31
C NMR of compound (4)

- 140 -

19
F NMR of compound (4)

31
P NMR of compound (4)

- 141 -

1
H NMR of compound (6)

13
C NMR of compound (6)

- 142 -

19
F NMR of compound (6)

31
P NMR of compound (6)

- 143 -

1
H NMR of compound (9)

13
C NMR of compound (9)

- 144 -

19
F NMR of compound (9)

31
P NMR of compound (9)

- 145 -

1
H NMR of compound (12)

19
F NMR of compound (12)

- 146 -

31
P NMR of compound (12)

1
H NMR of compound (14)

- 147 -

13
C NMR of compound (14)

31
P NMR of compound (14)

- 148 -

1
H NMR of compound (15)

13
C NMR of compound (15)

- 149 -

31
P NMR of compound (15)

1
H NMR of teraethyl-3,4-bis(methylene)phosphonate-cyclobut-3-ene-1,2-dione (
3,4
BM-β-
SQTPE) (17)

- 150 -

13
C NMR of teraethyl-3,4-bis(methylene)phosphonate-cyclobut-3-ene-1,2-dione (
3,4
BM-β-
SQTPE) (17)

31
P NMR of teraethyl-3,4-bis(methylene)phosphonate-cyclobut-3-ene-1,2-dione (
3,4
BM-β-
SQTPE) (17)

- 151 -

1
H NMR of (2-hydroxy-3,4-dioxocyclobut-1-enyl)methylphosphonic acid diethyl ester
(MPESQA) (28)

13
C NMR of (2-hydroxy-3,4-dioxocyclobut-1-enyl)methylphosphonic acid diethyl ester
(MPESQA) (28)

- 152 -

31
P NMR of (2-hydroxy-3,4-dioxocyclobut-1-enyl)methylphosphonic acid diethyl ester
(MPESQA) (28)

1
H NMR of (2-hydroxy-3,4-dioxocyclobut-1-enyl)methylphosphonic acid
triethylammonium salt (MPSQA) (36)

- 153 -

13
C NMR of (2-hydroxy-3,4-dioxocyclobut-1-enyl)methylphosphonic acid
tetrabutylammonium salt (MPSQA) (36)

31
P NMR of (2-hydroxy-3,4-dioxocyclobut-1-enyl)methylphosphonic acid
tetrabutylammonium salt (MPSQA) (36)

- 154 -

1
H NMR of (2-chloro-3,4-dioxocyclobut-1-enyl)methylphosphonic acid diethyl ester
(MPESQCl) (29)

13
C NMR of (2-chloro-3,4-dioxocyclobut-1-enyl)methylphosphonic acid diethyl ester
(MPESQCl) (29)

- 155 -

31
P NMR of (2-chloro-3,4-dioxocyclobut-1-enyl)methylphosphonic acid diethyl ester
(MPESQCl) (29)

1
H NMR of compound (30)

- 156 -

13
C NMR of compound (30)

31
P NMR of compound (30)

- 157 -

1
H NMR of (
2,4
BMPE-β-SQ) (31)

13
C NMR of (
2,4
BMPE-β-SQ) (31)

- 158 -

31
P NMR of (
2,4
BMPE-β-SQ) (31)

- 159 -

4.5 REFERENCES
1. Cohen, S.; Lacher, J. R.; Park, J. D., J. Am. Chem. Soc. 1959, 81, 3480.
2. Cohen, S.; Cohen, S. G., J. Am. Chem. Soc. 1966, 88, 1533.
3. West, R., Oxocarbons, Academic Press, New York, 1980, pp.1.
4. Ohno, M.; Eguchi, S., Bioactive Heterocycles I, Topics in Heterocyclic Chemistry,
SPRINGER-VERLAG BERLIN: Berlin, 2006, 6, 1.
5. Storer, R. I.; Aciro, C.; Jones, L. H., Chem. Soc. Rev. 2011, 40, 2330.
6. Marson, C. M., Chem. Soc. Rev. 2011, 40, 5514.
7. West, R.; Niu, H. Y., J. Am. Chem. Soc. 1963, 85, 2589.
8. Schleyer, P. V.; Maerker, C.; Dransfeld, A.; Jiao, H. J.; Hommes, N., J. Am. Chem.
Soc.1996, 118, 6317.
9. Quinonero, D.; Frontera, A.; Ballester, P.; Deya, P. M., Tetrahedron Lett. 2000, 41, 2001.
10. Liebeskind, L. S.; Mitchell, D.; Foster, B. S., J. Am. Chem. Soc. 1987, 109, 7908.
11. Liebeskind, L. S.; Bombrun, A., J. Org. Chem. 1994, 59, 1149.
12. Perri, S. T.; Rice, P.; Moore, H. W., Org. Synth.1990, 69, 220.
13. Moore, H. W.; Yerxa, B. R., Adv. Strain. Org. Chem.1995, 4, 81.
14. Negri, J. T.; Morwick, T.; Doyon, J.; Wilson, P. D.; Hickey, E. R.; Paquette, L. A., J. Am.
Chem. Soc. 1993, 115, 12189.
15. Mukkanti, A.; Periasamy, M., Arkivoc 2005, 48.
16. Dehmlow, E. V., Chem. Ber.-Recl. 1967, 100, 3829.
17. Knoche, H., Annalen Der Chemie-Justus Liebig 1969, 722, (MAY), 232.
18. Cohen, S.; Lacher, J. R.; Park, J. D., J. Am. Chem. Soc. 1959, 81, 3480.
19. Park, J. D.; Lacher, J. R.; Cohen, S., J. Am. Chem. Soc. 1962, 84, 2919.
20. Scharf, H. D., Angew. Chem.-Int. Edit. Engl. 1974, 13, 520.
21. Camps, F.; Llebaria, A.; Moreto, J. M.; Ricart, S.; Vinas, J. M., Tetrahedron Lett. 1990,
31, 2479.
22. Suzuki, Y.; Takizawa, T., J. Chem. Soc.-Chem. Commun. 1972, 837.
23. Hoberg, H.; Herrera, A., Angew. Chem.-Int. Edit. Engl. 1980, 19, 927.
24. Hoberg, H.; Herrera, A., Angew. Chem.-Int. Edit. Engl 1981, 20, 876.
- 160 -

25. Periasamy, M.; Radhakrishnan, U.; Brunet, J. J.; Chauvin, R.; ElZaizi, A. W., Chem.
Commun. 1996, 1499.
26. Breslow, R.; Altman, L. J.; Krebs, A.; Mohacsi, E.; Murata, I.; Peterson, R. A.; Posner, J.,
J. Am. Chem. Soc. 1965, 87, 1326.
27. Schmidt, A. H.; Ried, W., Synthesis 1978, 1.
28. Knorr, H.; Ried, W., Synthesis 1978, 649.
29. Schmidt, A. H.; Ried, W., Synthesis 1978, 869.
30. Schmidt, A. H., Synthesis 1980, 961.
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Chapter 5 Investigations toward synthesis of phosphoramidates of
2'-deoxy- ψ-isocytidine
5.1 INTRODUCTION
The origin and development of cancer are associated not only with the genetic modifications,
but also with the epigenetic changes to the genome that do not alert the nucleotide sequence. In
recent times there is an increased interest in the area of epigenetic modifications, which are
believed to play crucial role in the cause of cancer and it progression [1]. DNA methylation is the
epigenetic process that occurs on the cytosine bases playing an important role in gene expression
[2]. Methylation abnormalities deactivate tumor suppressor genes blocking their expression.
Therefore, finding inhibitor that can disable DNA methylation process and reactivate tumor
suppressor genes is considered essential for preventing the development of cancer [3]. In our
collaborative effort with the USC / Norris Comprehensive Cancer Center we attempted the
development of novel DNA methyltransferase inhibitor based on 2'-deoxy-ψ-isocytidine (dpψiC)
(Figure 5.1).
O
OH
HO
N NH
NH
2
O

Figure 5.1. 2'-deoxy-ψ-isocytidine
To date, a number of active compounds that can target DNA methyltransferases were
introduced, including 5-azacytosine nucleosides and zebularine (Figure 5.2). Even though these
compounds were found to be very potent inhibitors, they appeared to be unstable and toxic which
is undesirable for drug candidates [4].

- 165 -

N
O N
O
X OH
HO
N N
NH
2
O N
O
X OH
HO
X = OH
X = H
Zebularine
5-azacytosine nucleosides

Figure 5.2. Structures of 5-azacytosine and zebularine nucleosides.
Initially, zebularine was not designed to be DNA methylation inhibitor and it has different
mechanism of action that is associated with the absence of the 4-amino group compared to the
natural cytosine [3].This increases electrophilicity of the C4 and C6 position of the resulting 2-
oxypyrimidine ring. Subsequently, after incorporation of zebularine into DNA, its electrophilic
sites undergo attack from cysteine residue of DNA methylthransferases and covalently bind to it
reactivating hypermethylated genes. However, in order to achieve significant effect, it requires
administering elevated dosage of zebularine, which limits its use as a succeseful clinical
candidate. Azacytidine even though exhibit great inhibitory effect of DNA methylation it
appeared to be toxic due to the lack of stability of the triazinone ring system. Therefore, to
improve stability of the nucleoside, the structure of 2'-deoxy-ψ-isocytidine was proposed as more
stable analogue of azacytidine (Figure 5.1), where nitrogen atom of the triazinone ring linked to
the deoxyribose is replaced with the carbon atom [5-7].
Nucleosides, which are potential DNA methyltransferase inhibitors, in order to be potent,
have to be incorporated into DNA strain [3]. The incorporation process involves deoxynucleotide
triphosphate (NTP) analogue, which can be prepared by subsequent intracellular phosphorylation
of deoxynucleotide diphosphate (dNDP) and deoxynucleotide monophosphate (dNMP)
analogues. Unfortunately, nucleosides of interest cannot be phosphorylated inside the cells and
dNMP cannot be formed. Nucleotide monophosphate derivatives are very polar compounds
possessing poor bioavailability. Therefore, they cannot penetrate membrane cell walls and if
synthesized, cannot be delivered to the target cells [3].

- 166 -

Scheme 5.1. Mechanism of action of the phosphoramidates promoiety.
O
P O NUC O Ar
NH
EtO
O
O
P O NUC O Ar
NH
O
O
Esterase
O
P O NUC
O
NH O
O
P O NUC O
NH
O
O
Phosphoramidase
O
P O NUC O
OH
+
O
O
NH
2

To overcome these challenges, McGuigan and coworkers have developed the approach that
increases lipophilicity of the nucleotide monophosphates allowing the corresponding “ProTides”
to penetrate the interior of the cells [8, 9]. Once inside the cell the “ProTide” moiety decomposes
releasing the dNMP (Scheme 5.1) [8-10]. Below, the study toward preparation of the
phosphoramidates 21a-23a of the 2'-deoxy-ψ-isocytidine is described (Figure 5.3).
O
OH
O
N NH
NH
2
O
P
O
HN
O
BnO
O
22a
O
OH
O
N NH
NH
2
O
P
O
HN
O
EtO
O
21a
O
O
O
N NH
NH
2
O
P
O
NH
23a
P
O
O
O
H
N
EtO
O
EtO
O

Figure 5.3. Target phosphoramidates 21a-23a.
5.2 RESULTS AND DISCUSSION
Preparation of the 2'-deoxy- ψ-isocytidine
Even though 2'-deoxy-ψ-isocytidine is commercially available, it is very expensive ($600 for
100 mg) and could not be purchased in large quantity. As we recognized in the course of our
investigations, the preparation of the corresponding phosphoamidate derivatives is not a trivial
challenge. It could not be achieved from the small amount of the nucleoside on hand because it
- 167 -

requires screening of the various reaction conditions and synthetic methods. Therefore, in order
to start our investigation, firstly we had to attempt the synthesis of the 2'-deoxy-ψ-isocytidine
and we decided to follow the route developed by Leumann and coworkers in 2002 [11].
The convergent synthesis of the 2'-deoxy-ψ-isocytidine predominantly consists of two routes.
First part of the synthetic approach, as shown in Scheme 5.2, involves preparation of the
protected furanoid glycal 5 suitable for the Heck coupling reaction. Second pathway, represented
in Scheme 5.3, includes synthesis of the benzoyl N-protected iodoisocytosine 8, which is another
substrate for palladium coupling reaction.
The Heck-coupling reaction and the subsequent diastereoselective reduction in presence of
NaBH(OAc)
3
are the crucial steps in this synthetic approach shown in scheme 5.4.
The N-benzoyl protection was removed by treating compound 11 with the 20% ammonium
hydroxide in methanol and the target dψiC was purified using reverse phase column
chromatography.
Scheme 5.2. Preparation of modified saccharide - substrate for Heck-coupling reaction.
O
OH
HO
HO
Br
2
, H
2
O
5 days
O
HO
HO
O
TBDMSCl
N
H
N
O
TBDMSO
TBDMSO
O
DIBALH
O
TBDMSO
TBDMSO
OH
O
S
O
H
3
C Cl
Et
3
N
O
TBDMSO
TBDMSO
1 2 3
4 5

Scheme 5.3. Synthesis appropriate of N-protected pseudoisocytosine base for Heck coupling
reaction.
NH
N
O
NH
2
NH
N
O
NH
2
I
O
O
N I
O
Bz
O
Bz
O
100
o
C, DMF
NH
N
O
NH
I
Bz
6 7 8

- 168 -

Scheme 5.4. Synthetic route toward dψiC: Heck coupling reaction and following
diastereoselective reduction of carbonyl group.
NH
N
O
NH
I
Bz
O
TBDMSO
TBDMSO
+
O
TBDMSO
TBDMSO
N
H
N
H
N
O
Bz
O
O
HO
N
H
N
H
N
O
Bz
Pd(OAc)
2
Olah's reagent
NaBH(OAc)
3
O
HO
N
H
N
H
N
O
Bz
HO
O
HO
N
H
N
NH
2
O
HO
NH
4
OH
5 8 9
10 11
12

Preparation of the phosphorochloridates
Phosphorochloridates were prepared according to the general synthetic procedure reported by
McGuigan [12] (Scheme 5.5).
Scheme 5.5. Synthesis of phosphorochloridates 13 and 14.
O
P
H
N Cl
O
OR
O
OH
+
P
O
Cl Cl
Cl
Et
3
N
Et
2
O
P
O
Cl Cl
O
Et
3
N
NH
3
OR
O
Cl
13, R=Et, (92 %)
14, R=Bn (33 %)

- 169 -

Synthetic study toward phosphoramidates of 2'-deoxy- ψ-isocytidine
Usually, preparation of the phosphoramidates of different nucleosides is conducted in THF.
However, general poor solubility of the nucleosides leads to lower yields (5-10%) of the desired
analogues. Extremely poor solubility of the dψiC in THF and necessity to work with the limited
amount of starting material urged us to introduce suitable protecting groups. Protected
nucleosides exhibit much greater solubility in organic solvents including THF [13]. Benzyl
carbamate protecting group (Cbz) is one of the few protecting groups that can be removed in
presence of phosphoramidate moiety under mild conditions. In our initial approach to access
target phosphoramidates 21a-23a, we decided to prepare carboxybenzyl protected dψiC (Scheme
5.6).
Firstly, 5‟-hydroxyl group of the 2'-deoxy-ψ-isocytidine was protected with TBDMS group by
treating the nucleoside with corresponding silane in pyridine in the presence of imidazole. The
resulting nucleoside 16 underwent the reaction with the benzyl chloroformate affording
corresponding protected nucleosides 17 and 18. Subsequent reaction with Et
3
N:(HF)
3
in THF
completely removed silyl protecting group and Cbz-protected dψiC was purified by column
chromatography on silica gel (Scheme 5.6).
2'-Deoxy-ψ-isocytidine protected with benzyl carbamate was subjected to the coupling
reaction with the phosphochloridate 13 in THF in presence of N-methylimidazole. After the
reaction was complete, the product was purified by flash chromatography on silica gel and
compound containing phosphoramidate moiety attached to the dψiC was isolated. To our
surprise, no trace of the desired compound 21a was identified in the reaction mixture instead
only isomer 21b was isolated (Scheme 5.7).

- 170 -

Scheme 5.6. Synthesis of carboxybenzyl protected dψiC.
O
OH
O
N NH
NH
2
O
Si
O
OH
HO
N NH
NH
2
O
Si Cl
HN
N
, Py
71 %
O
OCbz
O
N NH
NHCbz
O
Si
O
OH
O
N NH
NHCbz
O
Si +
O
OCbz
HO
N NH
NHCbz
O
O
OH
HO
N NH
NHCbz
O
HF-Et
3
N HF-Et
3
N
THF THF
12 16 17 18
19 20
O
O
Cl
DMAP, CH
2
Cl
2

Scheme 5.7. First attempts to prepare target phosphoramidates.
O
OCbz
HO
N NH
NHCbz
O
O
P
H
N Cl
O
OEt
O
+
O
OH
O
N NH
NH
2
O
P
O
HN
O
EtO
O
1. N-Methylimidazol/THF
2. 1 4-cyclohexadiene, Pd/C, DMF
O
OH
HO
N N
NH
2
O P
O
O
HN
EtO O
19 13
21a
21b

Reaction with phosphoramidate 14 was carried out in pyridine with unprotected dψiC and
corresponding isomer 22b was isolated after purification on silica gel. Product bearing two
phosphoramidate moieties 23b was obtained in 2.3 % yield when 6 eq. of phosphochloridate
were used and tert-butyl magnesium chloride was employed instead of N-methylimidazole.
Thus, after detailed analysis of the NMR spectra, we concluded that the NMR data support the
structures of compounds 21b, 22b and 23b (Figure 5.4).
- 171 -

O
OH
O
N N
NH
2
O
P
O
HN
O
O
EtO
P
O
O
HN
EtO O
O
OH
HO
N N
NH
2
O P
O
O
HN
EtO O
O
OH
HO
N N
NH
2
O P
O
O
HN
O O
22b 21b 23b

Figure 5.4. Structures of isolated phosphoramidates 21b-23b.
Figure 5.5 and 5.6 highlight the key features of the NMR spectra of isomers 22b and 23b,
which helped to determine their structures. The Figure 5.5 represents comparative analysis of the
1
H and
13
C NMR spectra of phosphoramidate 22b and starting 2'-deoxy-ψ-isocytidine. From the
1
H NMR, it is clear that 5‟-methylene group appears in the same region around 3.5-3.7 ppm as
that of 5‟ methylene group of the starting material indicating that adjacent oxygen atom does not
bear any phosphorous (V) containing group. In the latter case, 5‟-methylene group was supposed
to show an upfield shift and appeared around 4.1 - 4.4 ppm. Likewise,
13
C NMR of the
phosphoramidate 22b does not show any upfield shift for the 5‟ carbon and it appears at 62 ppm
similarly to the 5‟ carbon in dψiC. Moreover, no splitting from the carbon phosphorus coupling
was observed.

1
H NMR

13
C NMR
Figure 5.5. Analysis of the
1
H and
13
C spectra of phosphoramidate 22b relative to the dψiC.
- 172 -

To provide additional support for structural assignment,
31
P spectra of phosphoramidate 22b
and 23b were compared (Figure 5.6). The
31
P spectrum of the compound 22b shows two peacks
near - 4 ppm corresponding to the two diastereomers formed by different orientation of the chiral
phosphoramidate functional group. This chemical shift is typical for the phosphoramidate moiety
attached to the aromatic alcohols or phenols. However, signal of the phosphoramidate motif
connected to the aliphatic alcohol moiety such as 3‟ and 5‟-hydroxyl groups of the dψiC is
supposed to appear around 2 - 4 ppm. As shown in Figure 5.5, one phosphoramidate motif of the
compound 23b appears close to 4 ppm whereas another one connected to the 5‟- hydroxyl group
of the dψiC shifted much further to the high field and comes at about 2.7 ppm. Proton and carbon
spectra of the compound 23b (not shown here) also indicate that one of the phosphorus
containing group is attached to the 5‟-hydroxyl group.

Figure 5.6. Analysis of the
31
P spectra of phosphoramidate 22b relative to that of 23b.
Since initial coupling reaction of the dψiC with the phosphorochloridate 13 occurs exclusively
on 4-hydroxyl group of the isocytosine, we focused our attempts to develop a protecting group
that can prevent reaction on base site. We started our investigation with the protection both 3‟
- 173 -

and 5‟ hydroxyl group by treating 2'-deoxy-ψ-isocytidine with di-tert-butylsilyl
bis(trifluoromethanesulfonate) [14] (Scheme 5.8). However, finding the right protecting group
turned out to be a challenging task. Since we have the necessity to work with the small quantity
of dψiC, the reactions for accurate positioning and removal of the protecting group should be a
high yielding process. Additionally, the sensitive phosphoramidate prodrug motif must sustain
the deprotection conditions. Usually, phosphoramidate prodrugs decompose with the slight
increase of the pH (pH = 7.4). However, it was shown that they can tolerate considerable acidic
conditions (pH = 2.0) for a short period of time [15-17]. Unfortunately, very small amount of
existing protecting groups can satisfy these requirements.
Scheme 5.8. Failed attempts to protect carbonyl group of isocytosine moiety.
O
OH
HO
N NH
NH
2
O
Si
OTf
OTf
12
Et
3
N
O
O
O
N NH
NH
2
O
Si
N N
NH
2
O
N N
NH
2
O
Si
N N
NH
2
Cl
N N
HN
OH
Si
POCl
3
,
Dimethylaniline
BnOH, PPh
3
, DIAD
TBDMSCl, DMAP,
Imidazol, Py
Yield less than 25%

Benzyl type protecting group can be attached to the 4-hydroxyl group of the isocytosine via
Mitsunobu coupling reaction [18, 19] and can be easy removed with hydrogenolysis. However,
the yields of this reaction is unacceptable (do not exceed 25%) and this protection cannot be used
for preparation of phosphoramidate 22a, containing benzyl function (Scheme 5.8). Introduction
of the chloride function instead of obstructive hydroxyl can be achieved by reacting
corresponding isocytosine with the phosphorus oxychloride in presence of N,N-dimethyl aniline
[20, 21]. Unfortunately, similar condition applied to compound 24 led to complex mixture.
Probably di-tert-butylsilyl protecting group cannot sustain acidic conditions of this reaction.
Furthermore, we were not able to utilize silyl based protecting groups, because they react with
amino group of the base leaving the hydroxyl group free.
During our synthetic study toward the right protecting group for isocytosine, we noticed that
any kind of phosphochloridates react with the 4-hydroxyl group very smoothly. Generally,
- 174 -

protecting groups based on other elements do not perform well. Thus, when we conducted our
investigation in this direction, we came across the bis(dimethylamino)phosphorochloridate
reagent, which has been used [22-24] for directing the palladium coupling of series of aryl
iodides. To our delight, bis(dimethylamino)phosphorochloridate reacts smoothly with the
isocytosine ring protecting the 4-hydroxyl group. Further treatment with HF-Py removed di-tert-
butylsilyl group leaving phosphordiamide function intact (Scheme 5.9). The resulting compound
26 was purified by reverse phase chromatography and phosphordiamide derivative of dψiC was
isolated with an acceptable 33% yield after three steps.
Scheme 5.9. Preparation of 2-bis(dimethylamino)phosphordiamide-2'-deoxy-ψ-isocytidine.
O
OH
HO
N NH
NH
2
O
Si
OTf
OTf
12
Et
3
N
O
O
O
N NH
NH
2
O
Si
O
P N(CH
3
)
2
N(CH
3
)
2
Cl
O
O
O
N N
NH
2
O
Si
P
O
N(CH
3
)
2
N(CH
3
)
2
HF-Py
20 min
O
OH
HO
N N
NH
2
O P
O
N(CH
3
)
2
N(CH
3
)
2
24
25 26

With the protected 2'-deoxy-ψ-isocytidine 26 on hand we attempted the synthesis of the
desired phosphoramidate analogues (Scheme 5.10 and Scheme 5.11). The coupling reaction of
the phosphordiamide protected dψiC with the corresponding phosphochloridates 13-14 was
carried out in pyridine with employment of tert-butylmagnesium chloride as a base. Sometimes
an excess of pyridine was used to dissolve all precipitates, which is particularly important to
achieve acceptable yields of 3‟-5‟-bisphosphoramidate 23a. Protected phosphoramidate
derivatives 21c, 22c, 23c were isolated with acceptable yields and fully characterized.
Bis(dimethylamino)phosphordiamide protecting group was removed by treating the isolated
compounds with a 1:1 mixture of dioxane and 2 M hydrochloric acid. A suitable deprotection
- 175 -

protocol was developed based on the screening of various reaction conditions [22-26] (Table 5.1)
with the model isocytosine analog 27 prepared for this purpose (Scheme 5.12). The results from
the table 5.1 indicated that treatment of isocytidine derivative with 1:1 mixture of dioxane and
hydrochloric acid at room temperature (entries 6 and 7) leads to a mild as well as efficient
deprotection, whereas other conditions were found to be inefficient or harsh. Later, it was
established that the employment of 0.5 M HCl / dioxane leads to very slow deprotection of
phosphoramidates and 2 M HCl/dioxane is more efficient and can be used instead. This
significantly reduces reaction time and no degradation of target compounds was detected. Final
phosphoramidates 21a, 22a, 23a were purified using HP column chromatography on silicagel.
Scheme 5.10. Preparation of phosphoramidates 21a and 23a.
O
OH
HO
N N
NH
2
O P
O
N(CH
3
)
2
N(CH
3
)
2
26
O
P
H
N Cl
O
OEt
O
O
OH
O
N NH
NH
2
O
P
O
HN
O
EtO
O
13 27
O
O
O
N N
NH
2
O
P
O
NH
28
P
O
N(CH
3
)
2
N(CH
3
)
2
P
O
O
O
H
N
EtO
O
EtO
O
P
O
N(CH
3
)
2
N(CH
3
)
2 +
MgCl
Pyridine
+
1 : 1 2M HCl/Dioxane,
72 hours
O
OH
O
N NH
NH
2
O
P
O
HN
O
EtO
O
21a
O
O
O
N NH
NH
2
O
P
O
NH
23a
P
O
O
O
H
N
EtO
O
EtO
O
1 : 1 2M HCl/Dioxane,
96 hours

Scheme 5.11. Preparation of phosphoramidates 22a.
O
OH
HO
N N
NH
2
O P
O
N(CH
3
)
2
N(CH
3
)
2
26
O
P
H
N Cl
O
OBn
O
O
OH
O
N NH
NH
2
O
P
O
HN
O
BnO
O
13 29
P
O
N(CH
3
)
2
N(CH
3
)
2 +
MgCl
Pyridine
1 : 1 2M HCl/Dioxane
7 days, rt O
OH
O
N NH
NH
2
O
P
O
HN
O
BnO
O
22a

- 176 -

Scheme 5.12. Synthesis of 2-bis(dimethylamino)phosphordiamide derivative of isocytosine.
N N
HN
O P
O
N(CH
3
)
2
N(CH
3
)
2
O
I
N N
HN
OH
O
I
MgCl
P
O
N(CH
3
)
2
N(CH
3
)
2
Cl
N NH
HN
O
O
I
[H
+
]
27 8 8

Table 5.1. Optimization of reaction conditions for the deprotection of
bis(dimethylamino)phosphordiamide group
Reagent/Conditions
Time Conversion
HF-Et
3
N
60% CH
3
COOH, 90
o
C
60% CH
3
COOH, 60
o
C
60% CH
3
COOH, rt
10% CF
3
COOH MeOH/H
2
O
0.5 M HCl/Dioxane = 1:1
2 M HCl/Dioxane = 1:1
12% rt
100%
96%
43%
100%
15%
100%
90
o
C
60
o
C
rt
rt
rt
rt
entry
1
2
3
4
5
6
7

1
H NMR

13
C NMR
Figure 5.7. Relative comparison of
1
H and
13
C NMR spectra of phosphoramidates 22a and 22b.
- 177 -

The spectral data in Figures 5.7 and 5.8 provide the evidence for the structures of
phosphoramidates 21-23a. Figure 5.7 shows the comparative analysis of the
1
H and
13
C NMR
spectra of phosphoramidate isomers 22a and 22b. From the comparison of
1
H NMR data, it is
obvious that the signal of 5‟-methylene group of the compound 22a shifted to up field and appear
at 4.2 ppm, which corresponds to the presence of the electron withdrawing group such as
phosphoramidate moiety at 5‟-position. The
13
C NMR spectrum shows that the peak
corresponding to the 5‟-carbon of aglycon also showed an up field shift appearing at 68 ppm
compared to that of 22b at 62 ppm. This shift also clearly indicates the presence of the
phosphoramidate function. Moreover, phosphorus–carbon (P-C) splitting can be observed in this
case with J
CP
≈ 6 Hz.

Figure 5.8. Relative comparison
31
P NMR spectrum of phosphoramidate 22a and 22b.
The comparative analysis of the
31
P NMR spectra of phosphoramidate 22b and 22a provide
additional support for the confirmation of the assigned structures (Figure 5.8). Two peaks that
correspond to the phosphoramidate diasteriomers 22a appeared near 2 - 3 ppm indicating that the
desired functional group is attached to the aliphatic hydroxyl group such as 5‟-hydroxyl of the 2'-
- 178 -

deoxy-ψ-isocytidine. At the same time, phosphorus signal of the phosphoramidate 22b comes
around -4 to -5 ppm, which points out that this functional group is adjacent to the aromatic ring.
5.3 CONCLUSIONS
A new efficient approach towards the preparation of the 5‟-subtibuted phosphoramidate
derivatives of 2'-deoxy-ψ-isocytidine was developed. Three target phosphoramidates 21a, 22a,
23a were synthesized with acceptable yields. The structures of compounds 21a-23a were
ascertained by
1
H,
13
C,
31
P NMR spectroscopy and high resolution mass spectrometry.
Employment of the protection groups based on bis(dimethylamino)phosphordiamide represent
novel mild and efficient approach for protection of the carbonyl group of isocytosine base.
Simple and effective conditions for deprotection of bis(dimethylamino)phosphordiamide group
in presence of phosphoramidate moiety were also developed. Currently, the synthesized
compounds are being tested as potential DNA-methylation inhibitors in Dr. Jones laboratory
located in USC/Norris Comprehensive Cancer Center.
5.4 EXPERIMENTAL PART
Preparation of 3’, 5’-Di-tert-butylsilylene-2'-deoxy-ψ-isocytidine 24
O
O
O
N NH
NH
2
O
Si

2'-Deoxy-ψ-isocytidine (250 mg, 1.1 mmol) was dried several times by azeotropic
evaporation with pyridine. It was then dissolved in anhydrous DMF (10 mL), transferred in to the
flask filled with argon and the solution was cooled in an ice bath. Subsequently t-Bu
2
Si(OTf)
2

(513 mg, 0.379 ml, 1.16 mmol) was slowly introduced and the reaction mixture was stirred for
30 min at 0 °C. The solution temperature was slowly brought to room temperature, E
3
N (330 mg,
3.3 mmol) was added and the mixture was stirred for another 30 min. When the reaction was
complete (tlc), all volatiles were removed under reduced pressure and the reaction mixture was
purified by column chromatography on silica gel using 22 % MeOH in CH
2
Cl
2
as eluent. The
product was isolated as a white foam 339 mg (82 %).
- 179 -

Mixture of epimers. NMR of one epimer.
1
H NMR (400 MHz, CD
3
OD) δ 8.03 (s, 1H), 5.62 (dd, J= 9.5, 3.0 Hz, 1H), 5.00 – 4.86 (m, 1H),
4.80 – 4.48 (m, 2H), 4.25 – 4.14 (m, 1H), 2.89 – 2.72 (m, 2H), 1.62 – 1.49 (m, 18H).
13
C NMR
(100 MHz, CD
3
OD) δ 164.5, 156.2, 149.8, 111.98, 76.63, 75.42, 69.47, 68.22, 36.43, 26.64,
26.19, 22.11, 19.27.
Preparation of 3’, 5’-Di-tert-butylsilylene-[bis(dimethylamino)phosphoryl]oxy-2'-deoxy-ψ-
isocytidine 25
O
O
O
N N
NH
2
O
Si
P
O
N(CH
3
)
2
N(CH
3
)
2

Compound 24 (130 mg, 0.346 mmol) was dissolved in a 1:1 mixture of anhydrous THF and
pyridine (10 mL). Solution of t-BuMgCl 1M in THF (0.35mL, 0.35 mmol, 1.01 eq) was slowly
added to reaction mixture and stirred for 30 min at room temperature. Next, N,N,N’,N’-
tetramethylphosphorodiamidic chloride (58.8 mg, 0.05 mL,0.346 mmol) was added dropwise
and reaction was left under stirring for 24 hours. After completion of the reaction all solvents
were removed by evaporation and the residue was dissolved in ethyl acetate followed by washing
three times with water. The resulting solution was dried under MgSO
4
and the ethyl acetate was
evaporated under reduced pressure. The reaction mixture was purified by column
chromatography on silica gel with 15 % MeOH in EtOAc as eluent. The desired product was
obtained as a white solid 126.5 mg (73 %).
Mixture of epimers, NMR of one epimer.
31
P NMR (162 MHz, CDCl
3
) δ 20.79 (s).
1
H NMR (400 MHz, CDCl
3
) δ 8.00 (s, 1H), 5.12 (dd, J
= 8.0, 4.7 Hz, 1H), 4.35 – 4.28 (m, 1H), 4.03 – 3.77 (m, 2H), 3.64 – 3.54 (m, 1H), 2.68 – 2.57
(m, 12H), 2.26 – 2.13 (m, 1H), 1.86 – 1.75 (m, 1H), 0.98 – 0.83 (m, 18H).
13
C NMR (101 MHz,
CDCl
3
) δ 166.31, 165.98 (d, J = 6.6 Hz), 160.37, 116.66 (d, J = 6.7 Hz), 80.49, 79.5, 76.2, 71.64,
42.77, 40.12 (m), 31.0, 30.73, 26.3, 23.76.

- 180 -

Preparation of [bis(dimethylamino)phosphoryl]oxy-2'-deoxy-ψ-isocytidine 26
O
OH
HO
N N
NH
2
O P
O
N(CH
3
)
2
N(CH
3
)
2

Compound 25 (90 mg, 0.180 mmol) was dissolved in THF and placed in a Teflon coated flask
cooled down to 0 °C. In a separate flask Olah‟s reagent (HF-Py, 0.1 mL) was carefully diluted
with pyridine (2 mL) and also cooled to 0 °C. Subsequently HF-Py solution was added dropwise
to the solution of compound 25. The reaction mixture was stirred at 0 °C for 20 min and
quenched with the addition of water (10 mL). The solvents were concentrated in vacuum and the
residue was purified by reverse phase chromatography on a C18 column using 35 % MeOH in
water as the eluent. The protected 2'-deoxy-ψ-isocytidine 26 was isolated as a colorless solid
36.4 mg (56 %).
Mixture of epimers, NMR of one epimer.
31
P NMR (162 MHz, CD
3
OD) δ 16.90 (s).
1
H NMR (400 MHz, CD
3
OD) δ 8.30 – 8.18 (m, 1H),
5.17 – 5.02 (m, 1H), 4.32 – 4.16 (m, 1H), 3.83 – 3.75 (m, 1H), 3.62 – 3.48 (m, 2H), 2.70 – 2.59
(m, 12H), 2.17 – 2.07 (m, 1H), 1.89 – 1.76 (m, 1H).
13
C NMR (101 MHz, CD
3
OD) δ 162.67,
162.47 (d, J = 6.6 Hz), 157.30, 111.46 (d, J = 6.4 Hz), 87.35, 72.88, 72.65, 62.37, 41.7, 35.55 (d,
J = 4.1 Hz).
General procedure for preparation of phosphoramidates 27-29
2'-Deoxy-ψ-isocytidine was dried three times by azeotropic evaporation with pyridine,
dissolved in anhydrous pyridine and placed under argon atmosphere. Solution of
t-butylmagnesium chloride (1 M in THF, 3 eq for mono, 6 eq for bis) was added dropwise and
the mixture was allowed to stir at room temperate for 30 minutes. Subsequently, the solution of
the appropriate phosphochloridate (3 eq. for 5‟-NUC; 6 eq. for 3‟,5‟-NUC) in freshly distilled
THF was slowly added and the reaction mixture was left under stirring for 24 hours. All volatiles
were removed under reduced pressure and the residue was purified by column chromatography
on silica gel (MeOH / CH
2
Cl
2
).
- 181 -

General procedure for deprotection of phosphoramidates 27-29
Protected phosphoramidates 27-29 (≈100 mg) were treated with a 1:1 mixture of 2 M
hydrochloric acid / dioxane (10 mL) for several days (from 3 - 7 days) at room temperature. The
progress of the reaction was monitored by
31
P NMR. After starting material was completely
consumed, all volatiles were removed under vacuum and the residue was purified either by flash
chromatography on silica gel with (MeOH / CH
2
Cl
2
) or using reverse phase chromatography
with a mixture of MeOH / H
2
O as the eluent to obtain the desired phosphoramidate.
2'-Deoxy-ψ-isocytidine 5’-[naphthyl(ethoxydimethylglycinyl)]-phosphate 21a
O
OH
O
N NH
NH
2
O
P
O
NH
O
EtO
O

Mixture of 2 diasteriomers 21a.
31
P NMR (162 MHz, CD
3
OD) δ 2.77 (s), 2.61 (s).
1
H NMR (400 MHz, CD
3
OD) δ 8.13 – 8.05
(m, 1H), 7.80 – 7.71 (m, 1H), 7.55-7.6 (m, 1H), 7.50 – 7.36 (m, 4H), 7.33-7.26 (m, 1H), 4.90 –
4.83 (m, 1H), 4.23 – 4.11 (m, 2H), 4.10 – 3.99 (m, 3H), 3.94 – 3.85 (m, 1H), 2.05 – 1.97 (m,
1H), 1.94-1.84 (m, 1H), 1.42-1.40 (m, 6H), 1.13 (m, 3H).
13
C NMR (100 MHz, CD
3
OD) δ 175.36, 155.69, 146.8, 134.80, 132.03, 127.14, 126.25, 125.91,
125.86, 125.02(m), 124.39(m), 124.34 (m), 121.58, 115.01, 84.76 (m), 74.62, 72.66, 66.95 (m),
61.13, 56.58, 40.11 (m), 26.50-26.39 (m), 26.14-26.03 (m), 12.99.

- 182 -

2'-Deoxy-ψ-isocytidine 3’,5’-bis[naphthyl(ethoxydimethylglycinyl)]-phosphate 23a
O
O
O
N NH
NH
2
O
P
O
NH
P
O
O
O
H
N
EtO
O
EtO
O

Mixture of 4 diasteriomers 23a.
31
P NMR (162 MHz, CD
3
OD) δ 2.91 – 2.39 (m), 2.30 – 1.53 (m).
1
H NMR (400 MHz, CD
3
OD)
δ 8.25 – 8.07 (m, 2H), 7.91 – 7.76 (m, 2H), 7.74 – 7.57 (m, 2H), 7.57 – 7.25 (m, 9H), 5.20 – 5.03
(m, 1H), 5.01 – 4.87 (m, 1H), 4.46 – 4.28 (m, 2H), 4.26 – 4.17 (m, 1H), 4.17 – 3.99 (m, 5H),
2.59 – 2.46 (m, 1H), 2.46 – 2.38 (m, 1H), 2.26 – 2.14 (m, 1H), 1.88 – 1.74 (m, 1H), 1.57 – 1.37
(m, 12H), 1.22 – 1.09 (m, 6H).
13
C NMR (100 MHz, CD
3
OD) δ 175.73 – 174.82 (m), 157.09 –
154.59 (m), 147.56 – 145.86 (m), 134.99 – 134.63 (m), 127.44 – 217.38 (m), 126.66 – 126.38
(m), 126.37 – 126.22 (m), 126.08 – 125.83 (m), 125.24 – 124.96 (m), 124.62 – 124.31 (m),
121.92 – 121.36 (m).116.29 – 114.14 (m), 84.04 – 82.92 (m), 79.64 – 78.75 (m), 75.25 - 74.24
(m), 66.92 – 65.76 (m), 61.13 (s, J = 2.3 Hz), 57.28 – 56.39 (m), 38.83(m), 38.40(m), 26.53(m),
26.12(m), 13.02.
2'-Deoxy-ψ-isocytidine 5’-[naphthyl(benzoxydimethylglycinyl)]-phosphate 22a
O
OH
O
N NH
NH
2
O
P
O
HN
O
O
O

Mixture of 2 diasteriomers 22a.
31
P NMR (162 MHz, CD
3
OD) δ 2.73 (s), 2.55 (s).
1
H NMR (400 MHz, CD
3
OD) δ 8.20 – 8.12
(m, 1H), 7.89 – 7.81 (m, 1H), 7.70 – 7.61 (m, 1H), 7.57 – 7.43 (m, 4H), 7.39 – 7.23 (m, 6H),
- 183 -

5.19 – 5.07 (m, 2H), 5.01 – 4.90 (m, 1H), 4.26 – 4.14 (m, 2H), 4.13 – 4.08 (m, 1H), 3.98 – 3.88
(m, 1H), 2.12 – 1.92 (m, 2H), 1.57 – 1.49 (m, 6H).
13
C NMR (100 MHz, CD
3
OD) δ 175.79,
156.3 (m), 147.25 (m), 136.52, 135.42, 128.76, 128.53, 128.47, 127.97, 127.16, 127.10, 126.88,
126.55, 126.50, 125.65, 124.96, 122.21, 115.6 (m), 85.3(m), 75.2 (m), 73.2 (m), 67.6 (m), 67.43,
57.3, 40.83 (m), 27.1 (m) 26.7(m).
2-[Bis(dimethylamino)phosphoryl-2'-deoxy-ψ-isocytidine 5’-[naphthyl(ethoxydimethylglycinyl)]-
phosphate 27
O
OH
O
N NH
NH
2
O
P
O
HN
O
EtO
O
P
O
NMe
2
NMe
2

Mixture of 2 diasteriomers 27.
31
P NMR (162 MHz, CD
3
Cl) δ 16.81 (s), 16.77 (s), 2.90 (s), 2.68 (s).
1
H NMR (400 MHz,
CD
3
OD) δ 8.18 – 8.02 (m, 2H), 7.85 – 7.70 (m, 1H), 7.65 – 7.52 (m, 1H), 7.48 – 7.34 (m, 3H),
7.34 – 7.22 (m, 1H), 5.09 – 4.92 (m, 1H), 4.26 – 4.11 (m, 2H), 4.08 – 3.96 (m, 3H), 3.96 – 3.87
(m, 1H), 2.69 – 2.55 (m, 12H), 2.04 – 1.96 (m), 1.86 – 1.79 (m), 1.68 – 1.53 (m), 1.48 – 1.36 (m,
6H), 1.33 – 1.24 (m), 1.17 – 1.08 (m, 3H).
13
C NMR (100 MHz, CD
3
OD) δ 175.37, 171.55,
162.69, 162.41, 157.11, 157.01, 146.71 (m), 134.81, 127.37 (m), 126.51, 126.44, 126.32, 126.26,
126.03, 125.88, 125.08 (m), 124.44, 124.34, 121.52, 121.49, 114.94 (m), 110.77 (m), 85.29 (d, J
= 8.4 Hz), 85.06 (d, J = 8.3 Hz), 73.17, 73.13, 72.49, 72.45, 66.78 (d, J = 5.9 Hz), 66.64 (d, J =
5.9 Hz), 61.14, 60.11, 56.7, 56.6, 41.24, 41.16, 35.56, 35.52, 26.4 (m), 26.1 (m) 13.04, 13.00.

- 184 -

2-[Bis(dimethylamino)phosphoryl-2'-deoxy-ψ-isocytidine-3’,5’-
bis[naphthyl(ethoxydimethylglycinyl)]-phosphate 28
O
O
O
N NH
NH
2
O
P
O
NH
P
O
O
O
H
N
EtO
O
EtO
O
P
O
NMe
2
NMe
2

Mixture of 4 diasteriomers 28.
31
P NMR (162 MHz, CD
3
OD) δ 16.99 – 16.46 (m), 2.89 – 2.32 (m), 2.27 – 1.63 (m).
1
H NMR
(400 MHz, CD
3
OD) δ 8.21 – 7.94 (m, 3H), 7.88 – 7.66 (m, 3H), 7.68 – 7.50 (m, 3H), 7.49 – 7.13
(m, 8H), 5.18 – 4.95 (m, 1H), 4.93 – 4.79 (m, 1H), 4.35 – 4.09 (m, 3H), 4.06 – 3.90 (m, 4H),
2.68 – 2.39 (m, 12H), 2.27 – 2.04 (m, 1H), 1.6 – 1.48 (m, 1H) 1.45 – 1.26 (m, 12H), 1.12 – 1.02
(m, 6H).
2-[Bis(dimethylamino)phosphoryl-2'-deoxy-ψ-isocytidine 5’-
[naphthyl(benzoxydimethylglycinyl)]-phosphate 29
O
OH
O
N NH
NH
2
O
P
O
HN
O
O
O
P
O
NMe
2
NMe
2

Mixture of 2 diasteriomers 29.
31
P NMR (162 MHz, CD
3
OD) δ 16.78 (s), 16.74 (s), 2.85 (s), 2.62 (s).
1
H NMR (400 MHz,
CD
3
OD) δ 8.27 – 8.09 (m, 2H), 7.90 – 7.70 (m, 1H), 7.70 – 7.58 (m, 1H), 7.57 – 7.39 (m, 3H),
7.38 – 7.22 (m, 6H), 5.17 – 5.08 (m, 2H), 5.02 (s, 1H), 4.28 – 4.12 (m, 2H), 4.12 – 4.03 (m, 1H),
4.00 – 3.88 (m, 1H), 2.75 – 2.63 (m, 12H), 2.08 – 2.00 (m, 1H), 1.92 – 1.84 (m, 1H), 1.57 – 1.46
(m, 6H).
13
C NMR (100 MHz, CD
3
OD) δ 176.7 (m), 175.13, 175.10, 162.4 (m), 157.1 (m),
149.14, 148.78, 146.7 (m), 136.61, 136.11, 135.89, 135.87, 134.78 (m), 128.14, 128.12, 128.05,
- 185 -

127.95, 127.93, 127.89, 127.86, 127.79, 127.64, 127.57, 127.23, 127.04, 126.98, 126.91, 126.49,
126.43, 126.32, 126.27, 126.05, 125.91, 125.61, 125.56, 125.44, 125.38, 125.09 (m), 124.89,
124.75, 124.45, 124.35, 122.84 (m), 114.98 (m) 110.74 (m), 85.24 (d, J = 8.2 Hz), 85.05 (d, J =
8.3 Hz), 73.15, 73.10, 72.41, 66.81, 66.79, 66.69 (m), 56.71, 56.67, 41.21, 41.16, 35.58, 35.54,
35.52, 35.54, 26.11, 26.07.

- 186 -

5.5 REPRESENTATIVE NMR SPECTRA
1
H NMR of 3’, 5’-Di-tert-butylsilylene-2'-deoxy- ψ-isocytidine (24)

13
C NMR of 3’, 5’-Di-tert-butylsilylene-2'-deoxy- ψ-isocytidine (24)

- 187 -

1
H NMR of 3’, 5’-Di-tert-butylsilylene-[bis(dimethylamino)phosphoryl]oxy-2'-deoxy- ψ-
isocytidine (25)

13
C NMR of 3’, 5’-Di-tert-butylsilylene-[bis(dimethylamino)phosphoryl]oxy-2'-deoxy- ψ-
isocytidine (25)

- 188 -

31
P NMR of 3’, 5’-Di-tert-butylsilylene-[bis(dimethylamino)phosphoryl]oxy-2'-deoxy- ψ-
isocytidine (25)

1
H NMR of [bis(dimethylamino)phosphoryl]oxy-2'-deoxy- ψ-isocytidine (26)

- 189 -

1
H NMR of [bis(dimethylamino)phosphoryl]oxy-2'-deoxy- ψ-isocytidine (26)

1
H NMR of [bis(dimethylamino)phosphoryl]oxy-2'-deoxy- ψ-isocytidine (26)

- 190 -

1
H NMR 2'-Deoxy- ψ-isocytidine 5’-[naphthyl(ethoxydimethylglycinyl)]-phosphate (21a)

13
C NMR 2'-Deoxy- ψ-isocytidine 5’-[naphthyl(ethoxydimethylglycinyl)]-phosphate (21a)

- 191 -

31
P NMR 2'-Deoxy- ψ-isocytidine 5’-[naphthyl(ethoxydimethylglycinyl)]-phosphate (21a)

1
H NMR of 2'-Deoxy- ψ-isocytidine 3’,5’-bis[naphthyl(ethoxydimethylglycinyl)]-phosphate
(23a)

- 192 -

13
C NMR of 2'-Deoxy- ψ-isocytidine 3’,5’-bis[naphthyl(ethoxydimethylglycinyl)]-phosphate
(23a)

13
C NMR of 2'-Deoxy- ψ-isocytidine 3’,5’-bis[naphthyl(ethoxydimethylglycinyl)]-phosphate
(23a)

- 193 -

1
H NMR of 2'-Deoxy- ψ-isocytidine 5’-[naphthyl(benzoxydimethylglycinyl)]-phosphate
(22a)

13
C NMR of 2'-Deoxy- ψ-isocytidine 5’-[naphthyl(benzoxydimethylglycinyl)]-phosphate
(22a)

- 194 -

31
P NMR of 2'-Deoxy- ψ-isocytidine 5’-[naphthyl(benzoxydimethylglycinyl)]-phosphate
(22a)

1
H NMR of 2-[Bis(dimethylamino)phosphoryl-2'-deoxy- ψ-isocytidine 5’-
[naphthyl(ethoxydimethylglycinyl)]-phosphate (27)

- 195 -

13
C NMR of 2-[Bis(dimethylamino)phosphoryl-2'-deoxy- ψ-isocytidine 5’-
[naphthyl(ethoxydimethylglycinyl)]-phosphate (27)

31
P NMR of 2-[Bis(dimethylamino)phosphoryl-2'-deoxy- ψ-isocytidine 5’-
[naphthyl(ethoxydimethylglycinyl)]-phosphate (27)

- 196 -

1
H NMR of 2-[Bis(dimethylamino)phosphoryl-2'-deoxy- ψ-isocytidine-3’,5’-
bis[naphthyl(ethoxydimethylglycinyl)]-phosphate (28)

13
C NMR of 2-[Bis(dimethylamino)phosphoryl-2'-deoxy- ψ-isocytidine-3’,5’-
bis[naphthyl(ethoxydimethylglycinyl)]-phosphate (28)

- 197 -

31
P NMR of 2-[Bis(dimethylamino)phosphoryl-2'-deoxy- ψ-isocytidine-3’,5’-
bis[naphthyl(ethoxydimethylglycinyl)]-phosphate (28)

1
H NMR of 2-[Bis(dimethylamino)phosphoryl-2'-deoxy- ψ-isocytidine 5’-
[naphthyl(benzoxydimethylglycinyl)]-phosphate (29)

- 198 -

13
C NMR of 2-[Bis(dimethylamino)phosphoryl-2'-deoxy- ψ-isocytidine 5’-
[naphthyl(benzoxydimethylglycinyl)]-phosphate (29)

31
P NMR of 2-[Bis(dimethylamino)phosphoryl-2'-deoxy- ψ-isocytidine 5’-
[naphthyl(benzoxydimethylglycinyl)]-phosphate (29)

- 199 -

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Abstract (if available)

Abstract This dissertation focuses on the development of novel synthetic approaches for incorporation of different traditional as well as untraditional bioisosteres into the structure of nucleotide analogues. ❧ It is well known that many biologically active compounds containing polar ionic groups such as phosphates cannot be successful drag candidates because they exhibit poor bioavailability stability. One of the concepts that help to overcome these challenges called bioisosterism. According to this concept certain functional groups or a part of the molecule can be replaced with the bioisosteric group which possesses similar electronic distribution and spatial arrangement. This substitution significantly improves stability and bioavailability of the target molecule. ❧ Chapter 1 describes the synthetic approach towards non‐hydrolysable RNA‐based nucleotide analogues in which bridging oxygen atoms of the triphosphate replaced by difluoromethylene group. ❧ Chapter 2 summarized our investigations towards replacement of the phosphate hydroxyl group with the difluoromethyl group. ❧ Chapter 3 describes the development of new methodology for preparation of monofluoroalkenes via Julia‐Kosinski reaction. ❧ Chapter 4 explores the possibility for bioisosteric replacement of α,- β,- and γ- phosphate of trisphosphoric acid with the squaryl moiety. ❧ Chapter 5 deals with the synthesis of phosphoramidates of 2'-deoxy‐ψ‐isocytidine.

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Asset Metadata

Creator Shakhmin, Anton A. (author)

Core Title Synthesis of novel nucleotide analogues based on the traditional and nontraditional bioisosteres

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 03/11/2014

Defense Date 03/09/2014

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

Tag fluorinated phosphonate,isopolarity,isostericity,non-hydrolysable nucleotide analogues,OAI-PMH Harvest,ribonucleotides,single‐turnover gap filling assay,squaric acid

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Prakash, G. K. Surya (committee chair), Hogen-Esch, Thieo E. (committee member), Shing, Katherine (committee member)

Creator Email ashakhmin@yahoo.com,shakhmin@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-368481

Unique identifier UC11295343

Identifier etd-ShakhminAn-2288.pdf (filename),usctheses-c3-368481 (legacy record id)

Legacy Identifier etd-ShakhminAn-2288.pdf

Dmrecord 368481

Document Type Dissertation

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Rights Shakhmin, Anton A.

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

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