Preparation of fluorinated RNA nucleotide analogs potentially stable to enzymatic hydrolysis in RNA and DNA polymerase assays Dedicated to Dr. Teruo Umemoto on the occasion of receiving the ACS Award for Creative Work in Fluorine Chemistry.

Article abstract of DOI:10.1016/j.jfluchem.2014.07.019

Analogs of ribonucleotides (RNA) stable to enzymatic hydrolysis were prepared and characterized. Computational investigations revealed that this class of compounds with a modified triphosphate exhibits the correct polarity and minimal steric effects compa

Full text of DOI:10.1016/j.jfluchem.2014.07.019

Journal of Fluorine Chemistry 167 (2014) 226–230
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Preparation of fluorinated RNA nucleotide analogs potentially stable to
enzymatic hydrolysis in RNA and DNA polymerase assays
Anton Shakhmin a, John-Paul Jones a, Inessa Bychinskaya a, Mikhail Zibinsky a,
Keriann Oertell b, Myron F. Goodman b, G.K. Surya Prakash a,
*
a Loker Hydrocarbon Research Institute, Department of Chemistry, University of Southern California, 837 Bloom Walk, Los Angeles, CA 90089-1661, United
States
b Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-1661, United States
A R T I C L E I N F O
A B S T R A C T
Article history:
Analogs of ribonucleotides (RNA) stable to enzymatic hydrolysis were prepared and characterized.
Computational investigations revealed that this class of compounds with a modified triphosphate
exhibits the correct polarity and minimal steric effects compared to the natural molecule. Non-
hydrolysable properties as well as the ability of the modified nucleotide to be recognized by enzymes
were probed by performing single-turnover gap filling assays with T7 RNA polymerase and DNA
Received 18 July 2014
Accepted 23 July 2014
Available online 1 August 2014
Dedicated to Dr. Teruo Umemoto on the
occasion of receiving the ACS Award for
Creative Work in Fluorine Chemistry.
polymerase
b.
ß 2014 Elsevier B.V. All rights reserved.
Keywords:
Non-hydrolysable nucleotide analogs
Isostericity and isopolarity
Fluorinated phosphonate
Ribonucleotides
Single-turnover gap filling assay
1. Introduction
been achieved by substituting bridging oxygen atoms of the
triphosphate unit with bioisosteric difluoromethylene groups [9–
11]. A computational study demonstrated that such a replacement
provides minimal steric perturbation and retains the polarity of the
natural triphosphate.
Nucleotides are central molecules in biology and have high
importance in biochemical and medicinal investigations [1].
Hydrolytically stable analogs modified at the
a,b-bridging oxygen
position in the triphosphate linkage cannot be utilized in enzymatic
nucleotidyl transfer reactions. Owing to such hydrolytic stability,
such a family of nucleotide analogs is increasingly used for studying
active sites of various enzymes, as well as their functions and
mechanisms [2–4]. Toward this endeavor, a number of modifica-
tions at the triphosphate fragment have been explored, allowing the
study of steric and electronic effects with relation to the activity of
the resulting analogs [5–8]. However, only a few analogs with a
modified triphosphate demonstrate properties similar to natural
nucleotides. At the same time, considering the high sensitivity of
enzymes toward electronic and steric parameters of the active site
binders, these properties of the modified nucleotides are extremely
important to effectively mimic of the natural substrates.
Recently, we reported a simple and efficient synthesis of
bis(difluoromethylene)triphosphoric acid (BMF4TPA)
4
[12]
(Scheme 1). Correct polarity, low steric demand of the bridging
groups, and notable stability of BMF4TPA under a wide range of
temperatures and pH prompted us to explore this unique set of
properties in biochemical assays via replacement of the triphos-
phate group in ribonucleotides with the bis(difluoromethylene)
analog 4. Both non-specific and enzyme catalyzed hydrolytic
stability of such synthetic analogs were expected to be greater
when compared to natural nucleotides.
2. Results and discussion
Herein, we report the synthesis of novel fluorinated ribonucle-
otide analogs potentially stable to enzymatic hydrolysis. This has
2.1. Synthesis
Generally, the BMF4TPA moiety was attached to a nucleoside
via nucleophilic displacement of 50-tosylate in protected nucleo-
sides by the tetra-n-butylammonium salt of the acid [13,14]. To
prepare the nucleoside tosylates, the A, C and U nucleosides were
*
Corresponding author.
E-mail address: gprakash@usc.edu (G.K. Surya Prakash).
http://dx.doi.org/10.1016/j.jfluchem.2014.07.019
0022-1139/ß 2014 Elsevier B.V. All rights reserved.

A. Shakhmin et al. / Journal of Fluorine Chemistry 167 (2014) 226–230
227
Scheme 1. Preparation of BMF4TPA [Bu4NH+]5. Reaction conditions: (a) LTMPA, THF,
ꢀ78 8C; (b) m-CPBA; (c) TMSBr; (d) Bu4NOH.
first directly acylated with benzoyl chloride at the N-base, 20- and
30-positions [15] (Scheme 2). This step was followed by treatment
with p-toluenesulfonyl chloride in anhydrous pyridine for 7 days at
ꢀ20 8C to give access to the protected 50-tosylates 9–11 in good
yields. However, the described route could not be applied to obtain
protected guanosine tosylate directly.
Scheme 3. Preparation of protected 50-tosylguanosine. Reaction conditions: (a)
DMF-DMA, MeOH; (b) TBDPSCl, DMAP, Py; (c) BzCl; (d) TBAF, THF, AcOH; (e) TsCl,
Py, ꢀ24 8C, 7 days.
2.2. Computational results
Instead, the solubility of guanosine had to be enhanced by
protecting the 50-hydroxyl with TBDMS and the N2-amino group
with a dimethylaminomethylene group [16]. Subsequently, a
facile reaction with benzoyl chloride in situ afforded the
protection of the 20- and 30-hydroxyl groups of 12. Deprotection
of 50-hydroxyl group was achieved by treating 12 with TBAF/THF/
AcOH mixture [17]. The corresponding guanosine tosylate 14 was
prepared via the well-established reaction with TsCl in pyridine
(Scheme 3).
A DFT computational study using Gaussian 09 [18] supported
the hypothesis that the incorporation of difluoromethylene groups
into the (
a,b) and (b,g) positions of triphosphoric acid would be
bioisosteric in nature.
Although in the free acid forms these analogs acquire different
conformations, the presence of a magnesium dication simulating
interactions in enzyme precatalytic state results in similar
structures coordinated around the ion for both acids.
The target nucleotide analogs were prepared (Scheme 4) via the
reaction of benzoyl protected 50-tosyl ribonucleosides 9–11 and 14
with the BMF4TPA tetra-n-butylammonium salt (5). It is not
surprising that due to the strong electron withdrawing effect of the
difluoromethylene bridging groups, BMF4TPA acts as a poor
nucleophile. However, conversion of BMF4TPA into the tetra-
butylammonium salt significantly enhanced its solubility and
reactivity as a nucleophile.
The substitution reaction was carried out in anhydrous DMF at
110 8C for 1 h, then left stirring at room temperature overnight. The
progress of the reaction can be monitored using a weak ion-
exchange HPLC using a DEAE-5PW column. The BMF4TPA fragment
can sustain harsh reaction conditions providing acceptable yields
of the products 15–18. Two equivalents of the tosyl nucleoside
were required to ensure maximum consumption of BMF4TPA
[Bu4NH+]5.
The investigation clearly shows that bioisosteric incorporation
of difluoromethylene group instead of labile phosphoanhydride
oxygen atoms leads to a minimal steric perturbation and retains
similar charge distribution compared to the natural triphosphoric
acid. Previous studies have revealed that the pKa values of the
BMF4TPA (pKa4 = 5.33, pKa5 = 7.23) are lower than corresponding
values of natural triphosphoric acid (5.83–6.50 and 8.73–9.24),
[10] but possibly close enough to be considered isoacidic [19].
Natural bond order (NBO) analysis of the BMF4TPA analog
relative to the natural triphosphoric acid (TPA) showed similar
charge distribution. Dipole moments of 4.42 Debye and 4.38 Debye
were calculated for the BMF4TPA analog and the TPA, respectively,
further demonstrating that the overall electrostatic properties of
the molecule have not been altered dramatically (Fig. 1B).
O
P
O
O
P
O
O
P
O
All ribonucleotide analogs exhibited similar retention times
and were eluted within 28–35 min using 1 M TEAB and water as
eluent. After reactions reached maximum conversions, all nucleo-
tide analogs were subjected to deprotection using water/methanol
F2
C
F2
C
Base(PG)
TsO
Base(PG)
HO
O
O
O
a
(Bu4N+)4
OBz OBz
9-11, 14
OBz OBz
solution of ammonium hydroxide. In the case of (
ATP, ( ),( )-bisCF2 CTP, and ( ),( )-bisCF2 UTP, we were
able to achieve conversions close to 90%, although in case of the
),( )-bisCF2 GTP conversion did not exceed 20%.
a,b),(b,g)-bisCF2
15-18
a
,
b
b
,
g
a,b b,g
(a,b b,g
O
P
O
O
P
O
P
F2
C
F2
C
Base
O
O
b
O
O
O
(Et3NH+)4
OH OH
19-22
Scheme 4. Preparation of (
DMF, 110 8C; (b) NH4OH, MeOH. Yields of bisCF2-NTP analogs: (
ATP (19) 29%, ( ),( )-bisCF2-CTP (20) 33%, ( ),( )-bisCF2-UTP (21) 22%,
),( )-bisCF2-GTP (22) 6%.
a
,
b
),(
b
,
g
)-bisCF2-NTP analogs. Reaction conditions: (a)
Scheme 2. Synthesis of benzoyl protected tosylnucleosides. 6, 9 base = N6-Bz2-
adenine, 7, 10 base = N4-Bz-cytosine, 8, 11 base = N3-Bz-uracyl. Reaction
conditions: (a) TBDMSCl, Py; (b) BzCl, (c) TFA, H2O, THF; (d) TsCl, Py, ꢀ24 8C, 7 days.
a,b),(b,g)-bisCF2-
a,
b
b,g
a,b b,g
(a,b b,g

228
A. Shakhmin et al. / Journal of Fluorine Chemistry 167 (2014) 226–230
Fig. 1. Comparative computational study of natural triphosphoric acid and BMF4TPA analog. (A) Bond scan at B3LYP/cc-PVDZ. (B) Charge density maps of the TPA and BMF4TPA
analog generated by Molden Program [20].
By increasing the bond length of the bridging P–O or P–C bonds
of the optimized structure incrementally, bond strength was
estimated to be over 80 kcal/mol for the BMF4TPA analog
compared to approximately 40 kcal/mol for the natural tripho-
sphoric acid (Fig. 1A). The calculated ‘‘bond strength’’ of these
analogs also suggests that the isopolar and isosteric replacement
makes the molecule significantly more stable and resistant toward
potential enzymatic hydrolysis. These computational data, in a
preliminary study, encouraged us to evaluate the behavior of
prepared nucleotides exposed to biological systems.
labeled above the corresponding lane. The assay was run for
30 min and then analyzed. As predicted, the reaction shown in the
left lane, containing the natural substrate, was able to proceed to
the full length. The reaction in the right lane, however, stalled due
to the inability of the T7 RNA pol to utilize the bis-CF2-UTP analog.
However, this study alone does not demonstrate that the
nonhydrolyzable derivative is incorporated at the enzymatic site.
Studies to obtain crystal structures of such potential ternary
complexes are under way.
Subsequently, we decided to test our nucleotide analogs with
another class of polymerases. Even though the Tyr271 residue in
2.3. Preliminary biological results
DNA polymerase
moderate degree and can discriminate between dNTP and rNTP
[21], it is known that pol does not exhibit high fidelity with
respect to base pairing and recognizing ribo- and deoxyribonu-
cleotides. Thus, we tested the hydrolytic stability of our compound
b prevents the incorporation of rNTPs to a
The non-hydrolysable nature of prepared ribonucleotide
analogs was probed by performing a single-turnover gap-filling
assay. Experiments with incorporation of UTP or bis-CF2-UTP (21)
by T7 RNA pol are shown in Fig. 2A. The position of the first
templating dA in a dsDNA substrate following the T7 promoter
sequence is labeled on the left side of the gel as U ! A as the
location of the incorporation of either natural UTP, left lane, or bis-
CF2-UTP (21), right lane. FL refers to the full length RNA. Each
reaction contained CTP, GTP, ATP, and either UTP or bis-CF2-UTP, as
b
with DNA polymerase
UTP (21) analog into a single-gapped DNA substrate with dA at the
first templating position, using DNA pol , indeed verified the non-
b. Incorporation of dTTP, UTP, and bis-CF2-
b
hydrolysable nature of the bis-CF2-UTP (21) analog. The position of
the unextended primer is labeled P on the left side of the gel, and
the incorporation of a single dNTP or NTP base is shown at P + 1.
Fig. 2. Biochemical assays with bis-CF2-UTP analog. (A) Single turnover assay with T7 RNA Polymerase. (B) Single turnover gap filling assay with DNA polymerase
b.

A. Shakhmin et al. / Journal of Fluorine Chemistry 167 (2014) 226–230
229
ThedNTPorNTP beingincorporated,alongwiththeconcentration,is
labeled across the top of the gel, and the reaction time below each
lane. Both experiments indicate that unlike natural UTP and dTTP
substrates, the difluoromethylene groups of the bis-CF2-UTP inhibit
incorporation into an RNA (Fig. 2A) or DNA (Fig. 2B) oligonucleotide
and such nucleotides are not utilized by polymerases. These studies
alone do not demonstrate their inhibitory nature in the active site.
We believe that such nucleotide analogs stable to enzymatic
hydrolysis will help uncover additional details of the mechanism of
enzyme catalyzed processes and may be useful to the study of
biochemical processes that rely on triphosphate concentration
sensing. Such focused studies are underway.
collected and washed three times with water and dried over
MgSO4. The solvent was evaporated and without further purifica-
tion the residue was dissolved in 100 mL of THF and 2.1 mL
(36.5 mmol) of acetic acid. Subsequently 14.6 mL (14.6 mmol) of
1 M solution of TBAF in THF was added dropwise to the stirred
solution. Reaction mixture was allowed to stir at room tempera-
ture overnight. Next all volatiles were evaporated and the residue
was dissolved in dichloromethane (200 mL) and washed three
times with water and dried over MgSO4. The solvent was
evaporated and the reaction mixture was subjected to chro-
matographic separation on silica gel with CH2Cl2/MeOH (9/1) as
eluent affording 2.05 g of 13 (47%).
1H NMR (400 MHz, CDCl3)
d: 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). 13C NMR
3. Conclusion
(100 MHz, CDCl3) d: 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.
Analogs of ribonucleotides (RNA) stable to potential enzymatic
hydrolysis were synthesized and characterized. Computational
investigations revealed that this class of compounds witha modified
triphosphate exhibits the correct polarity and minimal steric effects
compared to the natural molecule. Preliminary non-hydrolysable
properties as well as the ability of the modified nucleotide to be
recognized by enzymes were probed by performing single-turnover
4.2. General procedure for preparation of nucleosides-50-tosylates 9–
11 and 14
Benzoyl protected respective ribonucleosides (500 mg) were
dried three times by azeotropic evaporation of pyridine on a rotary
evaporator under vacuum. After that, the flask was filled with
argon and the ribonucleoside was redissolved in 25 mL of
anhydrous pyridine. Subsequently, equimolar solution of p-
toluenesulfonyl chloride in dry pyridine was added to the stirred
solution of ribonucleoside precooled to 0 8C. The reaction mixture
was removed from the ice and placed in the freezer (ꢀ20 8C) 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
CH2Cl2/MeOH (1:10) as eluent.
gap filling assays with T7 RNA polymerase and DNA polymerase b.
Furthercrystallizationstudy ofenzyme–substratecomplexes and X-
ray crystal structure characterization will be required to show
incorporation of the non-hydrolyzable RNA analogs in the active
sites. Such studies are underway.
4. Experimental
Unless otherwise mentioned, all reagents were purchased from
commercial sources. All NMR spectra were recorded on a Varian
400 MHz NMR spectrometer. Chemical shifts (d) are reported in
part per million (ppm) relative to internal residual CHCl3 in CDCl3
(
d
7.25, 1H), internal residual HDO in D2O (pH ꢁ8,
d
d
4.8, 1H),
0.00, 19F),
external chlorotrifluoromethane as the 19F standard (
4.2.1. 50-Tosyl-N,N,20,30-tetrabenzoyladenosine 9
and external phosphoric acid (
d
0.00, 31P) as a standard for 31P
Yield: 61%. 1H NMR (400 MHz, CDCl3)
d: 2.34 (s, 3H), 4.44 (dd,
experiments. High resolution mass spectra were recorded in ESI+
mode on a high resolution mass spectrometer at the Mass
Spectrometry facility, University of Arizona. HPLC analysis and
purification of the nucleotide analogs 32–35 were performed on a
Shimadzu HPLC system (SCL-10A VP, SPD-10A VP, and LC-8A) with
J = 11.2 Hz, 4.8 Hz, 1H), 4.5 (dd, J = 10 Hz, 2.8 Hz, 1H), 4.66 (m, 1H),
5.93 (dd, J = 4.8 Hz, J = 5.6 Hz, 1H), 6.11 (dd, J = 6 Hz, 5.2 Hz, 1H),
6.44 (d, J = 5.6 Hz, 1H), 7.22–7.56 (m,14H), 7.23–7.93 (m, 10H),
8.29 (s, 1H), 8.54 (s, 1H) ppm. 13C NMR (100 MHz, CDCl3)
d 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.
Tosoh Bioscience DEAE-5PW 21.5 cm ꢂ 15 cm, 13
m
m (0–60% 1 M
TEAB, pH 8.0) and Shimadzu Premier C18 5
preparative column.
m
m 250 mm ꢂ 23 mm
Anhydrous THF was prepared by distillation over sodium wire.
Diethyl difluoromethylphosphonate was obtained from triethyl-
phosphite and chlorodifluoromethane according to a well-estab-
lished protocol [22,23]. BMF4TPA tetrabutylammonium salt 5 was
prepared according to protocol developed by Prakash et al. [12].
For the preparation of protected tosylnucleosides 9–11, we utilized
reported procedure [24]. For synthesis of protected guanosine 13
we employed published protocols [16,17], Nucleoside tosylates 9–
11 and 14 were prepared based on procedure reported by Burton
and Flynn [23]
4.2.2. 50-Tosyl-20,30-O,N4-tribenzoylcytidine 10
Yield: 43%. 1H NMR (400 MHz, CDCl3)
d: 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). 13C NMR
(100 MHz, CDCl3)
d 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.
4.2.3. 50-Tosyl-20,30-O,N3-tribenzoyluridine 11
Yield: 55%. 1H NMR (400 MHz, CDCl3)
d: 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. 13C NMR (100 MHz, CDCl3)
d 21.7, 68.6, 71.2, 73.5, 80.6, 87.5,
4.1. Preparation of N2-[(dimethylamino)methylene] 20,30-
dibenzoylguanosine 13
Benzoyl chloride (2.33 g, 1.7 mmol) was added to the stirred
solution of 4.4 g (7.58 mmol) of 50-O-[(tert-butyl)diphenylsilyl]-
N2-[(dimethylamino)methylene]-guanosine 26 in 80 mL of anhy-
drous pyridine cooled to 0 8C. The resulting mixture was stirred at
room temperature for 8 h. All volatiles were removed under
reduced pressure and the residue was distributed between water
(300 mL) and dichloromethane (300 mL). The organic fraction was
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.
4.2.4. N-[(dimethylamino)methylene]-,20,30-dibenzoylguanosine 14
Yield: 59%. 1H NMR (400 MHz, CDCl3)
d
: 2.2 (s, 3H), 3.03 (s, 3H),
3.18 (s, 3H), 4.27 (dd, J = 11.6 Hz, 4 Hz, 1H), 4.4 (dd, J = 11.6 Hz,

230
A. Shakhmin et al. / Journal of Fluorine Chemistry 167 (2014) 226–230
2.4 Hz, 1H), 4.5 (m, 1H), 6 (d, J = 2.4 Hz 1H), 6.2 (m, 1H), 6.3 (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). 13C NMR (100 MHz, CDCl3)
d: 21.6, 35.3, 41.3, 67.6, 69.8, 73.9,
ꢀ119.36 (t, J = 72.94 Hz), ꢀ119.37 (t, J = 76.7 Hz) ppm. 31P NMR
(162 MHz, D2O): 2.32–3.9 (m, 2P), 9.64–12.05 (m, 1P) ppm.
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.
4.3.4. (a,b),(b,g)-bis(CF2) dGTP 22
Yield: 6%. 1H NMR (400 MHz, D2O): 1.1 (t, J = 7.3 Hz, 27H,
Et3NH+), 3.02 (q, J = 7.3 Hz, 18H, Et3NH+) 4.11–4.22 (m, 4H), 4.36–
4.41 (m, 1H), 5.72–5.81 (m, 1H), 8.11 (s, 1H) ppm. 19F NMR
(376 MHz, D2O): ꢀ118.9 (t, J = 76.7 Hz) ppm. 31P NMR (162 MHz,
D2O): 2.37–4.25 (m, 2P), 11.7–14.4 (m, 1P) ppm. HRMS: calculated
for [M+H+] C12H15F4N5O12P3 589.9872, found 589.9875.
4.3. General procedure for preparation of nucleotide analogs by using
reaction of (Bu4N+)5BMF4TPA salt with benzoyl protected nucleoside
50-tosylates
A 10 mL flask under argon atmosphere containing 2 mL of dry
DMF was charged with 100 mg (0.12 mmol) of 20-deoxy-50-
tosylnucleoside and 91.5 mg (0.6 mmol) of (Bu4N+)5BMF4TPA salt.
The resulting mixture was stirred for 1 h at 100 8C and then left at
Acknowledgments
Research support provided by the National Cancer Institute of
the National Institute of Health via Grant 5-U19-CA105010 and the
Loker Hydrocarbon Institute is greatly appreciated.
room temperature for two days. The conversion was checked by 19
F
NMR and HPLC using DEAE-5PW weak ion-exchange column with
1 M Et3NH+HCO3ꢀ/H2O as eluent. After the 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 H2O/MeOH/NH4OH (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 under vacuum, then re-dissolved in
water and subjected to HPLC purification on a preparative DEAE-
References
[1] W.A. Beard, S.H. Wilson, Chem. Rev. 106 (2006) 361.
[2] C.A. Sucato, T.G. Upton, B.A. Kashemirov, V.K. Batra, V. Martinek, Y. Xiang, W.A.
Beard, L.C. Pedersen, S.H. Wilson, C.E. McKenna, J. Florian, A. Warshel, M.F.
Goodman, Biochemistry 46 (2007) 461.
[3] C.A. Sucato, T.G. Upton, B.A. Kashemirov, J. Osuna, K. Oertell, W.A. Beard, S.H.
Wilson, J. Florian, A. Warshel, C.E. McKenna, M.F. Goodman, Biochemistry 47
(2008) 870.
[4] C.E. McKenna, B.A. Kashemirov, T.G. Upton, V.K. Batra, M.F. Goodman, L.C.
Pedersen, W.A. Beard, S.H. Wilson, J. Am. Chem. Soc. 129 (2007) 15412.
[5] Q.-F. Ma, G.L. Kenyon, G.D. Markham, Biochemistry 29 (1990) 1412.
[6] Q.-F. Ma, I.C. Bathurst, P.J. Barr, G.L. Kenyon, J. Med. Chem. 35 (1992) 1938.
[7] G.M. Blackburn, S.P. Langston, Tetrahedron Lett. 32 (1991) 6425.
[8] R. Li, A. Muscate, G.L. Kenyon, Bioorg. Chem. 24 (1996) 251.
[9] T.G. Upton, B.A. Kashemirov, C.E. McKenna, M.F. Goodman, G.K.S. Prakash, R.
Kultyshev, V.K. Batra, D.D. Shock, L.C. Pedersen, W.A. Beard, S.H. Wilson, Org. Lett.
11 (2009) 1883.
[10] G. Hirai, T. Watanabe, K. Yamaguchi, T. Miyagi, M. Sedeoka, J. Am. Chem. Soc. 129
(2007) 15420.
[11] G.M. Blackburn, H. Turkmen, Org. Biomol. Chem. 3 (2005) 225.
[12] G.K.S. Prakash, M. Zibinsky, T.G. Upton, B.A. Kashemirov, C.E. McKenna, K. Oertell,
M.F. Goodman, V.K. Batra, L.C. Pedersen, W.A. Beard, D.D. Shock, S.H. Wilson, G.A.
Olah, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 15693.
[13] G.M. Blackburn, M.-J. Guo, S.P. Langston, G.E. Taylor, Tetrahedron Lett. 31 (1990) 5637.
[14] V.J. Davisson, D.R. Davis, V.M. Dixit, C.D. Poulter, J. Org. Chem. 52 (1987) 1794.
[15] X. Zhu, A.I. Scott, Synth. Commun. 38 (2008) 1346.
5PW weak ion-exchange column with water/Et3NH+HCO3 as
ꢀ
eluent (0–60% gradient regime). The buffer solution was removed
by evaporation and desired nucleotide analogs were dried under
high vacuum.
4.3.1. (a,b),(b,g)-bis(CF2) ATP 19
Yield: 29%. 1H NMR (400 MHz, D2O): 1.05 (t, J = 7.6 Hz, 27H,
Et3NH+), 2.97 (q, J = 7.6 Hz, 18H, Et3NH+) 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. 19F NMR
(376 MHz, D2O): ꢀ119.2 (t, J = 72.2 Hz), ꢀ120.3 (t,
J = 83.8 Hz) ppm. 31P NMR (162 MHz, D2O): 1.8–4.8 (m, 2P),
10.9–13.2 (m, 1P) ppm. HRMS: calculated for [M+Na+]
C
12H16F4N5O11P3Na 597.9887, found 597.9891.
[16] Z.Y. Cui, L. Zhang, B.L. Zhang, Tetrahedron Lett. 42 (2001) 561.
[17] J. Matulic-Adamic, L. Beigelman, Helv. Chim. Acta 82 (1999) 2141.
[18] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li,
H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J.
Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, T. Keith, R. Kobayashi, J. Normand,
K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega,
N.J. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W.
Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador,
J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J.
Cioslowski, D.J. Fox, Gaussian 09, rev. A.02, Gaussian, Inc., Wallingford, CT, 2009.
[19] D.B. Berkowitz, M.J. Bose, Fluor. Chem. 112 (2001) 13.
4.3.2. ( ),( )-bis(CF2) CTP 20
a,b b,g
Yield: 33%. 1H NMR (400 MHz, D2O): 1.1 (t, J = 7.2 Hz, 27H,
Bu3NH+), 2.98 (q, J = 7.2 Hz, 18H, Et3NH+), 4.07–4.11 (m, 2H), 4.15–
4.2 (m, 2H), 4.23–4.27 (m, 1H), 5.86 (d, J = 4.8 Hz, 1H), 5.89 (d,
J = 7.6 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H) ppm. 19F NMR (376 MHz,
D2O): ꢀ118.3 (t, J = 75.2 Hz), ꢀ118.8 (t, 76.7 Hz) ppm. 31P NMR
(162 MHz, D2O): 2.56–4.42 (m, 2P), 11.87–14.58 (m, 1P) ppm.
HRMS: calculated for [M+Na+] C11H15F4N3O11P3 573.9775, found
573.9780.
4.3.3. (a,b),(b,g)-bis(CF2) UTP 21
[20] G. Schaftenaar, J.H. Noordik, J. Comput.-Aided Mol. Des. 14 (2000) 123.
[21] N.A. Cavanaugh, W.A. Beard, V.K. Batra, L. Perera, L.G. Pedersen, S.H. Wilson, J. Biol.
Chem. 286 (2011) 31650.
[22] L.Z. Soborovskii, N.F. Baina, Zh. Obshch. Khim. 29 (1959) 1144.
[23] D.J. Burton, R.M. Flynn, J. Fluorine Chem. 10 (1977) 329.
Yield: 22%. 1H NMR (400 MHz, D2O): 1.12 (t, J = 7.3 Hz, 27H,
Et3NH+), 3.04 (q, J = 7.3 Hz, 18H, Et3NH+), 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. 19F NMR (376 MHz, D2O):
[24] X.F. Zhu, A.I. Scott, Synth. Commun. 38 (2008) 1346.