A novel method for the preparation of nucleoside triphosphates from activated nucleoside phosphoramidates

Article abstract of DOI:10.1021/ol049267j

A novel method for the preparation of nucleoside triphosphates has been developed. The strategy employs a highly reactive pyrrolidinium phosphoramidate zwitterion intermediate that undergoes efficient coupling with tris(tetra-n-butylammonium) hydrogen pyrophosphate to generate nucleoside triphosphate.

Full text of DOI:10.1021/ol049267j

ORGANIC
LETTERS
2004
Vol. 6, No. 13
2257-2260
A Novel Method for the Preparation of
Nucleoside Triphosphates from
Activated Nucleoside Phosphoramidates
Weidong Wu, Caren L. Freel Meyers, and Richard F. Borch*
Department of Medicinal Chemistry and Molecular Pharmacology and Cancer Center,
Purdue UniVersity, West Lafayette, Indiana 47907
rickb@pharmacy.purdue.edu
Received April 21, 2004
ABSTRACT
A novel method for the preparation of nucleoside triphosphates has been developed. The strategy employs a highly reactive pyrrolidinium
phosphoramidate zwitterion intermediate that undergoes efficient coupling with tris(tetra-n-butylammonium) hydrogen pyrophosphate to generate
nucleoside triphosphate.
Nucleoside 5′-triphosphates are a class of very important
compounds in biological systems. Naturally occurring deoxy-
ribo- and ribonucleoside triphosphates are the basic building
blocks for enzymatic synthesis of DNA and RNA in vivo
and in vitro. Modified nucleoside triphosphates have received
much attention in searches for potential therapeutic and
diagnostic agents and in the study of numerous biochemical
and pharmacological processes. As a result, a number of
methods have been developed for the preparation of nucleo-
side triphosphates and their analogues (see ref 1 for a
review).1 The widely used method of “one-pot, three-step”
nucleoside triphosphate synthesis developed by Ludwig2 and
others3 involves generation of nucleoside dichlorophosphate
via Yoshikawa’s procedure,4 followed by reaction with bis-
(tri-n-butylammonium) pyrophosphate. However, it is not
applicable to all nucleoside derivatives, especially for triphos-
phorylation of nucleoside analogues bearing modified bases
sensitive to Yoshikawa’s phosphorylation procedure.5 Other
widely used multistep methods rely upon the use of activated
nucleoside monophosphates such as unsubstituted phosphor-
amidate,6 morpholidate,7 or imidazolidate8 that undergo
subsequent displacement with pyrophosphate. In most cases,
the final transformation to triphosphate is slow (one to several
days), and the yields are generally moderate. We have
developed a novel method for the synthesis of nucleoside
triphosphates employing a highly reactive pyrrolidinium
phosphoramidate zwitterion intermediate 3 that undergoes
fast and efficient coupling with tris(tetra-n-butylammonium)
(5) Wu, W.; Bergstrom, D. E.; Davisson, V. J. J. Org. Chem. 2003, 68,
3860-5.
(6) (a) Tomasz, J.; Simoncsits, A.; Kajtar, M.; Krug, R. M.; Shatkin, A.
J. Nucleic Acids Res. 1978, 5, 2945-57. (b) Simoncsits, A.; Tomasz, J.
Nucleic Acids Res. 1975, 2, 1223-33.
(7) (a) Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961, 83, 649-
58. (b) Moffatt, J. G. Can. J. Chem. 1964, 42, 599-604. (c) van Boom, J.
H.; Crea, R.; Luyten, W. C.; Vink, A. B. Tetrahedron Lett. 1975, 16, 2779-
82.
(8) (a) Hoard, D. E.; Ott, D. G. J. Am. Chem. Soc. 1965, 87, 1785-8.
(b) Shimazu, M.; Shinozuka, K.; Sawai, H. Tetrahedron Lett. 1990, 31,
235-8.
(1) Burgess, K.; Cook, D. Chem. ReV. 2000, 100, 2047-60.
(2) Ludwig, J. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131-
3.
(3) Ruth, J. L.; Cheng, Y. C. Mol. Pharmacol. 1981, 20, 415-22.
(4) Yoshikawa, M.; Kato, T.; Takenishi, T. Tetrahedron Lett. 1967, 50,
5065-8.
10.1021/ol049267j CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/02/2004

Scheme 1
Table 1. Reaction Conditions and Yields for Nucleoside
Triphosphates Synthesis
entry
1
starting cpd
reaction conditions
product, yield
4a
Pd/C, THF, 2 h;
5a , 85%a
(HNBu3)2H2P2O7, 4 h
Pd/C, THF, 2 h;
2
3
4
5
6
7
4a
4a
4c
4b
4d
4e
5a , 100%a
5a , 77%b
5c, 69%b
5b, 75%b
5d , 71%b
5e, 55%b
(NBu4)3HP2O7, 10 min
Pd/C, THF, 2 h;
(NBu4)3HP2O7, 30 min
Pd/C, THF, 2 h;
(NBu4)3HP2O7, 30 min
Pd/Al2O3, DMF, 1 h;
(NBu4)3HP2O7, 30 min
Pd/Al2O3, DMF, 1 h;
(NBu4)3HP2O7, 30 min
Pd/Al2O3, DMF,c 1 h;
(NBu4)3HP2O7, 30 min
a Conversion based on 31P NMR. b Isolated yield. c 5% (v/v) water was
added to the reaction mixture following catalytic hydrogenolysis
hydrogen pyrophosphate to generate the corresponding
nucleoside triphosphates (Scheme 1).
phosphoramidate zwitterion intermediate in situ was ac-
complished quantitatively by catalytic hydrogenolysis with
palladium on activated carbon in anhydrous THF at room
temperature (Scheme 2 and spectra a and b in Figure 1).10,11
Following filtration of the catalyst, the pyrophosphate
coupling reaction was carried out by the addition of either
bis(tri-n-butylammonium) pyrophosphate7b,12 or tris(tetra-n-
butylammonium) hydrogen pyrophosphate13 (TBAPP) to the
above phosphoramidate zwitterion solution. The coupling
reaction with bis(tri-n-butylammonium) pyrophosphate took
4 h to complete, and thymidine triphosphate was formed in
85% conversion judged by 31P NMR (Table 1, entry 1). The
major byproduct was thymidine monophosphate, formed by
reaction of the phosphoramidate zwitterion intermediate with
trace water present in the reaction mixture. However, the
reaction carried out with tris(tetra-n-butylammonium) hy-
drogen pyrophosphate was very rapid, resulting in quantita-
tive triphosphate conversion within 10 min with no formation
of hydrolysis product (Table 1, entry 2; also see spectrum c
in Figure 1).14,15 This observation is in accordance with the
report of Davisson et al.13 that the nucleophilicity of
trialkylammonium pyrophosphates is reduced compared with
that of the tetraalkylammonium salts.
This approach is based upon the detailed investigation of
the activation mechanisms of nucleoside phosphoramidate
prodrugs developed in this lab for the intracellular delivery
of anticancer and antivirus nucleotides.9 The reactivity of
the zwitterionic phosphoramidate intermediate 3 toward
different nucleophiles following chemical activation of the
phosphoramidate ester 1 has also been studied and has led
to the development of a novel strategy leading to the
formation of nucleotides,9 nucleoside sugar diphosphates,10
and the immobilization of nucleotides on solid supports11
(Scheme 1). The strategy is now extended to the preparation
of nucleoside triphosphates, and we report herein the
preparation of triphosphates of both natural and modified
nucleosides from the corresponding nucleoside phosphor-
amidates.
Preliminary experiments were carried out with thymidine
phosphoramidate 4a as the model compound, and the results
are shown in Figure 1 and Table 1. Generation of the
(9) (a) Freel Meyers, C. L.; Hong, L.; Joswig, C.; Borch, R. F. J. Med.
Chem. 2000, 43, 4313-8. (b) Freel Meyers, C. L.; Borch, R. F. J. Med.
Chem. 2000, 43, 4319-27. (c) Tobias, S. C.; Borch, R. F. J. Med. Chem.
2001, 44, 4475-80. (d) Tobias, S. C.; Borch, R. F. Mol. Pharm. 2004, 1,
112-6.
(10) Freel Meyers, C. L.; Borch, R. F. Org. Lett. 2001, 3, 3765-8.
(11) Freel Meyers, C. L.; Borch, R. F. Org. Lett. 2003, 5, 341-4.
(12) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631-5.
(13) Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremler, K. E.;
Muehlbacher, M.; Poulter, C. D. J. Org. Chem. 1986, 51, 4768-79.
(14) It is noteworthy that tris(tetra-n-butylammonium) hydrogen pyro-
phosphate contains water of hydration (at least 3 equiv);13 the absence of
hydrolysis byproduct indicates that nucleophilicity of this pyrophosphate
salt is much greater toward the phosphoramidate zwitterion intermediate
than that of water.
(15) It is important to use only 1 equiv of pyrophosphate reagent in these
reactions, because excess pyrophosphate is very difficult to separate from
nucleoside triphosphate during purification by anion exchange chromatog-
raphy on Q Sepharose FF.
Figure 1. 31P NMR of nucleoside triphosphate formation from
nucleoside phosphoramidate at room temperature (ref ) tri-
phenylphosphine oxide): (a) nucleoside phosphoramidate; (b)
activated phosphoramidate after catalytic hydrogenolysis; and (c)
10 min after addition of TBAPP.
2258
Org. Lett., Vol. 6, No. 13, 2004

Scheme 2a
Scheme 3a
a Reagent and conditions: (a) for 4a and 4c, H2, Pd/C, THF, 2
h; for 4b, 4d, and 4e, H2, Pd/alumina, DMF, 1 h; (b) for 4a and
4c, (NBu4)3HP2O7, THF, 30 min; for 4b, 4d, and 4e, (NBu4)3HP2O7,
DMF, 30 min.
On the basis of the above results, the scope and applicabil-
ity of this approach toward preparation of triphosphates of
both natural and modified nucleoside analogues were ex-
amined (Scheme 2 and Table 1). Among the nucleotide
analogues shown in Scheme 2, ribavirin (1-â-D-ribofurano-
syl-1,2,4-trizaole-3-carboxamide) triphosphate (RTP, 5d) and
Ara-C (1-â-D-arabinofuranosyl cytosine, Cytarabine) triphos-
phate (Ara-CTP, 5e) were chosen because of their importance
in the antiviral16 and antitumor17 therapeutic treatment as well
as for the study of numerous biochemical and pharmacologi-
cal processes. Application of this approach to the synthesis
of these nucleoside triphosphates was successful for uridine
(Table 1, entry 4) but met with limited success in the case
of 2′-deoxycytidine, ribavirin, and Ara-C. Poor mass recov-
eries were obtained presumably due to poor solubility of the
starting phosphoramidates and products and the significant
adsorption on Pd/C catalyst. Use of palladium on alumina
as hydrogenolysis catalyst resulted in both decreased adsorp-
tion of the nucleoside analogue and an increase in the rate
a Reagent and conditions: (a) HOBT, pyridine, THF, 4 h, rt; (b)
N-methylimidazole, pyridine/THF, 6-24 h, rt; (c) for 9c, C6H5-
CH2OH, LiHMDS, THF, -70 to -40 °C, 1 h; for 9d and 9e,
C6H5CH2OH, DMAP, THF, overnight, rt; (d) HCl, pH 2.0, CH3CN/
H2O (1:1), 4-24 h, rt; then NH3HCO3, pH 8.0, CH3CN/H2O (1:1),
2-24 h, rt; (e) Pd(PPh3)4, p-C6H4SO2Na, THF/H2O (2:1), 1 h, rt.
of hydrogenolysis (Table 1, entries 5, 6, and 7). The reaction
solvent was changed to DMF in an effort to increase the
solubility of the cytosine analogues. Both hydrogenolysis and
pyrophosphate coupling proceeded smoothly in DMF, and
gave essentially quantitative conversion to nucleoside tri-
phosphate as judged by 31P NMR.18 Addition of water (5%
v/v) to the reaction mixture following catalytic hydrogenoly-
sis of the Ara-C phosphoramidate was necessary to facilitate
filtration of the reaction intermediate, and the pyrophosphate
coupling still proceeded smoothly and afforded only a small
amount of hydrolysis product.
(16) (a) Crotty, S.; Maag, D.; Arnold, J. J.; Zhong, W.; Lau, J. Y.; Hong,
Z.; Andino, R.; Cameron, C. E. Nat. Med. 2000, 6, 1375-9. (b) Crotty, S.;
Cameron, C. E.; Andino, R. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6895-
900. (c) Maag, D.; Castro, C.; Hong, Z.; Cameron, C. E. J. Biol. Chem.
2001, 276, 46094-8. (d) Crotty, S.; Cameron, C.; Andino, R. J. Mol. Med.
2002, 80, 86-95.
A simple purification procedure employing Q Sepharose
FF, a medium pressure anion exchanger, was developed to
(17) (a) Furth, J. J.; Cohen, S. S. Cancer Res. 1968, 28, 2061-7. (b)
Graham, F. L.; Whitmore, G. F. Cancer Res. 1970, 30, 2636-44. (c) Kufe,
D. W.; Major, P. P.; Egan, E. M.; Beardsley, G. P. J. Biol. Chem. 1980,
255, 8997-900. (d) Major, P. P.; Egan, E. M.; Beardsley, G. P.; Minden,
M. D.; Kufe, D. W. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 3235-9.
(18) We also explored this catalyst and solvent (Pd/alumina/DMF) on
the coupling reaction of â-D-glucose-1-phosphate with thymidine phosphor-
amidate in the presence of tetrabutylammonium chloride. The coupling
reaction gave a very clean 31P NMR with no formation of hydrolysis
byproduct even in the presence of water.
Org. Lett., Vol. 6, No. 13, 2004
2259

purify the triphosphate. All triphosphates described in this
study were rapidly and easily purified by using a linear
gradient of 50 to 500 mM ammonium bicarbonate (pH 7.8)
phosphoramidates 4c and 4d in 69% yield (two steps). Ara-C
phosphoramidate 4e was synthesized by using the same
approach starting from N-allyloxycabonyl (AOC) protected
nucleoside 6e.9d Selective phosphorylation of 6e on the 5′-
hydroxyl group with reagent 8 was achieved to afford
benzotriazolyl phosphoramidate 9e in 71% yield. Displace-
ment of the OBT group with benzyl alcohol in the presence
of DMAP gave phosphoramidate 10e in 68% yield. Finally,
compound 10e was deprotected by using palladium chemistry9d
to give the desired phosphoramidate 4e in 84% yield (Scheme
3).
In summary, a novel method for the synthesis of nucleo-
side triphosphates has been developed. The approach de-
scribed here employs a highly reactive pyrrolidinium phos-
phoramidate zwitterion intermediate that undergoes an
efficient coupling reaction with tris(tetra-n-butylammonium)
hydrogen pyrophosphate to generate the corresponding
nucleoside triphosphate. Reaction conditions for activation
of the nucleoside phosphoramidates and subsequent coupling
with pyrophosphate have been optimized, and this method
has been applied to the synthesis of triphosphates of both
natural and unnatural nucleoside analogues. This approach
would have advantage in the synthesis of triphosphates of
unnatural nucleosides with modified bases, particularly azoles
bearing a carboxamide substituent, where side reactions occur
and yields are poor when using nonenzymatic methods.5 It
should complement the existing methods for the synthesis
of nucleoside triphosphates, an important class of compounds
for which there is no broadly applicable methodology.1
1
at room temperature and were judged pure by HPLC, H,
13C, and 31P NMR. Where available, commercial materials
were used for comparisons with synthetic materials by HPLC
and spectral analysis. The isolated yields of purified nucleo-
side triphosphates ranged from 55% to 77% (Table 1).
Thymidine and deoxycytidine phosphoramidates 4a and
4b were synthesized according to the reported phosphor-
amidite procedure;9a,c however, the same method failed to
give reasonable results for synthesis of ribonucleoside
phosphoramidates due to poor solubility and serious com-
plications when starting from unprotected ribonucleosides.9d
Thus, protection of the 2′,3′ hydroxyl groups of the ribose
moiety was necessary for synthesis of uridine and ribavirin
phosphoramidates 4c and 4d. The 2′,3′-O-methoxymeth-
ylidene protective group19 proved to be an ideal choice,
because it is stable under the reaction conditions and can be
removed without cleaving the acid labile phosphoramidate.
2′,3′-O-protected uridine 6c and ribavirin 6d were prepared
according to the reported procedure5,20 in 92% and 84% yield,
respectively. The protected nucleosides 6c and 6d were
reacted with phosphorylating agent 8,21 generated in situ from
phosphorodichloridate 7 and 1-hydroxybenzotriazole (HOBT),
to give benzotriazolyl phosphoramidate 9c and 9d in 75%
yield. The OBT moiety was displaced by benzyl alcohol in
the presence of LiHMDS (for 9c) or DMAP (for 9d) to give
10c and 10d. 2′,3′-O-Methoxymethylidene protecting groups
were removed under very mild conditions involving hy-
drolysis at pH 2.0 and then at pH 8.019,20 to afford the desired
Acknowledgment. We thank Dr. Sandra C. Tobias for
synthesis of deoxycytidine phosphoramidate 4b. Financial
support from the National Cancer Institute (CA34619) is also
gratefully acknowledged.
(19) (a) Griffin, B. E.; Jarman, M.; Reese, C. B.; Sulston, J. E.
Tetrahedron 1967, 23, 2301-13. (b) Jones, S. S.; Rayner, B.; Reese, C.
B.; Ubasawa, A.; Ubasawa, M. Tetrahedron 1980, 36, 3075-85.
(20) Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. J. Org.
Chem. 1987, 52, 1794-801.
(21) (a) van der Marel, G. A.; van Boeckel, C. A.; Wille, G.; van Boom,
J. H. Nucleic Acids Res. 1982, 10, 2337-51. (b) van der Marel, G. A.; van
Boeckel, C. A.; Wille, G.; van Boom, J. H. Tetrahedron Lett. 1981, 22,
3887-90. (c) van Boeckel, C. A. A.; van der Marel, G.; Wille, G.; van
Boom, J. H. Chem. Lett. 1981, 1725-8.
Supporting Information Available: Experimental pro-
cedures and NMR spectra for 4c-e and 5d,e. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL049267J
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Org. Lett., Vol. 6, No. 13, 2004