A P(V)-N activation strategy for the synthesis of nucleoside polyphosphates

Article abstract of DOI:10.1021/jo4011156

A general and high-yielding synthesis of nucleoside 5′-triphosphates (NTPs) and nucleoside 5′-diphosphates (NDPs) from protected nucleoside 5′-phosphoropiperidates promoted by 4,5-dicyanoimidazole (DCI) has been developed. ³¹P NMR tracing experiments showed that the sequential deprotection and coupling reactions were exceptionally clean. The phosphoropiperidate exhibited superior reactivity to the conventional phosphoromorpholidate toward DCI-promoted NTP/NDP synthesis. The experimental results suggested that the mechanism of DCI activation could be distinctive for NTP and NDP synthesis, depending on the different nucleophilicity of pyrophosphate and phosphate.

Full text of DOI:10.1021/jo4011156

Article
pubs.acs.org/joc
A P(V)−N Activation Strategy for the Synthesis of Nucleoside
Polyphosphates
Qi Sun,* Shanshan Gong, Jian Sun, Si Liu, Qiang Xiao, and Shouzhi Pu
Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and Technology Normal University, 605 Fenglin Avenue, Nanchang,
Jiangxi 330013, P. R. China
S
* Supporting Information
ABSTRACT: A general and high-yielding synthesis of nucleoside
5′-triphosphates (NTPs) and nucleoside 5′-diphosphates (NDPs)
from protected nucleoside 5′-phosphoropiperidates promoted by
4,5-dicyanoimidazole (DCI) has been developed. 31P NMR
tracing experiments showed that the sequential deprotection and
coupling reactions were exceptionally clean. The phosphoropiper-
idate exhibited superior reactivity to the conventional phosphor-
omorpholidate toward DCI-promoted NTP/NDP synthesis. The
experimental results suggested that the mechanism of DCI
activation could be distinctive for NTP and NDP synthesis,
depending on the different nucleophilicity of pyrophosphate and
phosphate.
claimed by this method. On the other hand, the sulfonyl
imidazolium triflate method also emphasized that no protection
is required for the synthesis of nucleoside polyphosphate and
related conjugates. However, this advantage is based on the
prerequisite that nucleoside 5′-monophosphate (NMP) is
available. In case that NTP synthesis has to start from specific
nucleoside substrates, the high yielding and regioselective
phosphorylation at the 5′-OH remains a bottleneck as
exemplified by a recent report using this method (29% yield
for a NMP synthesis from a nucleoside substrate).9
As a major type of NTP synthetic precursors, phosphor-
amidates, such as phosphoromorpholidates10 and phosphor-
imidazolides,11 have been utilized to synthesize NTPs in
moderate yields, but these reactions are typically sluggish and
require days to complete. To accelerate the reaction rate, more
reactive zwitterionic phosphoramidate intermediates such as N-
methylimidazolium,12 N-methylpyrrolidinium,13 and pyridi-
nium phosphoramidates5 were generated in situ to promote
their coupling with pyrophosphate. But a concomitant issue was
the formation of more polyphosphate byproducts.
Unlike the tetracoordinated phosphoramidates, tricoordi-
nated phosphoramidites are much more reactive to nucleophilic
displacement at the phosphorus center due to the free electron
pair and trigonal pyramid geometry.14 In oligonucleotide
synthesis, phosphoramidites have been extensively used as
phosphitylating regents to link two nucleoside units with a
phosphite triester in the presence of an acidic activator.15 In
contrast, research on the activation of the more stable P(V)−N
bond in phosphoramidate for coupling reactions has been
INTRODUCTION
■
Natural nucleoside polyphosphates (NPPs) including nucleo-
side 5′-triphosphates and 5′-diphosphates (NTPs and NDPs)
play pivotal roles in numerous biological processes, such as
energy transfer, gene replication and expression, intracellular
signaling, and regulation of protein activity. Natural and
modified NTPs are indispensable substrates for a myriad of
techniques in modern biological research, ranging from
polymerase chain reaction to DNA/RNA labeling, random
mutagenesis, and DNA sequencing.1 The high demand for
NPPs in biology and medicine has always been a stimulus to
establishment of general and practical synthetic routes for these
compounds.2
Currently, the traditional “one-pot, three-step”3 and salicyl
phosphorochlorodite methods4 are still in wide use, but their
yields and generality are far from satisfactory. Although NTP
synthesis experienced a low-tide period of nearly two decades,
the past few years have witnessed revived interest in this area.
Novel synthetic methods based on H-phosphonate,5 cyclo-
saligenyl phosphate triester,6 cyclic phosphite,7 and sulfonyl
imidazolium triflate8 were brought to the arsenal. Among these
new reports, the cyclic phosphite approach presented a
straightforward and protection-free method, which is highly
desired for NTP synthesis. But the reported isolated yields of
dNTPs were low (19−46%). Moreover, the H and 31P NMR
1
spectra of their dNTPs showed that the samples were
contaminated with significant amount of impurities. In a
recently published paper employing this method,9 a very low
yield of only 7% was obtained for a dNTP compound, and two
distinctive sets of peaks on its 1H and 31P NMR spectra clearly
indicated the existence of undesired 3′-triphosphate byproduct,
which is contradictory to the high regioselectivity at the 5′-OH
Received: May 21, 2013
Published: August 5, 2013
© 2013 American Chemical Society
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Table 1. Synthesis of Protected Nucleoside 5′-Phosphoropiperidates (11−19)
limited up to now. On the basis of the labile nature of
phosphoramidates at low pH condition, we hypothesized that a
proper acidic activator may weaken the P(V)−N bond in situ
and promote its specific coupling with pyrophosphate/
phosphate in the absence of H2O. Furthermore, precedent
reports16 of the catalytic effects of 1H-tetrazole and N-
methylimidazolium chloride on the synthesis of NDP-sugars,
lipid polyphosphate-sugars, and lipid diphosphate conjugates
strongly inspired our rationale for investigating this P(V)−N
activation approach for NPP synthesis. We report here a novel
and highly efficient method for the synthesis of both NTPs and
NDPs from protected nucleoside 5′-phosphoropiperidates
promoted by 4,5-dicyanoimidazole (DCI).
Moreover, the Cbz-protected nucleoside substrates (1−9)
could be efficiently prepared in multigram quantities and
excellent yields according to the reported procedures.17
Phosphitylation of 1−9 with benzyl N,N-diisopropylchloro-
phosphoramidite (10), acid-catalyzed hydrolysis, and oxidative
coupling with CCl4/Et3N/piperidine afforded 11−19 in
excellent yields ranging from 75% to 85% over three
consecutive steps. The preparation of 11−19 could be easily
scaled up to multigram quantities without decrease in yields.
Preparation of NTPs and NDPs. The conversion of 11−
19 to NPPs 23−40 was conducted in two consecutive steps
(Table 2). First, hydrogenation with Pd/C yielded fully
deprotected nucleoside 5′-phosphoropiperidates in 3 h. After
Pd/C was removed by filtration, addition of 6 equiv of DCI
(20) and 2 equiv of tris(tetra-n-butylammonium) hydrogen
pyrophosphate (21) or 8 equiv of DCI and 3 equiv of bis(tetra-
n-butylammonium) hydrogen phosphate (22) converted the
phosphoropiperidate intermediates to NPP products in 6 h at
20 °C. Ethanol precipitation followed by ion exchange
chromatography afforded 23−40 in excellent isolated yields
(68−81%) and high purity. It is noteworthy that this method
exhibits no sensitivity to either furanose or nucleobase moieties,
and the efficacy of coupling is equally high for both NTPs and
NDPs.
RESULTS AND DISCUSSION
■
Preparation of Protected Nucleoside 5′-Phosphoro-
piperidates. As shown in Table 1, the protected phosphor-
opiperidates of eight natural ribo- and 2′-deoxyribonucleosides
(11−14, 16−19) and an antiviral nucleoside ribavirin (15)
were rationally designed and synthesized. Compared to acetyl
and benzoyl groups conventionally used in NTP synthesis, the
combination of carboxybenzyl (Cbz) and benzyl ester enables
simultaneous and quantitative deprotection of both nucleoside
and phosphoramidate moieties by mild catalytic hydrogenation
prior to the coupling reaction.
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Table 2. A DCI-Promoted Methodology for Conversion of 11−19 to NPPs 23−40
Proof of Concept and Optimization of the P(V)−N
Activation Strategy for NTP Synthesis. To prove the
concept of P(V)−N activation strategy for NTP synthesis,
protected uridine 5′-phosphoromorpholidate (41) was first
synthesized as a prototype compound due to the precedents of
phosphoromorpholidate10,16 in the synthesis of NTPs and
NDP-sugars. 31P NMR tracing experiments showed that 41 was
quantitatively transformed into uridine 5′-phosphoromorpho-
lidate (42) by catalytic hydrogenation in 3 h. However, to our
surprise, addition of 1 equiv of pyrophosphate 21 according to
Moffatt’s method10b yielded no UTP (23) at all, even after 5
days. At this point, 4 equiv of 1H-tetrazole was added to the
unreacted 42 and 21 according to the method described by
Wong et al. for NDP-sugar synthesis.16a 42 gradually
disappeared in 72 h, and the desired triphosphate 23 was
obtained in 57% yield (Figure 1).
Inspired by this result, a series of weakly acidic activators
commonly used in phosphoramidite methodology (pKa (H2O)
= 4.9−9.2) was screened under the same conditions.15 When
DCI and pyridinium chloride were applied, significantly
shortened reaction time (6 h) and comparable yields (67%
for DCI and 56% for pyridinium chloride) were obtained. The
precipitation of pyrophosphate upon addition of HCl salts of
imidazole, N-methylimidazole, and DMAP precluded their
application as activators. Due to its excellent solubility in
organic solvents, nonhydroscopic nature, and commercial
availability, DCI was selected as the best activator for further
optimization.
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Figure 1. Proof of concept of the P(V)−N activation strategy for NTP synthesis (A) and 31P NMR tracing of the reaction with 1H-tetrazole as an
a
activator (B). (a) 41; (b) 42, 3 h after hydrogenation; (c) 120 h after addition of 21; (d) 72 h after addition of 1H-tetrazole. 31P NMR yield.
Analysis of the 31P NMR spectrum of DCI-catalyzed
coupling reaction with 1 equiv of 21 showed that Up4U was
generated because of the competition of triphosphate product
23. Thus, a higher excess of 21 was added to improve coupling
specificity. As shown in Figure 2A, 2 equiv of 21 completely
suppressed the side reaction and increased the yield of 23 to
88%. The only byproduct was a small amount of uridine 5′-
monophosphate (UMP) derived from the hydrolysis of 42.
Meanwhile, it was noticed that if the amount of 21 was
increased to 2 equiv but that of DCI remained unchanged (4
equiv), the reaction rate dropped remarkably (10 h). This was
possibly due to the neutralization of more DCI by additional
21. The data in Figure 2B showed that 6 equiv of DCI restored
the reaction time to 6 h, but further increasing the amount of
DCI exhibited no significant improvement.
Our design of the protected P(V)−N precursors enabled
diversity-oriented synthesis of a series of phosphoramidates
with different amino leaving groups (11, 41, 43−45) in good
yields (Figure 3A). As shown in Figure 3B, side by side
comparison of their couplings with 21 (2 equiv) in the presence
of DCI (6 equiv) showed that the reaction of phosphoropiper-
idate 11 with 21 (93% yield, 6 h) resulted in a yield higher than
that of the conventional phosphoromorpholidate 41 (88%
yield, 6 h), which was possibly due to the stronger protonation
capability of the N atom of piperidine (pKa (H2O) = 11.22)
than that of morpholine (pKa (H2O) = 8.36). Though Moffatt
and Khorana also found that phosphoropiperidate exhibited
reactivity better than that of phosphoromorpholidate, the very
low yield of phosphoropiperidate by the DCC coupling method
(20%) precluded its application as a P(V)−N precursor in their
method.10a It is worth noting that all three phosphoramidates
with a cyclic secondary amino leaving group (11, 41, and 45)
achieved high NTP conversion (>80% yield). In contrast, the
reactions of the ones with a noncyclic amino leaving group (43
and 44) were sluggish (<60%, 48 h) regardless of their pKas.
The effect of temperature was also evaluated for this reaction.
11 was first subjected to catalytic hydrogenation. Then, the
reactions of uridine 5′-phosphoropiperidate (46) with 21 (2
equiv) and DCI (6 equiv) were performed at 20 °C, 30 °C, 40
°C, and 50 °C, respectively. The consumption of 46 was much
faster at higher temperature (50 °C, 30 min), but the formation
of UMP (hydrolysis of 46), UDP (decomposition of 23), and
Up4U (competition reaction of 23 with 46) became
pronounced at the same time (Figure 4). The best compromise
between reaction time and yield was found at 20 °C.
Optimization of the P(V)−N Activation Strategy for
NDP Synthesis. On the basis of the newly established
phosphoropiperidate/DCI system for NTPs, its application in
NDP synthesis was explored. 31P NMR tracing experiments
determined that 8 equiv of DCI and 3 equiv of 22 were optimal
to maximize the reaction rate and eliminate the formation of
Up3U byproduct (Figure S1 and S2, Supporting Information).
Comparison of the reactions of 11 and 41 showed that the
difference in the reactivity of phosphoropiperidate (91% yield,
6 h) and phosphoromorpholidate (76% yield, 12 h) became
pronounced in the presence of less nucleophilic phosphate
(Figure S3, SI).
Mechanisms of DCI-Promoted NTP and NDP Syn-
thesis. As illustrated in the proposed reaction path for 23
(Figure 5A), protonation of the inactive monoionic phosphor-
amidate 46 (P−O−/P−NR2) generates the neutral phosphor-
amidate 47a (P−OH/P−NR2) and its zwitterionic tautomer
47b (P−O−/P−NH+R2).18 Based on Michielssens and
Lonnberg’s research on the hydrolysis of nucleoside phosphor-
̈
amidates,19 the subsequent P−O coupling with 21 is proposed
to proceed via an associative pathway with the more reactive
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Figure 3. Synthesis of protected uridine 5′-phosphoramidates 11, 41,
43−45 (A) and effect of the amino leaving groups on UTP (23)
a
formation (B). 31P NMR yield.
Figure 2. Effects of the amounts of 21 (A) and DCI (B) on UTP (23)
a
formation. 31P NMR yield.
CONCLUSION
■
In summary, our P(V)−N activation strategy presents a novel
and highly efficient approach for the synthesis of NTPs and
NDPs from nucleoside 5′-phosphoropiperidates promoted by
4,5-dicyanoimidazole (DCI). This novel method features (1)
easily accessible Cbz/Bn-protected nucleoside phosphoropiper-
idate precursors, which could be prepared in excellent yields
(75−85%) over three consecutive steps on multigram scale, (2)
one-pot removal of all protecting groups by catalytic hydro-
genation before coupling with pyrophosphate and phosphate,
(3) no sensitivity to nucleobase and furanose (18 ribo- and 2′-
deoxyribonucleoside examples, including ribavirin triphosphate
and diphosphate), (4) short reaction time and mild conditions
(6 h at 20 °C), and (5) excellent isolated yields (68−81%) and
high purity (as exemplified by the NMR spectra and HPLC of
all 18 examples in Supporting Information).
tautomer 47b. It was observed that if Et3N was not added in
hydrogenation, the resulting 47 was highly susceptible to
hydrolysis during filtration. In a control experiment, addition of
DCI to the mixture of 11 (P−OBn/P−NR2) and 21 exerted no
activation effect, indicating that zwitterionic 47b is essential for
P(V)−N bond activation.
In addition to acid catalysis, nucleophilic catalysis of the
conjugate base of DCI may also be involved.15c−f,20 But during
the reactions, no peaks other than those of the starting
materials (47, 21/47, 22) and product (23/32) were observed
on the 31P NMR spectra of the tracing experiments (Figure 5B
and 5C), which may possibly be ascribed to the overlap of the
peak of phosphorodicyanoimidazolide intermediate (48) with
that of the α-P of 23 and 32 (−11.5 ppm).21 Therefore, 2,6-
lutidinium chloride, which has a non-nucleophilic conjugate
base, was tested for the synthesis of UTP (23) and UDP (32),
respectively, to elucidate the role of the conjugate base of DCI.
While 2,6-lutidinium chloride-promoted synthesis of 23 (91%
yield, 6 h) was fairly close to that of DCI, the synthesis of 32
was dramatically affected (37%, 48 h) when 2,6-lutidinium
chloride was used instead of DCI. The above experimental
results indicated that nucleophilic catalysis of the conjugate
base of DCI is trivial to the coupling reaction with highly
nucleophilic pyrophosphate 21. In contrast, nucleophilic
catalysis via intermediate 48 is essential to promote the
formation of diphosphate 32 when less nucleophilic phosphate
22 was used (Figure 5A).
Moreover, 31P NMR tracing spectra revealed that the
conversion of fully deprotected nucleoside 5′-phosphoropiper-
idates to the desired NPPs in the presence of DCI were
exceptionally smooth and clean (e.g., 93% for UTP, 91% for
UDP). Our experimental results showed that phosphoropiper-
idate has superior reactivity toward the DCI-promoted P(V)−
N bond activation and subsequent P(V)−O bond coupling
reactions compared with that of the conventional phosphor-
omorpholidate and suggested that, other than acid catalysis,
nucleophilic catalysis of the conjugate base of DCI may also
play an important role in NPP synthesis, depending on the
distinctive nucleophilic nature of pyrophosphate and phos-
phate. The application of this method to improve the synthesis
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Figure 5. Proposed reaction mechanisms (A) and 31P NMR tracing
spectra of UTP (23) synthesis (B) and UDP (32) synthesis (C).
Benzyl N,N-Diisopropylchlorophosphoramidite (10). To a
solution of PCl3 (59.2 mL, 682 mmol) in CH2Cl2 (200 mL) at 0 °C
was added a solution of benzyl alcohol (14.2 mL, 136 mmol) in
CH2Cl2 (50 mL) dropwise over 3 h. The reaction was stirred at 20 °C
for 2 h. Concentration in vacuo afforded benzyl dichlorophosphite as
light yellow oil (28.5 g, 99%). To a solution of benzyl
dichlorophosphite (28.5 g, 134 mmol) in ether (120 mL) was added
a solution of diisopropylamine (19.3 mL, 136 mmol) and triethyl-
amine (19.0 mL, 136 mmol) in ether (50 mL) dropwise at −20 °C
over 3 h. The reaction was stirred overnight at 20 °C. The
triethylammonium chloride was removed by filtration, and the filtrate
was concentrated in vacuo. Vacuum distillation (90 °C, 8 mmHg) of
Figure 4. 31P NMR spectra of UTP (23) synthesis at 20 °C and 50 °C
(A) and effect of reaction temperature on UTP (23) formation (B).
a
31P NMR yield.
of NDP-sugars and dinucleoside polyphosphates is currently
underway and will be reported in due course.
EXPERIMENTAL SECTION
■
General Methods. Chemical reagents and solvents were obtained
from commercial suppliers. Natural ribonucleosides (A, C, G, U), 2′-
deoxyribonucleosides (dA, dC, dG, dT), and ribavirin (R) were
protected with Cbz groups as previously reported.1,2 Tris(tetra-n-
butylammonium) hydrogen pyrophosphate (21) and bis(tetra-n-
butylammonium) hydrogen phosphate (22) were prepared according
to known procedures.3,4 All reactions were performed under an
atmosphere of dry argon and monitored by analytical thin-layer
chromatography on plates coated with 0.25 mm silica gel 60 F254.
TLC plates were visualized by UV irradiation (254 nm). Flash column
chromatography employed silica gel (particle size 32−63 μm). Ion
exchange chromatography employed DEAE A-25 exchanger. All NMR
spectra were obtained with a 400 MHz instrument with chemical shifts
reported in parts per million (ppm, δ) referenced to CDCl3 or D2O.
IR spectra were recorded on a FT-IR spectrometer. Low-resolution
and high-resolution mass spectra were obtained with an ion trap and a
TOFQ mass spectrometer, respectively, and reported as m/z. HPLC
traces of 23 to 40 were recorded on an analytical instrument equipped
with a C18 analytical column (4.6 × 150 mm, 5 μm) [flow rate = 1.0
mL/min; linear gradient of 5% to 80% MeOH in TEAB buffer (10
mM, pH 8.5) over 20 min; UV detection at 230 and 254 nm].
1
the residue oil afforded 10 (28.4 g, 82%) as colorless oil; H NMR
(400 MHz, CDCl3): δ 7.37 (s, 4H), 7.33−7.32 (m, 5H), 4.99−4.91
(m, 2H), 3.90−3.84 (m, 2H), 1.35 (d, J = 6.0 Hz, 6H), 1.27 (d, J = 5.6
Hz, 6H) ppm; 13C NMR (100 MHz, CDCl3): δ 137.5, 128.5, 127.9,
127.3, 67.6, 46.1, 46.0, 24.1, 23.3 ppm; 31P NMR (162 MHz, CDCl3):
δ 181.57 ppm.
5′-O-[Benzyloxy(piperidin-1-yl)]phosphinyl-2′,3′-O-bis-
(benzyloxycarbonyl)uridine (11). General procedure for the
synthesis of protected nucleoside 5′-phosphoramidates (11−19, 41,
43−45): To a solution of Cbz-protected uridine (1, 500 mg, 0.98
mmol) and Et3N (0.42 mL, 3.0 mmol) in CH2Cl2 (20 mL) was added
a solution of benzyl N,N-diisopropylchlorophosphoramidite (10, 548
mg, 2.0 mmol) in CH2Cl2 (10 mL) dropwise. The reaction was stirred
at ambient temperature for 30 min and concentrated in vacuo. The
residue was coevaporated with CH3CN (10 mL × 2) and redissolved
in EtOAc (5 mL). Et3N·HCl salt was removed by filtration, and the
filtrate was concentrated to afford the crude phosphoramidite
intermediate. To a solution of the intermediate in CH3CN (20 mL)
were added 1H-tetrazole (140 mg, 2.0 mmol) and deionized H2O (0.2
mL). The reaction was stirred for 5 min and concentrated in vacuo.
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The residue was dissolved in CH2Cl2 (50 mL) and washed with HCl
aqueous solution (0.5 M, 30 mL) and deionized H2O (30 mL). The
organic phase was dried with anhydrous Na2SO4 and concentrated in
vacuo to give the crude H-phosphonate diester intermediate. To an
ice-cooled solution of piperidine (120 μL, 1.2 mmol), Et3N (0.05 mL),
and CCl4 (0.15 mL) in CH3CN (20 mL) were added a solution of the
intermediate in CH3CN (5 mL) dropwise. The reaction was stirred at
ambient temperature for 30 min and concentrated in vacuo. The
residue was dissolved in EtOAc (5 mL). Et3N·HCl salt was removed
by filtration, and the filtrate was concentrated to give the crude
product. Flash column chromatography (petroleum ether/EtOAc 1:2
to CH2Cl2/MeOH 50:1) on silica gel afforded 11 (615 mg, 84%) as a
5′-O-[Benzyloxy(piperidin-1-yl)]phosphinyl-2′,3′-O-bis-
(benzyloxycarbonyl)ribavirin (15). Starting from Cbz-protected
ribavirin (5, 500 mg), flash column chromatography afforded 15 (573
mg, 78%) as a white foam, mp: 44−45 °C; H NMR (400 MHz,
1
CDCl3): δ 8.41 (d, J = 8.0 Hz, 1H), 7.35−7.32 (m, 15H), 6.11−6.07
(m, 2H), 5.74 (m, 1H), 5.57 (m, 1H), 5.11 (m, 4H), 4.99 (m, 2H),
4.50 (s, 1H), 4.22 (m, 2H), 3.00 (m, 4H), 1.49 (m, 2H), 1.40 (m, 4H)
ppm; 13C NMR (100 MHz, CDCl3): δ 160.4, 157.7, 153.8, 153.5,
144.7, 136.3, 134.5, 128.8, 128.6, 128.5, 128.4, 128.3, 127.9, 127.8,
89.6, 81.4, 77.3, 73.6, 70.7, 70.5, 68.1, 64.4, 45.3, 25.9, 24.2 ppm; 31P
NMR (162 MHz, CDCl3): δ 9.11, 8.84 ppm; IR (KBr): νmax 3459,
3067, 2961, 2857, 2377, 2316, 1760, 1696, 1608, 1500, 1460, 1141,
1112, 1065, 1008, 974, 910, 828, 783, 746, 699, 586 cm−1; HRMS (ESI
+): m/z calcd for C36H41N5O11P [M + H]+ 750.2535; found 750.2539.
5′-O-[Benzyloxy(piperidin-1-yl)]phosphinyl-2′-deoxy-3′-O-
(benzyloxycarbonyl)thymidine (16). Starting from Cbz-protected
thymidine (6, 400 mg), flash column chromatography afforded 16
(552 mg, 85%) as a white foam, mp: 39−40 °C; 1H NMR (400 MHz,
CDCl3): δ 9.02 (s, 1H), 7.48 (s, 1H), 7.38−7.35 (m, 10H), 6.37−6.29
(m, 1H), 5.22 (d, J = 5.9 Hz, 1H), 5.17 (s, 2H), 5.09−4.95 (m, 2H),
4.27−4.20 (m, 3H), 3.05 (m, 4H), 2.50, 2.43 (dd, J1 = 6.9 Hz, J2 = 14.4
Hz, 1H), 2.07−1.92 (m, 1H), 1.86, 1.83 (s, 3H), 1.54 (m, 2H), 1.45
(m, 4H) ppm; 13C NMR (100 MHz, CDCl3): δ 163.6, 154.3, 150.2,
136.1, 134.9, 134.6, 128.8, 128.7, 128.6, 128.5, 128.4, 128.0, 127.8,
111.5, 111.4, 84.9, 84.6, 82.8, 78.2, 78.0, 77.2, 70.2, 68.1, 65.7, 45.4,
45.3, 37.7, 37.6, 26.0, 24.2, 12.3 ppm; 31P NMR (162 MHz, CDCl3): δ
9.49, 9.24 ppm; IR (KBr): νmax 3425, 3067, 2939, 2852, 2377, 2348,
2317, 2251, 1749, 1691, 1543, 1460, 1403, 1262, 1123, 1058, 1006,
964, 912, 738, 697 cm−1; HRMS (ESI+): m/z calcd for C30H37N3O9P
[M + H]+ 614.2262; found 614.2269.
1
white foam, mp: 44−45 °C; H NMR (400 MHz, CDCl3): δ 9.02 (s,
1H), 7.59, 7.54 (d, J = 8.2 Hz, 1H), 7.37−7.33 (m, 15H), 6.18, 6.13 (d,
J = 6.0 Hz, 1H), 5.64, 5.49 (d, J = 8.0 Hz, 1H), 5.36 (s, 1H), 5.23 (m,
1H), 5.14−4.96 (m, 6H), 4.36 (s, 1H), 4.27−4.17 (m, 2H), 3.06 (s,
4H), 1.53 (s, 2H), 1.45 (s, 4H) ppm; 13C NMR (100 MHz, CDCl3): δ
161.8, 152.9, 152.8, 149.0, 138.5, 135.4, 135.0, 133.5, 127.7, 127.6,
127.5, 127.4, 126.9, 102.3, 102.1, 85.5, 85.2, 79.6, 79.5, 76.2, 74.9, 74.8,
72.4, 69.5, 69.3, 67.2, 63.8, 63.7, 44.4, 25.0, 23.2 ppm; 31P NMR (162
MHz, CDCl3): δ 9.37, 8.96 ppm; IR (KBr): νmax 3441, 2933, 2854,
2354, 1759, 1693, 1636, 1497, 1458, 1386, 1264, 1118, 1057, 1015,
966, 905, 812, 782 cm−1; HRMS (ESI+): m/z calcd for C37H41N3O12P
[M + H]+ 750.2422; found 750.2411.
5′-O-[Benzyloxy(piperidin-1-yl)]phosphinyl-N4-2′,3′-O-tris-
(benzyloxycarbonyl)cytidine (12). Starting from Cbz-protected
cytidine (2, 600 mg), flash column chromatography afforded 12 (649
1
mg, 80%) as a white foam, mp: 46−47 °C; H NMR (400 MHz,
CDCl3): δ 8.05, 8.01 (d, J = 7.8 Hz, 1H), 7.76 (s, 1H), 7.39−7.31 (m,
20H), 7.18 (t, J = 8.0 Hz, 1H), 6.16, 6.11 (d, J = 2.8 Hz, 1H), 5.38−
5.33 (m, 2H), 5.22−4.95 (m, 8H), 4.42−4.22 (m, 3H), 3.04 (m, 4H),
1.52 (m, 2H), 1.43 (m, 4H) ppm; 13C NMR (100 MHz, CDCl3): δ
162.5, 154.6, 153.8, 152.0, 144.1, 136.1, 134.9, 134.6, 128.7, 128.6,
128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 95.2, 89.0, 88.8,
80.1, 77.2, 76.8, 72.7, 70.4, 68.2, 68.0, 64.9, 45.4, 26.0, 24.2 ppm; 31P
NMR (162 MHz, CDCl3): δ 9.50, 9.15 ppm; IR (KBr): νmax 3424,
2939, 2854, 2349, 2317, 1755, 1675, 1629, 1558, 1382, 1266, 1233,
1121, 1058, 966, 908, 844, 785, 742, 697 cm−1; HRMS (ESI+): m/z
calcd for C45H48N4O13P [M + H]+ 883.2950; found 883.2955.
5′-O-[Benzyloxy(piperidin-1-yl)]phosphinyl-2′,3′-O-bis-
(benzyloxycarbonyl)adenosine (13). Starting from Cbz-protected
adenosine (3, 500 mg), flash column chromatography afforded 13
(589 mg, 82%) as a white foam, mp: 46−47 °C; 1H NMR (400 MHz,
CDCl3): δ 8.28, 8.26 (s, 1H), 8.02, 7.99 (s, 1H), 7.40−7.31 (m, 10H),
6.23, 6.19 (d, J = 5.2 Hz, 1H), 5.98−5.94 (m, 3H), 5.72−5.68 (m,
1H), 5.13−5.00 (m, 6H), 4.47 (s, 1H), 4.37−4.26 (m, 2H), 3.02 (s,
4H), 1.48 (s, 2H), 1.39 (s, 4H) ppm; 13C NMR (100 MHz, CDCl3) δ
155.5, 153.9, 153.7, 153.4, 149.9, 149.8, 139.2, 139.1, 136.4, 136.3,
134.6, 134.5, 128.7, 128.6, 128.5, 128.4, 128.3, 127.9, 127.8, 120.1,
120.0, 86.0, 85.6, 80.8, 77.2, 76.1, 76.0, 73.8, 70.5, 70.4, 68.1, 68.0,
64.7, 64.4, 45.3, 25.9, 24.2 ppm; 31P NMR (162 MHz, CDCl3): δ 9.27,
9.11 ppm; IR (KBr): νmax 3457, 3207, 3035, 2937, 2853, 2377, 2316,
1759, 1644, 1503, 1460, 1370, 1278, 1241, 1059, 1006, 965, 905, 845,
741 cm−1; HRMS (ESI+): m/z calcd for C38H42N6O10P [M + H]+
773.2695; found 773.2703.
5′-O-[Benzyloxy(piperidin-1-yl)]phosphinyl-2′-deoxy-N4-3′-
O-bis(benzyloxycarbonyl)cytidine (17). Starting from Cbz-pro-
tected 2′-deoxycytidine (7, 500 mg), flash column chromatography
afforded 17 (599 mg, 81%) as a white foam, mp: 45−46 °C; 1H NMR
(400 MHz, CDCl3): δ 8.13 (m, 2H), 7.36−7.34 (m, 14H), 7.17 (m,
1H), 6.23 (m, 1H), 5.19−5.15 (m, 5H), 5.02−4.92 (m, 2H), 4.36 (s,
1H), 4.24 (m, 2H), 3.00 (m, 4H), 2.83 (m, 1H), 1.96 (m, 1H), 1.50
(m, 2H), 1.40 (m, 4H) ppm; 13C NMR (100 MHz, CDCl3): δ 162.4,
154.7, 154.3, 152.3, 143.8, 136.1, 135.1, 134.7, 128.8, 128.7, 128.5,
128.4, 128.3, 127.9, 127.8, 94.9, 87.3, 83.6, 78.1, 70.1, 68.1, 67.9, 65.5,
45.3, 39.2, 26.0, 24.2 ppm; 31P NMR (162 MHz, CDCl3): δ 9.35, 9.14
ppm; IR (KBr): νmax 3459, 1747, 1660, 1562, 1501, 1450, 1392, 1331,
1258, 1120, 966, 742, 697 cm−1; HRMS (ESI+): m/z calcd for
C37H42N4O10P [M + H]+ 733.2633; found 733.2621.
5′-O-[Benzyloxy(piperidin-1-yl)]phosphinyl-2′-deoxy-3′-O-
(benzyloxycarbonyl)adenosine (18). Starting from 2′-deoxyade-
nosine (8, 400 mg), flash column chromatography on silica gel
afforded 18 (517 mg, 80%) as a white foam, mp: 46−47 °C; 1H NMR
(400 MHz, CDCl3): δ 8.28 (s, 1H), 8.07 (d, J = 4.0 Hz, 1H), 7.34−
7.29 (m, 10H), 6.71 (s, 2H), 6.42 (m, 1H), 5.43, 5.36 (d, J = 4.0 Hz,
1H), 5.15 (s, 2H), 5.13−4.93 (m, 2H), 4.36 (m, 1H), 4.26−4.21 (m,
2H), 2.98 (m, 4H), 2.89−2.58 (m, 2H), 1.44 (m, 2H), 1.36 (m, 4H)
ppm; 13C NMR (100 MHz, CDCl3): δ 155.7, 154.3, 153.2, 149.7,
138.7, 136.3, 134.7, 128.8, 128.7, 128.6, 128.5, 128.3, 127.9, 127.8,
119.9, 84.6, 84.1, 83.2, 78.4, 70.2, 68.0, 65.4, 45.3, 37.8, 26.0, 24.2
ppm; 31P NMR (162 MHz, CDCl3): δ 9.35 ppm; IR (KBr): νmax 3337,
2924, 2854, 1747, 1648, 1595, 1463, 1380, 1255, 1064, 741, 697, 651,
567 cm−1; HRMS (ESI+): m/z calcd for C30H36N6O7P [M + H]+
623.2378; found 623.2388.
5′-O-[Benzyloxy(piperidin-1-yl)]phosphinyl-2′,3′-O-bis-
(benzyloxycarbonyl)guanosine (14). Starting from Cbz-protected
guanosine (4, 500 mg), flash column chromatography afforded 14
(552 mg, 77%) as a white foam, mp: 54−55 °C; 1H NMR (400 MHz,
CDCl3): δ 11.86 (d, J = 24.0 Hz, 1H), 7.33−7.13 (m, 15H), 6.58 (m,
1H), 6.33−6.26 (m, 2H), 6.04−6.00 (m, 2H), 5.18−5.10 (m, 4H),
4.99 (d, J = 12.0 Hz, 2H), 4.64−4.42 (m, 2H), 4.24, 4.22 (m, 1H),
2.99 (m, 4H), 1.45 (m, 2H), 1.39 (m, 4H) ppm; 13C NMR (100 MHz,
CDCl3): δ 157.9, 153.9, 153.7, 153.6, 152.2, 136.6, 134.6, 134.5, 133.7,
128.6, 128.5, 128.3, 128.2, 127.8, 115.9, 87.0, 86.6, 80.1, 75.3, 74.8,
74.1, 73.6, 70.5, 68.1, 64.0, 45.3, 25.9, 24.2 ppm; 31P NMR (162 MHz,
CDCl3): δ 9.70 ppm; IR (KBr): νmax 3324, 3169, 2940, 2854, 1760,
1691, 1638, 1590, 1278, 1002, 965, 843, 783, 697 cm−1; HRMS (ESI
+): m/z calcd for C38H42N6O11P [M + H]+ 789.2644; found 789.2635.
5′-O-[Benzyloxy(piperidin-1-yl)]phosphinyl-2′-deoxy-3′-O-
(benzyloxycarbonyl)guanosine (19). Starting from Cbz-protected
2′-deoxyguanosine (9, 400 mg), flash column chromatography
1
afforded 19 (479 mg, 75%) as a white foam, mp: 91−92 °C; H
NMR (400 MHz, CDCl3) δ 7.76 (d, J = 12.0 Hz, 1H), 7.37−7.29 (m,
10H), 6.59−6.52 (m, 2H), 6.19 (m, 1H), 5.47, 5.36 (d, J = 8.0, 1H),
5.17 (m, 2H), 5.01 (m, 2H), 4.56−4.31 (m, 2H), 4.24, 4.16 (m, 1H),
3.01 (m, 4H), 2.79−2.46 (m, 3H), 1.48 (m, 2H), 1.40 (m, 4H) ppm;
13C NMR (100 MHz, CDCl3) δ 158.9, 154.2, 153.9, 151.3, 136.3,
135.8, 134.8, 128.7, 128.6, 128.5, 128.4, 127.8, 117.6, 84.8, 84.1, 82.9,
78.5, 70.1, 68.1, 65.2, 45.3, 36.7, 25.9, 24.2 ppm; 31P NMR (162 MHz,
8423
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Article
CDCl3) δ 9.55 ppm; IR (KBr): νmax 3426, 3197, 2940, 2864, 1748,
1690, 1638, 1535, 1383, 1264, 1171, 1069, 1001, 744, 698 cm−1;
HRMS (ESI+): m/z calcd for C30H36N6O8P [M + H]+ 639.2327;
found 639.2316.
Uridine-5′-triphosphate, Tetrasodium Salt (23). General
procedure for the synthesis of NTPs (23−31): To a solution of 11
(50 mg, 0.067 mmol) and Et3N (9 μL, 0.067 mmol) in DMF (1.5 mL)
was added 5% Pd/C (5 mg). The reaction was stirred under a
hydrogen atmosphere at 20 °C for 3 h. The catalyst was removed with
a syringe filter (0.45 μm pore size) and washed with DMF (0.2 mL ×
2) under an atmosphere of argon. To the combined DMF solution
were added tris(tetra-n-butylammonium) hydrogen pyrophosphate
(21, 123 mg, 0.136 mmol) and 4,5-dicyanoimidazole (DCI, 48 mg,
0.41 mmol). The reaction was stirred at 20 °C for 6 h and
concentrated in vacuo. The residue was dissolved in NaOAc aqueous
solution (10 M, 0.5 mL), and EtOH (50 mL) was added. The resulting
white precipitate was collected by centrifuge. The crude product was
dissolved in deionized H2O (0.5 mL) and loaded on a DEAE
Sephadex A-25 ion exchange column (1.6 × 25 cm). Elution with
NH4HCO3 buffer (linear gradient 0.2 to 0.6 M), combination of
appropriate fractions, and lyophilization afforded the product in
ammonium salt form. Passage of the solution of the ammonium salt in
deionized H2O through a bed of Dowex 50W-X8 ion-exchange resin
(Na+ form) and lyophilization afforded 23 (31 mg, 81%) as the
tetrasodium salt, a white solid. 1H NMR (400 MHz, D2O): δ 7.95 (d, J
= 8.1 Hz, 1H), 5.97−5.94 (m, 2H), 4.41−4.37 (m, 2H), 4.26−4.23 (m,
3H) ppm; 13C NMR (100 MHz, D2O): δ 165.3, 151.0, 140.8, 101.8,
87.4, 82.5, 72.8, 68.7, 64.0 ppm; 31P NMR (162 MHz, D2O): δ −7.89
5′-O-[Benzyloxy(morpholin-4-yl)]phosphinyl-2′,3′-O-bis-
(benzyloxycarbonyl)uridine (41). Starting from Cbz-protected
uridine (1, 200 mg), flash column chromatography afforded 41 (240
1
mg, 82%) as a white foam, mp: 54−55 °C; H NMR (400 MHz,
CDCl3): δ 7.40, 7.32 (d, J = 8.2 Hz, 1H), 7.29−7.16 (m, 15H), 6.00,
5.93 (d, J = 5.7 Hz, 1H), 5.56, 5.44 (d, J = 8.1 Hz, 1H), 5.29, 5.18 (q, J
= 4.0 Hz, 1H), 5.04−4.93 (m, 6H), 4.25−4.10 (m, 3H), 3.48−3.40 (m,
4H), 3.01−2.93 (m, 4H) ppm; 13C NMR (100 MHz, CDCl3): δ 162.7,
153.9, 153.8, 150.0, 139.8, 139.7, 135.8, 134.5, 134.4, 128.8, 128.7,
128.6, 128.5, 128.4, 128.2, 128.1, 103.2, 87.5, 86.9, 80.4, 80.3, 77.3,
75.8, 75.7, 73.2, 70.6, 70.5, 68.8, 68.7, 68.6, 66.9, 66.8, 64.9, 44.5 ppm;
31P NMR (162 MHz, CDCl3): δ 8.19, 7.79 ppm; IR (KBr): νmax 3445,
2967, 2857, 2347, 2063, 1759, 1692, 1636, 1499, 1459, 1386, 1264,
1112, 1012, 974, 909, 815, 782, 741, 698, 608 cm−1; HRMS (ESI+):
m/z calcd for C36H39N3O13P [M + H]+ 752.2215; found 752.2227.
5′-O-[Benzyloxy(cyclohexylamino)]phosphinyl-2′,3′-O-bis-
(benzyloxycarbonyl)uridine (43). Starting from Cbz-protected
uridine (1, 200 mg), flash column chromatography afforded 43 (202
1
mg, 68%) as a white foam, mp: 49−51 °C; H NMR (400 MHz,
CDCl3): δ 7.54, 7.48 (d, J = 8.2 Hz, 1H), 7.37−7.33 (m, 15H), 6.14,
6.09 (d, J = 7.8 Hz, 1H), 5.64, 5.49 (d, J = 8.2 Hz, 1H), 5.38 (t, J = 4.4
Hz, 1H), 5.25 (t, J = 5.7 Hz, 1H), 5.14−5.00 (m, 6H), 4.34−4.21 (m,
3H), 2.97−2.88 (m, 2H), 1.64−1.86 (m, 6H), 1.25−1.12 (m, 4H)
ppm; 13C NMR (100 MHz, CDCl3): δ 162.4, 153.9, 153.8, 149.9,
139.6, 136.1, 136.0, 134.5, 128.8, 128.7, 128.6, 128.5, 128.4, 127.9,
127.8, 103.3, 103.2, 86.8, 86.4, 80.6, 80.5, 77.2, 75.8, 75.7, 73.4, 73.3,
70.6, 70.5, 68.4, 68.3, 68.2, 64.8, 64.7, 50.9, 50.8, 35.8, 35.7, 25.2, 24.9
ppm; 31P NMR (162 MHz, CDCl3): δ 8.93, 8.50 ppm; IR (KBr): νmax
3734, 2932, 2855, 2377, 2316, 1758, 1695, 1500, 1457, 1265, 1013,
973, 910, 866, 815, 740, 698 cm−1; HRMS (ESI+): m/z calcd for
C38H43N3O12P [M + H]+ 764.2579; found 764.2586.
(d, Jp,p = 17.4 Hz, 1P), −11.1 (d, Jp,p = 17.4 Hz, 1P), −22.0 (t, Jp,p
=
17.4 Hz, 1P) ppm; LRMS (ESI−): m/z calcd for C9H11N2O15P3 [M −
H]− 483.0; found 483.1.
Cytidine-5′-triphosphate, Tetrasodium Salt (24). Starting
from 12 (60 mg), CTP (27 mg, 70%) was obtained as the tetrasodium
1
salt, a white solid. H NMR (400 MHz, D2O): δ 7.95 (d, J = 8.0 Hz,
1H), 6.10 (d, J = 8.0 Hz, 1H), 5.94 (d, J = 4.0 Hz, 1H), 4.33 (m, 1H,),
4.27 (m, 1H), 4.21 (m, 3H) ppm; 13C NMR (100 MHz, D2O): δ
165.3, 156.7, 141.7, 96.5, 89.0, 82.9, 74.2, 69.2, 64.7 ppm; 31P NMR
(D2O, 162 MHz): δ −10.55 (d, JP,P = 19.4 Hz, 1P), −11.31 (d, JP,P
=
19.4 Hz, 1P), −22.94 (t, JP,P = 19.4 Hz, 1P) ppm; LRMS (ESI−) m/z
calcd for C9H15N3O14P3 [M − H]− 482.0; found 482.1.
5′-O-{Benzyloxy[(methoxycarbonyl)methylamino]}-
phosphinyl-2′,3′-O-bis(benzyloxycarbonyl)uridine (44). Start-
ing from Cbz-protected uridine (1, 200 mg), flash column
chromatography afforded 44 (217 mg, 74%) as a white foam, mp:
48−50 °C; 1H NMR (400 MHz, CDCl3): δ 9.45 (d, J = 8.8 Hz, 1H),
7.51, 7.48 (d, J = 8.2 Hz, 1H), 7.38−7.32 (m, 15H), 6.10, 6.06 (d, J =
5.7 Hz, 1H), 5.65, 5.53 (d, J = 8.1 Hz, 1H), 5.42−5.30 (m, 2H), 5.13−
5.04 (m, 6H), 4.39−4.02 (m, 3H), 3.92, 3.82 (m, 1H), 3.72−3.63 (m,
5H) ppm; 13C NMR (100 MHz, CDCl3): δ 171.4, 171.3, 162.9, 162.8,
153.9, 153.8, 153.7, 150.2, 150.1, 140.0, 139.9, 135.8, 135.7, 134.5,
134.4, 128.7, 128.6, 128.4, 128.3, 128.0, 127.9, 103.4, 103.2, 87.1, 86.8,
80.3, 80.2, 77.2, 75.7, 75.5, 73.1, 73.0, 70.5, 70.4, 68.6, 68.5, 64.9, 64.8,
64.7, 52.4, 42.7, 42.6 ppm; 31P NMR (162 MHz, CDCl3): δ 8.73, 8.42
ppm; IR (KBr): νmax 3210, 2956, 2378, 2349, 2316, 1756, 1694, 1498,
1459, 1694, 1459, 1389, 1265, 1007, 906, 816, 783, 740, 697 cm−1;
HRMS (ESI+): m/z calcd for C35H37N3O14P [M + H]+ 754.2008;
found 754.2017.
Adenosine-5′-triphosphate, Tetrasodium Salt (25). Starting
from 13 (52 mg), ATP (30 mg, 75%) was obtained as the tetrasodium
salt, a white solid. 1H NMR (400 MHz, D2O): δ 8.45 (s, 1H), 8.17 (s,
1H), 6.04 (d, 1H), 4.68 (t, 1H,), 4.50 (m, 1H), 4.34 (m, 1H), 4.20 (m,
2H) ppm; 13C NMR (100 MHz, D2O): δ 154.1, 150.8, 148.9, 140.6,
118.6, 87.1, 84.1, 74.5, 70.4, 65.3 ppm; 31P NMR (162 MHz, D2O): δ
−6.26 (d, JP,P = 19.4 Hz, 1P), −11.00 (d, JP,P = 19.4 Hz, 1P), −21.80
(t, JP,P = 19.4 Hz, 1P) ppm; LRMS (ESI−) m/z calcd for
C10H15N5O13P3 [M − H]− 506.0; found 506.1.
Guanosine-5′-triphosphate, Tetrasodium Salt (26). Starting
from 14 (53 mg), GTP (30 mg, 73%) was obtained as the tetrasodium
salt, a white solid. 1H NMR (400 MHz, D2O): δ 8.01 (s, 1H), 5.81 (d,
J = 4.0 Hz, 1H), 4.63 (t, J = 4.0 Hz, 1H), 4.48 (dd, 1H), 4.27 (s, 1H),
4.16 (m, 2H) ppm; 13C NMR (100 MHz, D2O): δ 158.8, 153.8, 151.6,
137.5, 116.1, 86.8, 83.8, 73.9, 70.2, 65.2 ppm; 31P NMR (162 MHz,
D2O): δ −8.98 (d, JP,P = 17.8 Hz, 1P), −11.13 (d, JP,P = 17.8 Hz, 1P),
−22.54 (t, JP,P = 17.8 Hz, 1P) ppm; LRMS (ESI−) m/z calcd for
C10H15N5O14P3 [M − H]− 522.0; found 522.2.
5′-O-{Benzyloxy[(2S)-methoxycarbonyl-pyrrolidin-1-yl]}-
phosphinyl-2′,3′-O-bis(benzyloxycarbonyl)uridine (45). Start-
ing from Cbz-protected uridine (1, 200 mg), flash column
chromatography afforded 45 (195 mg, 63%) as a white foam, mp:
Ribavirin-5′-triphosphate, Tetrasodium Salt (27). Starting
from 15 (50 mg), RTP (28 mg, 73%) was obtained as the tetrasodium
salt, a white solid. 1H NMR (400 MHz, D2O): δ 8.74 (s, 1H), 5.96 (d,
J = 4.0 Hz, 1H), 4.62 (s, 1H), 4.51 (s, 1H), 4.30 (s, 1H), 4.12 (m, 2H)
ppm; 13C NMR (100 MHz, D2O): δ 162.9, 156.4, 145.9, 92.0, 84.2,
1
46−48 °C; H NMR (400 MHz, CDCl3): δ 8.66 (s, 1H), 7.76, 7.55
(d, J = 8.2 Hz, 1H), 7.43−7.33 (m, 15H), 6.27, 6.14 (d, J = 6.6 Hz,
1H), 5.70, 5.44 (d, J = 8.2 Hz, 1H), 5.41, 5.36 (m, 1H), 5.29, 5.23 (t, J
= 6.2 Hz, 1H), 5.18−5.00 (m, 6H), 4.44−4.21 (m, 4H), 3.71, 3.68 (s,
3H), 3.31−2.99 (m, 2H), 2.18−1.62 (m, 5H) ppm; 13C NMR (100
MHz, CDCl3): δ 174.2, 162.6, 162.4, 154.0, 153.8, 150.1, 150.0, 139.9,
139.6, 136.2, 136.1, 136.0, 134.6, 134.5, 128.7, 128.6, 128.4, 128.2,
128.1, 103.4, 103.2, 86.2, 85.4, 80.8, 80.7, 77.2, 75.7, 75.6, 73.7, 73.5,
70.6, 70.5, 70.4, 68.4, 64.9, 60.3, 60.2, 52.3, 47.2, 46.7, 31.3, 31.0, 25.1
ppm; 31P NMR (162 MHz, CDCl3): δ 6.72, 5.85 ppm; IR (KBr): νmax
3425, 3065, 3036, 2956, 2883, 2378, 2349, 2316, 1755, 1695, 1459,
1395, 1265, 1120, 1003, 783, 744, 698 cm−1; HRMS (ESI+): m/z
calcd for C38H41N3O14P [M + H]+ 794.2321; found 794.2310.
74.8, 70.2, 65.3 ppm; 31P NMR (162 MHz, D2O): δ −8.49 (d, JP,P
=
19.4 Hz, 1P), −9.08 (d, JP,P = 19.4 Hz, 1P), −20.68 (t, JP,P = 19.4 Hz,
1P) ppm; LRMS (ESI−) m/z calcd for C8H14N4O14P3 [M − H]−
483.0; found 483.1.
Thymidine-5′-triphosphate, Tetrasodium Salt (28). Starting
from 16 (40 mg), dTTP (29 mg, 78%) was obtained as the
1
tetrasodium salt, a white solid. H NMR (400 MHz, D2O): δ 7.72 (s,
1H), 6.32 (t, J = 8.0 Hz, 1H), 4.64 (d, 1H), 4.17 (m, 3H), 2.36−2.31
(m, 2H), 1.89 (s, 3H) ppm; 13C NMR (100 MHz, D2O): δ 166.6,
151.8, 137.3, 111.8, 85.4, 84.9, 70.8, 65.4, 38.5, 11.6 ppm; 31P NMR
(162 MHz, D2O): δ −8.54 (d, JP,P = 17.8 Hz, 1P), −11.38 (d, JP,P
=
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17.8 Hz, 1P), −22.58 (t, JP,P = 17.8 Hz, 1P) ppm; LRMS (ESI−) m/z
calcd for C10H16N2O14P3 [M − H]− 481.0; found 481.1.
4.14 (m, 2H) ppm; 13C NMR (100 MHz, D2O): δ 155.6, 152.8, 149.1,
140.0, 118.7, 87.1, 84.0, 74.5, 70.2, 64.9 ppm; 31P NMR (162 MHz,
D2O): δ −8.52 (d, JP,P = 19.4 Hz, 1P), −10.88 (d, JP,P = 19.4 Hz, 1P)
ppm; LRMS (ESI−) m/z calcd for C10H14N5O10P2 [M − H]− 426.0;
found 426.2.
Guanosine-5′-diphosphate, Trisodium Salt (35). Starting from
14 (53 mg), GDP (25 mg, 73%) was obtained as the trisodium salt, a
white solid. 1H NMR (400 MHz, D2O): δ 8.01 (s, 1H), 5.82 (d, J = 8.0
Hz, 1H), 4.64 (m, 1H), 4.45 (t, J = 4.0 Hz, 1H), 4.27 (s, 1H), 4.14 (m,
2H) ppm; 13C NMR (100 MHz, D2O): δ 158.8, 153.9, 151.6, 137.5,
116.1, 86.9, 83.8, 73.8, 70.3, 65.1 ppm; 31P NMR (162 MHz, D2O): δ
−10.53 (d, 1P), −11.32 (d, 1P) ppm; LRMS (ESI−): m/z calcd for
C10H14N5O11P2 [M − H]− 442.0; found 442.2.
Ribavirin-5′-diphosphate, Trisodium Salt (36). Starting from
15 (50 mg), RDP (22 mg, 70%) was obtained as the trisodium salt, a
white solid. 1H NMR (400 MHz, D2O): δ 8.75 (s, 1H), 5.97 (s, 1H),
4.64 (dd, 1H), 4.51 (d, 1H), 4.31 (s, 1H), 4.13 (m, 2H) ppm; 13C
NMR (100 MHz, D2O): δ 162.9, 156.4, 145.8, 92.1, 84.0, 74.7, 70.0,
64.8 ppm; 31P NMR (162 MHz, D2O): δ −9.30 (d, 1P), −10.78 (d,
1P) ppm; LRMS (ESI−): m/z calcd for C8H13N4O11P2 [M − H]−
403.0; found 403.2.
2′-Deoxycytidine-5′-triphosphate, Tetrasodium Salt (29).
Starting from 17 (49 mg), dCTP (26 mg, 70%) was obtained as the
tetrasodium salt, a white solid. 1H NMR (400 MHz, D2O): δ 7.89 (d, J
= 8.0 Hz, 1H), 6.24 (m, 1H), 6.06 (d, J = 8.0 Hz, 1H), 4.54 (s, 1H),
4.13 (s, 3H), 2.35−2.31 (m, 1H), 2.26−2.21 (m, 1H) ppm; 13C NMR
(100 MHz, D2O): δ 165.7, 157.1, 141,7, 96.5, 85.9, 85.5, 70.5, 65.2,
39.3 ppm; 31P NMR (162 MHz, D2O): δ −9.26 (d, JP,P = 17.8 Hz,
1P), −10.95 (d, JP,P = 17.8 Hz, 1P), −22.32 (t, JP,P = 17.8 Hz, 1P)
ppm; LRMS (ESI−) m/z calcd for C9H15N3O13P3 [M − H]− 466.0;
found 466.1.
2′-Deoxyadenosine-5′-triphosphate, Tetrasodium Salt (30).
Starting from 18 (42 mg), dATP (29 mg, 75%) was obtained as the
1
tetrasodium salt, a white solid. H NMR (400 MHz, D2O): δ 8.42 (s,
1H), 8.15 (s, 1H), 6.44 (t, J = 4.0 Hz, 1H), 4.71 (s, 1H), 4.24 (s, 1H),
4.20−4.08 (m, 2H), 2.80−2.73 (m, 1H), 2.55−2.52 (m, 1H) ppm; 13C
NMR (100 MHz, D2O): δ 155.6, 152.7, 148.8, 140.1, 118.7, 85.9, 83.7,
70.9, 65.3, 39.1 ppm; 31P NMR (162 MHz, D2O): δ −7.78 (d, JP,P
=
19.4 Hz, 1P), −10.61 (d, JP,P = 19.4 Hz, 1P), −21.89 (t, JP,P = 19.4 Hz,
1P) ppm; LRMS (ESI−) m/z calcd for C10H15N5O12P3 [M − H]−
490.0; found 490.1.
Thymidine-5′-diphosphate, Trisodium Salt (37). Starting from
16 (40 mg), dTDP (23 mg, 76%) was obtained as the trisodium salt, a
2′-Deoxyguanosine-5′-triphosphate, Tetrasodium Salt (31).
1
white solid. H NMR (400 MHz, D2O): δ 7.66 (s, 1H), 6.26 (t, 1H),
Starting from 19 (43 mg), dGTP (29 mg, 72%) was obtained as the
4.56 (s, 1H), 4.08 (m, 3H), 2.28 (m, 2H), 1.84 (s, 3H) ppm; 13C
1
tetrasodium salt, a white solid. H NMR (400 MHz, D2O): δ 7.97 (s,
NMR (100 MHz, D2O): δ 166.6, 151.8, 137.5, 111.8, 85.5, 85.0, 70.8,
1H), 6.15 (d, 1H), 4.66 (s, 1H), 4.14−4.04 (m, 3H), 2.65 (m, 1H),
2.42−2.36 (m, 1H); 13C NMR (100 MHz, D2O): δ 159.0, 153.9,
151.4, 137.8, 116.3, 85.7, 83.6, 71.1, 65.4, 38.5; 31P NMR (162 MHz,
D2O): δ −8.33 (d, JP,P = 19.4 Hz, 1P), −11.02 (d, JP,P = 19.4 Hz, 1P),
−22.32 (t, JP,P = 19.4 Hz, 1P); LRMS (ESI−): m/z calcd for
C10H15N5O13P3 [M − H]− 506.0; found 506.1.
65.1, 38.5, 11.7 ppm; 31P NMR (162 MHz, D2O): δ −9.04 (d, JP,P
=
21.1 Hz, 1P), −10.91 (d, JP,P = 21.1 Hz, 1P) ppm; LRMS (ESI−): m/z
calcd for C10H15N2O11P2 [M − H]− 401.0; found 401.2.
2′-Deoxycytidine-5′-diphosphate, Trisodium Salt (38). Start-
ing from 17 (49 mg), dCDP (21 mg, 68%) was obtained as the
trisodium salt, a white solid. 1H NMR (400 MHz, D2O): δ 7.90 (d, J =
4.0 Hz, 1H), 6.22 (dd, 1H), 6.03 (d, J = 4.0 Hz, 1H), 4.51 (d, 1H),
4.10−4.07 (m, 3H), 2.34−2.29 (m, 1H), 2.25−2.20 (m, 1H) ppm; 13C
NMR (100 MHz, D2O): δ 165.5, 156.7, 141.9, 96.4, 86.0, 85.5, 70.7,
65.1, 39.4 ppm; 31P NMR (162 MHz, D2O): δ −9.99 (d, JP,P = 21.1
Hz, 1P), −11.12 (d, JP,P = 21.1 Hz, 1P) ppm; LRMS (ESI−): m/z
calcd for C9H14N3O10P2 [M − H]− 386.0; found 386.2.
Uridine-5′-diphosphate, Trisodium Salt (32). General proce-
dure for the synthesis of NDPs (32−40): To a solution of 11 (50 mg,
0.067 mmol) and Et3N (9 μL, 0.067 mmol) in DMF (1.0 mL) was
added 5% Pd/C (5 mg). The reaction was stirred under a hydrogen
atmosphere at 20 °C for 3 h. The catalyst was removed with a syringe
filter (0.45 μm pore size) and washed with DMF (0.1 mL × 2) under
an atmosphere of argon. To the combined DMF solution were added a
solution of bis(tetra-n-butylammonium) hydrogen phosphate (22, 58
mg, 0.2 mmol) in DMF (1.0 mL) and 4,5-dicyanoimidazole (DCI, 63
mg, 0.54 mmol). The reaction was stirred at 20 °C for 6 h and
concentrated in vacuo. The residue was dissolved in NaOAc aqueous
solution (10 M, 0.5 mL), and EtOH (50 mL) was added. The resulting
white precipitate was collected by centrifuge. The crude product was
dissolved in deionized H2O (0.5 mL) and loaded on a DEAE
Sephadex A-25 ion exchange column (1.6 × 25 cm). Elution with
NH4HCO3 buffer (linear gradient 0.2 to 0.6 M), combination of
appropriate fractions, and lyophilization afforded the product in
ammonium salt form. Passage of the solution of the ammonium salt in
deionized H2O through a bed of Dowex 50W-X8 ion-exchange resin
(Na+ form) and lyophilization afforded 32 (25 mg, 79%) as the
trisodium salt, a white solid. 1H NMR (400 MHz, D2O): δ 7.92 (d, J =
8.0 Hz, 1H), 5.89−5.87 (m, 2H), 4.35−4.30 (m, 2H), 4.18−4.14 (m,
3H) ppm; 13C NMR (100 MHz, D2O): δ 166.4, 151.9, 141.9, 102.8,
88.7, 83.3, 74.0, 69.4, 64.4 ppm; 31P NMR (162 MHz, D2O): δ −6.30
(d, JP,P = 21.1 Hz, 1P), −10.40 (d, JP,P = 21.1 Hz, 1P) ppm; LRMS
(ESI−): m/z calcd for C9H13N2O12P2 [M − H]− 403.0; found 403.2.
Cytidine-5′-diphosphate, Trisodium Salt (33). Starting from
12 (60 mg), CDP (22 mg, 69%) was obtained as the trisodium salt, a
2′-Deoxyadenosine-5′-diphosphate, Trisodium Salt (39).
Starting from 18 (42 mg), dADP (23 mg, 72%) was obtained as the
1
trisodium salt, a white solid. H NMR (400 MHz, D2O): δ 8.32 (s,
1H), 8.03 (s, 1H), 6.33 (dd, 1H), 4.66 (d, 1H), 4.20 (s, 1H), 4.07 (m,
2H), 2.68 (m, 1H), 2.50 (m, 1H) ppm; 13C NMR (100 MHz, D2O): δ
154.9, 151.9, 148.5, 140.1, 118.4, 85.8, 83.8, 71.2, 65.3, 39.2 ppm; 31P
NMR (162 MHz, D2O): δ −10.18 (d, JP,P = 21.1 Hz, 1P), −10.96 (d,
JP,P = 21.1 Hz, 1P) ppm; LRMS (ESI−): m/z calcd for C10H14N5O9P2
[M − H]− 410.0; found 410.2.
2′-Deoxyguanosine-5′-diphosphate, Trisodium Salt (40).
Starting from 19 (43 mg), dGDP (24 mg, 73%) was obtained as the
1
trisodium salt, a white solid. H NMR (400 MHz, D2O): δ 7.97 (s,
1H), 6.16 (t, 1H), 4.65 (s, 1H), 4.13 (s, 1H), 4.03 (m, 2H), 2.65 (m,
1H), 2.41 (m, 1H) ppm; 13C NMR (100 MHz, D2O): δ 158.8, 153.8,
151.3, 137.6, 116.1, 85.6, 83.4, 71.1, 65.1, 38.6 ppm; 31P NMR (162
MHz, D2O): δ −6.30 (d, JP,P = 21.1 Hz, 1P), −10.40 (d, JP,P = 21.1 Hz,
1P) ppm; LRMS (ESI−): m/z calcd for C10H14N5O10P2 [M − H]−
426.0; found 426.2.
ASSOCIATED CONTENT
* Supporting Information
■
S
1
white solid. H NMR (400 MHz, D2O): δ 7.92 (d, J = 8.0 Hz, 1H),
Optimization data for NDP synthesis, NMR spectra for all
compounds, and HPLC traces for all 18 NPPs. This material is
available free of charge via the Internet at http://pubs.acs.org.
6.05 (d, J = 8.0 Hz, 1H), 5.91 (d, J = 4.0 Hz, 1H) 4.30 (d, 1H), 4.24 (s,
1H), 4.19−4.15 (m, 3H) ppm; 13C NMR (100 MHz, D2O): δ 166.0,
157.5, 141.7, 96.6, 89.3, 82.8, 74.3, 69.2, 64.4 ppm; 31P NMR (162
MHz, D2O): δ −9.52 (d, JP,P = 19.4 Hz, 1P), −11.13 (d, JP,P = 19.4 Hz,
1P) ppm; LRMS (ESI−): m/z calcd for C9H13N2O12P2 [M − H]−
402.0; found 402.2.
AUTHOR INFORMATION
Corresponding Author
*E-mail: sunqi96@tsinghua.org.cn.
■
Adenosine-5′-diphosphate, Trisodium Salt (34). Starting from
13 (52 mg), ADP (24 mg, 73%) was obtained as the trisodium salt, a
1
Notes
white solid. H NMR (400 MHz, D2O): δ 8.38 (s, 1H), 8.04 (s, 1H),
6.00 (d, J = 4.0 Hz, 1H), 4.64 (dd, 1H), 4.48 (s, 1H), 4.29 (s, 1H),
The authors declare no competing financial interest.
8425
dx.doi.org/10.1021/jo4011156 | J. Org. Chem. 2013, 78, 8417−8426

The Journal of Organic Chemistry
Article
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Montesarchio, D. Eur. J. Org. Chem. 2011, 1155.
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ACKNOWLEDGMENTS
■
We thank the National Natural Science Foundation of China
(Nos. 21002041, 21262014), Natural Science Foundation of
Jiangxi Province (No. 20114BAB203008), Project of the
Science Funds of Jiangxi Education Office (No. GJJ12589),
Key Project of Chinese Ministry of Education (No. 212092),
Scientific Research Foundation of Chinese Ministry of Human
Resources, and Social Security for Returned Chinese Scholars
for financial support to Q. S.
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