Efficient synthesis of nucleoside 5′-triphosphates and their β,γ-bridging oxygen-modified analogs from nucleoside 5′-phosphates

Article abstract of DOI:10.1016/j.tetlet.2014.02.031

Thirteen nucleoside 5′-triphosphates (NTPs) and their β,γ-bridging oxygen-modified analogs (β,γ-CX ₂-NTPs, X = H, F, Cl, and Br) have been efficiently synthesized from nucleoside 5′-phosphoropiperidates with 4,5-dicyanoimidazole as the activator. A high-yielding and chromatography-free protocol for the preparation of both natural and base-modified nucleoside 5′-phosphoropiperidates from the corresponding nucleoside 5′-phosphates was also developed.

Full text of DOI:10.1016/j.tetlet.2014.02.031

Tetrahedron Letters 55 (2014) 2114–2118
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Efficient synthesis of nucleoside 50-triphosphates and their
b,c
-bridging oxygen-modified analogs from nucleoside 50-phosphates
a
a
a
a
b
a
Qi Sun a,b, , Shanshan Gong , Jian Sun , Chengjun Wang , Si Liu , Guodong Liu , Cha Ma
⇑
a Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and Technology Normal University, 605 Fenglin Avenue, Nanchang, Jiangxi 330013, PR China
b High Level Engineering Research Center of Biopharmaceutical Molecules and Diagnostic Apparatuses, Jiangxi Provincial Colleges and Universities, 605 Fenglin Avenue, Nanchang,
Jiangxi 330013, PR China
a r t i c l e i n f o
a b s t r a c t
Thirteen nucleoside 50-triphosphates (NTPs) and their b,
c-bridging oxygen-modified analogs (b,c-CX2-
Article history:
NTPs, X = H, F, Cl, and Br) have been efficiently synthesized from nucleoside 50-phosphoropiperidates
with 4,5-dicyanoimidazole as the activator. A high-yielding and chromatography-free protocol for the
preparation of both natural and base-modified nucleoside 50-phosphoropiperidates from the correspond-
ing nucleoside 50-phosphates was also developed.
Received 15 December 2013
Revised 21 January 2014
Accepted 13 February 2014
Available online 21 February 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Nucleoside triphosphate
Phosphoropiperidate
4,5-Dicyanoimidazole
Diphosphonate
Disulfide
Nucleoside 50-triphosphates (NTPs) are a unique group of bio-
molecules that play key roles in a vast array of pivotal biological
processes, such as energy metabolism, polymerization of DNA
and RNA, signal transduction, and regulation of protein functions.1
Due to the existence of the high-energy phosphoanhydride bonds,
NTPs are highly susceptible to hydrolysis.2 Replacement of the la-
bile P–O–P linkage with an isosteric P–CXY–P (X, Y = H, F, Cl, Br,
Figure 1. The structures of b,c-bridging oxygen-modified NTP analogs.
and OH) unit at b,c-bridging position (Fig. 1) enhances the meta-
bolic stability of the modified NTP analogs.3 These hydrolysis-resis-
tant NTP analogs have been extensively utilized to probe the
catalytic mechanism of phosphoryl tranfer processes,4 and investi-
gated as enzyme inhibitors5 and receptor agonists6 in numerous
medicinal studies.
Due to their important roles in biological research and potential
pharmaceutical applications, various synthetic methods for NTPs
have been adopted for the preparation of b,c-bridging oxygen-
O/N-methylimidazole,8 and sulfonyl imidazolium salts.9 Moreover,
a solid-phase method has been reported for the synthesis of b,
CH2-NTPs.5a However, the preparation of special b,
-CH2-tri-
phosphitylating reagent greatly limits its practical application.
c-
c
More recently, we established a novel P(V)–N activation ap-
proach for the synthesis of nucleoside diphosphates (NDPs), tri-
phosphates (NTPs),10 and nucleoside diphosphate sugars (NDP-
sugars)11 from the fully protected phosphoropiperidates. Though
the Bn and Cbz protecting groups of the phosphoropiperidate pre-
cursors could be quantitatively removed by catalytic hydrogena-
tion, reducible functional groups, such as AN3 and AC@CA on
ribose moiety and halogen atoms (e.g., F, Cl, Br, and I) on nucleo-
bases could not be tolerated. Therefore, a direct and efficient access
to the unprotected phosphoropiperidates may significantly sim-
plify the original method and extend the applications of the
P(V)–N activation strategy to more diversified nucleoside sub-
strates. We report herein a high-yielding and chromatography-free
method for the preparation of nucleoside 50-phosphoropiperidates
modified NTP analogs (b,c-CX2-NTPs, X = H, F, Cl, and Br). The con-
ventional ‘one-pot, three-step’,6b–d salicyl chlorophosphite,5b and
phosphoromorpholidate3d,4a,6e methods have been employed to
synthesize b,c-CX2-NTPs in moderate yields. These compounds
have also been obtained from the coupling of nucleoside 50-phos-
phates (NMPs) with diphosphonates in the presence of condensing
reagents, such as diphenylphosphoryl chloride,4e CDI,4d,7 (CF3CO)2-
⇑
Corresponding author. Tel.: +86 791 8380 5183; fax: +86 791 8382 6894.
E-mail address: sunqi96@tsinghua.org.cn (Q. Sun).
http://dx.doi.org/10.1016/j.tetlet.2014.02.031
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

Q. Sun et al. / Tetrahedron Letters 55 (2014) 2114–2118
2115
Scheme 1. A general and optimized method for the synthesis of nucleoside 50-phosphoropiperidates (6–10) from nucleoside 50-phosphates. aIsolated yield.
from the corresponding NMPs. The obtained nucleoside 50-
phosphoropiperidates exhibited excellent reactivity toward
pyrophosphate and diphosphonate reagents in the presence of
4,5-dicyanoimidazole (DCI) and afforded NTP and b,c-CX2-NTP
products in high isolated yields.
As shown in Scheme 1, the phosphoropiperidates of four natural
nucleosides (6–9) and a base-modified nucleoside (5-bromouri-
dine, 10) were prepared from the corresponding NMP starting
materials (1–5). In contrast to the DCC condensation method,4a,12
the redox condensation approach based on 2,20-dipyridyldisul-
fide/PPh3 is amenable to amine nucleophiles with a wide range
of pKa values.13 In precedent reports, 2.0–5.0 equiv of 2,20-
dipyridyldisulfide/PPh3 in 1:1 molar ratio were used to give the
phosphoromorpholidates and phosphoropiperidates of adenosine
and guanosine in good yields (ꢀ70–80%).14 In the present work,
2,20-dithiodianiline, a less expensive disulfide reagent, was used
31
Figure 2. P NMR tracing of the redox condensation reaction mixture for the synthesis of uridine 50-phosphoropiperidate (6).

2116
Q. Sun et al. / Tetrahedron Letters 55 (2014) 2114–2118
instead of 2,20-dipyridyldisulfide. We found that when 1–5 were
treated with 1.5 equiv of the disulfide, 2.5 equiv of PPh3, and
5.0 equiv of piperidine in DMSO at 20 °C, the phosphoropiperidates
6–10 could be obtained almost quantitatively on 31P NMR in 3–
12 h (Fig. 2). Acetone precipitation afforded the sodium salts of
6–10 in 95–98% isolated yields and high purity.
6.0 equiv of DCI. The b,c-CX2-NTP products were obtained in
excellent isolated yields ranging from 71–84% in only 0.5–1 h.
The experimental results of 11–15 in Table 1 well demonstrated
the generality and reproducibility of the reported P(V)–N activa-
tion strategy for the synthesis of NTPs. To explore its application
in the preparation of b,c
-CX2-NTPs, uridine 50-phosphoropiperidate
To improve the solubility of P(V)–N precursors in DMF, the so-
dium salts of 6–10 were first converted to the triethylammonium
salts. Treatment of the phosphoropiperidates 6–10 with 2.0 equiv
of tris(tetra-n-butylammonium) hydrogen pyrophosphate and
6.0 equiv of DCI in DMF at 20 °C according to our previous report10
gave the desired NTPs in high isolated yields (Table 1), indicating
that the unprotected phosphoropiperidates synthesized by the re-
dox condensation method were as reactive as those obtained
in situ from the catalytic hydrogenation of the protected phosphoro-
piperidates. It is worth noting that 5-bromouridine 50-triphosphate
(15), which could not stand the catalytic hydrogenation condition in
the original method, was afforded in 80% yield via this approach. Due
(6) was treated with 2.0 equiv of tris(tetra-n-butylammonium)
hydrogen methylenediphosphonate and 6.0 equiv of acidic activa-
tors in DMF at 20 °C. As listed in Table 2, the data of 31P NMR trac-
ing experiments were in good accordance with those obtained for
NTPs. In the negative control experiment, no reaction was observed
in the absence of the acidic activator even after 72 h. While DCI
exhibited the highest reactivity and efficacy in the activation of
P(V)–N bond (96%, 3 h, Fig. 3), 2,6-lutidinium chloride yielded a
comparable result (90%, 4 h). Though the reaction promoted by
pyridinium chloride was as fast as that by 2,6-lutidinium chloride,
more byproducts were observed (75%, 4 h). In contrast, the reac-
tion with 1H-tetrazole achieved high yield, but it took much longer
to finish (95%, 24 h). Meanwhile, addition of N-methylimidazolium
chloride and imidazolium chloride resulted in the precipitation of
methylenediphosphonate.
to the higher stability of b,c-CX2-NTPs, the DCI-promoted coupling
reactions were conducted in DMF at 40 °C with 2.0 equiv of tris
(tetra-n-butylammonium) hydrogen diphosphonate reagents and
Table 1
DCI-promoted conversion of nucleoside 50-phosphoropiperidates to NTPs (11–15) and b,
c
-CX2-NTPs (16–23)
Compd
X
Base
Temp (°C)
Time (h)
Yielda (%)
11
12
13
14
15
16
17
18
19
20
21
22
23
O
O
O
O
U
C
A
G
5-BrU
U
C
A
G
5-BrU
U
U
U
20
20
20
20
20
40
40
40
40
40
40
40
40
6
6
6
6
6
1
1
1
1
1
1
1
81
74
77
76
80
79
71
72
74
78
71
78
84
O
CH2
CH2
CH2
CH2
CH2
CF2
CCl2
CBr2
0.5
a
Isolated yield.
Table 2
Effect of the acidic activator on the formation of b,c-CH2-UTP (16)
Activator
Time (h)
Yielda (%)
No activator
DCI
72
3
No reaction
96
1H-Tetrazole
2,6-LutidineꢁHCl
PyridineꢁHCl
N-MethylimidazoleꢁHCl
ImidazoleꢁHCl
24
4
4
N.A.
N.A.
95
90
75
Precipitation
Precipitation
a
31P NMR yield.

Q. Sun et al. / Tetrahedron Letters 55 (2014) 2114–2118
2117
Figure 3. The stacked 31P NMR tracing spectra of the DCI-promoted synthesis of b,
c-CH2-UTP (16) at 20 °C.
with methylenediphosphonate due to the electron-withdrawing
effect of halogen atoms.8 But in our experiments, we observed that
difluoro- and dichloromethylenediphosphonate generated the
Table 3
Effect of reaction temperature on the formation of b,c-CH2-UTP (16)
Temp (°C)
Time (h)
Yielda (%)
b,c-CX2-NTPs in comparable reaction time and yield to meth-
20
30
40
50
3
2
1
0.5
96
96
94
89
ylenediphosphonate at 40 °C (1 h). A plausible explanation was
that the elevated reaction temperature diminished the difference
in the nucleophilicity of the diphosphonate reagents. Surprisingly,
the reaction rate of dibromomethylenediphosphonate was even
faster at 40 °C (0.5 h, Fig. 4).
a
31P NMR yield.
In summary, we developed an efficient method for the synthesis
of NTPs and b,c-CX2-NTPs from NMP starting materials. The mod-
In the following research, we tested the possibility to reduce the
ified redox condensation method based on 2,20-dithiodianiline/
PPh3 provided a general, high-yielding, and chromatography-free
protocol for the preparation of unprotected nucleoside 50-phosp-
horopiperidate intermediates and extended the applicable scope
of the P(V)–N activation strategy to more nucleoside analogs with
reducible functional groups. While the subsequent DCI-promoted
coupling of the phosphoropiperidates with pyrophosphate fur-
nished clean and smooth transformation into the corresponding
NTPs, the reactions with different diphosphonates at elevated tem-
amount of methylenediphosphonate to 1.5 equiv without causing
the formation of symmetrical dinucleoside polyphosphate byprod-
uct (NppCH2ppN). But the experimental result showed that signif-
icant amount of 6 (ca. 11%) was transformed into UppCH2ppU,
indicating that b,c-CH2-UTP (16) was highly reactive toward
the P(V)–N phosphorylating reagent and 2.0 equiv of meth-
ylenediphosphonate was the minimum amount required to sup-
press the formation of NppCH2ppN.
Due to the enhanced stability of b,c-CH2-NTPs, the reaction
temperature was elevated to accelerate the reaction rate. As shown
in Table 3, the reaction time for the synthesis of 16 was signifi-
cantly shortened with increasing temperature (20–50 °C). But it
was also observed that when the reaction was performed at
50 °C, the amount of phosphate and polyphosphate byproducts be-
gan to increase due to the hydrolysis of 6. Therefore, 40 °C is opti-
perature (40 °C) afforded the b,c-bridging oxygen-modified NTP
analogs within 1 h in high isolated yields.
Acknowledgments
We thank the National Natural Science Foundation of China
(Nos. 21002041 and 21262014), 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 (2011), and Research Funds (No. ky2012zy08)
and Startup Funds for PhDs (2010) from JXSTNU for financial
support.
mal to maximize both the reaction rate and yield of b,c-CH2-NTPs.
With the optimized reaction conditions, the P(V)–N activation
method was applied to the coupling of 6 with difluoro-, dichloro-,
and dibromomethylenediphosphonate. It has been reported that
the reactions of halogen-substituted methylenediphosphonates
with phosphorylating reagents were typically slower than those
Figure 4. The 31P NMR spectrum of the crude reaction mixture of b,
c-CBr2-UTP (23).

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