Application of a solid-phase β-triphosphitylating reagent in the synthesis of nucleoside β-triphosphates

Article abstract of DOI:10.1021/jo0610107

A β-triphosphitylating reagent was subjected to reaction with aminomethyl polystyrene resin-bound p-acetoxybenzyl alcohol to yield the corresponding polymer-bound β-triphosphitylating reagent. The solid-phase reagent was reacted with unprotected nucleosides (e.g., 3′-azido-3′- deoxythymidine, cytidine, thymidine, uridine, inosine, or adenosine) in the presence of 1H-tetrazole. Polymer-bound nucleosides underwent oxidation with t-butyl hydroperoxide, deprotection of cyanoethoxy groups with DBU, and the acidic cleavage, respectively, to afford only monosubstituted 5′-O-β-triphosphorylated nucleosides.

Full text of DOI:10.1021/jo0610107

base4,5 or carbohydrate6-11 moieties of the nucleotides. Research
on designing nucleotides containing a modified triphosphate
group has been focused on developing triphosphate analogues
by replacing oxygens with heteroatoms, such as sulfur and
Application of a Solid-Phase â-Triphosphitylating
Reagent in the Synthesis of Nucleoside
â-Triphosphates
1
2-16
17
nitrogen in thiotriphosphates
and imidotriphosphates,
Yousef Ahmadibeni and Keykavous Parang*
respectively. These compounds are finding diverse applications
in pharmacology, biochemistry, molecular biology, and nucleic
acid research. Furthermore, unnatural triphosphate mimics that
can be easily synthesized are urgently needed for application
in these fields and antiviral research.
Department of Biomedical and Pharmaceutical Sciences,
College of Pharmacy, The UniVersity of Rhode Island, Kingston,
Rhode Island 02881
kparang@uri.edu
The synthesis of nucleoside â-triphosphates remains unex-
plored, possibly due to the challenges in their preparation. We
describe the synthesis of a solid-phase â-triphosphitylating
reagent and its application for the synthesis of nucleoside
â-triphosphates as part of our ongoing research to develop
nucleosides containing a modified triphosphate group. These
compounds can have diverse applications in studying the
enzymes that use natural nucleoside triphosphates. To the best
of our knowledge, this is the first paper on the synthesis of a
polymer-bound â-triphosphitylating reagent and nucleoside 5′-
O-â-triphosphates.
ReceiVed May 16, 2006
Only monosubstituted nucleoside 5′-O-â-triphosphates were
produced. The presence of a â-triphosphitylating reagent on a
hindered and rigid solid support only allowed the reaction with
the most reactive and exposed hydroxyl group in the nucleosides.
The solid-phase strategy also offered the advantage of facile
isolation and the recovery of products. The unprotected nucleo-
sides were mixed with the polymer-bound reagent. Washing
the support allowed for removal of unreacted reagents and
starting materials. Furthermore, in the final reaction, the linker
remained trapped on the resin, which facilitated the separation
of the monosubstituted final products by filtration.
Scheme 1 illustrates the synthesis of the â-triphosphitylating
reagent (7). Phosphorus trichloride (1, 10 mmol) was reacted
with 3-hydroxypropionitrile (1 or 2 equiv) in the presence of
triethylamine (1 or 2 equiv) to yield 2-cyanoethyl phosphoro-
dichloridite (2) or bis(2-cyanoethyl)phosphorochloridite (3),
A â-triphosphitylating reagent was subjected to reaction with
aminomethyl polystyrene resin-bound p-acetoxybenzyl al-
cohol to yield the corresponding polymer-bound â-triphos-
phitylating reagent. The solid-phase reagent was reacted with
unprotected nucleosides (e.g., 3′-azido-3′-deoxythymidine,
cytidine, thymidine, uridine, inosine, or adenosine) in the
presence of 1H-tetrazole. Polymer-bound nucleosides un-
derwent oxidation with t-butyl hydroperoxide, deprotection
of cyanoethoxy groups with DBU, and the acidic cleavage,
respectively, to afford only monosubstituted 5′-O-â-tri-
phosphorylated nucleosides.
(4) Kawate, T.; Allerson, C. R.; Wolfe, J. L. Org. Lett. 2005, 7, 3865-
868.
3
(
5) Hoffmann, C.; Genieser, H.-G.; Veron, M.; Jastorff, B. Bioorg. Med.
Chem. Lett. 1996, 6, 2571-2574.
6) Wu, W.; Meyers, C. L. F.; Borch, R. F. Org. Lett. 2004, 6, 2257-
2260.
(
Nucleoside triphosphates are the building blocks for the
synthesis of DNA and RNA. Naturally occurring deoxyribo-
and ribonucleoside triphosphates are synthesized intracellularly
(7) Camplo, M.; Faury, P.; Charvet, A. S.; Graciet, J. C.; Chermann, J.
C.; Kraus, J. L. Eur. J. Med. Chem. 1994, 29, 357-362.
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1
from nucleosides in the presence of kinases. Triphosphorylation
J. Med. Chem. 2003, 46, 3245-3256.
is also essential for the biological activities of antiviral nucleo-
(9) von Janta-Lipinski, M.; Costisella, B.; Ochs, H.; Hubscher, U.;
Hafkemeyer, P.; Matthes, E. J. Med. Chem. 1998, 41, 2040-2046.
sides.2,3
(10) Anastasi, C.; Quelever, G.; Burlet, S.; Garino, C.; Souard, F.; Kraus,
Modified nucleoside triphosphates have been investigated for
potential therapeutic applications and in the study of a number
of pharmacological and biochemical processes. Most of the
attention has been on modifying and/or substitution on the
J.-L. Curr. Med. Chem. 2003, 10, 1825-1843.
(11) Van Aerschot, A.; Herdewijn, P.; Balzarini, J.; Pauwels, R.; De
Clercq, E. J. Med. Chem. 1989, 32, 1743-1749.
(
12) Okruszek, A.; Olesiak, M.; Balzarini, J. J. Med. Chem. 1994, 37,
3
850-3854.
(13) Barai, V. N.; Zinchenko, A. I.; Kvach, S. V.; Titovich, O. I.;
*
Corresponding author. Tel.: (401) 874-4471; fax: (401) 874-5787;
e-mail: kparang@uri.edu.
1) Van Rompay, A. R.; Johansson, M.; Karlsson, A. Pharmacol. Ther.
000, 87, 189-198.
2) Parker, W. B.; White, E. L.; Shaddix, S. C.; Ross, L. J.; Buckheit,
Rubinova, E. B.; Kalinichenko, E. N.; Mikhailopulo, I. A. HelV. Chim. Acta
2003, 86, 2827-2832.
(
(14) Ludwig, J.; Eckstein, F. Nucleosides Nucleotides 1991, 10, 663-
664.
(15) Ludwig, J.; Eckstein, F. J. Org. Chem. 1991, 56, 5860-5865.
(16) Ludwig, J.; Eckstein, F. J. Org. Chem. 1991, 56, 1777-1783.
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1992, 35, 1938-1941.
2
(
R. W., Jr.; Germany, J. M.; Secrist, J. A., III; Vince, R.; Shannon, W. H.
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(3) De Clercq, E.; Field, H. J. Brit. J. Pharmacol. 2006, 147, 1-11.
1
0.1021/jo0610107 CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/24/2006
J. Org. Chem. 2006, 71, 5837-5839
5837

SCHEME 1. Synthesis of â-Triphosphitylating Reagent (7)
reacted with polymer-bound reagent 9 in the presence of 1H-
tetrazole to yield 10a-f. Oxidation with t-butyl hydroperoxide
followed by removal of the cyanoethoxy group with DBU
afforded the corresponding polymer-bound nucleoside 5′-O-â-
triphosphotriester (12a-f). The cleavage of polymer-bound
compounds was carried out under acidic conditions (TFA). The
intramolecular cleavage mechanism of final products from
(12a-f) is shown in Scheme 2. The linker-trapped resin (15)
was separated from the final products by filtration. The crude
products had a purity of 87-96% (Table 1) and were purified
by using small C18 Sep-Pak cartridges and appropriate solvents
to afford nucleoside 5′-O-â-triphosphates (14a-f) in 65-87%
overall yield (calculated from 9 in the four-step reaction
sequence) (Table 1). Only one type of monosubstituted com-
pound was produced with high selectivity as a result of this
sequence. The chemical structures of the final products (14a-
respectively. The parallel reaction of 1 (10 mmol) with
diisopropylamine (10 mmol, 1 equiv) afforded diisopropylphos-
phoramidodichloridite (4). The addition of water (2 equiv) gave
the compound 5 that was reacted with 3 (1 equiv) in the presence
of triethylamine (1 equiv) to afford 6. The reaction of equimolar
amounts of 2 and 6 produced a â-triphosphitylating reagent (7)
in 91% overall yield. The chemical structure of 7 was
1
f) were determined by nuclear magnetic resonance spectra ( H
1
3
31
NMR, C NMR, and P NMR), high-resolution time-of-flight
electrospray mass spectrometry, and quantitative phosphorus
1
analysis. Stability studies using H NMR showed that all final
compounds remained stable after 8 months storage in DMSO
at -20 °C.
1
determined by nuclear magnetic resonance spectra ( H NMR,
1
3
31
C NMR, and P NMR) and high-resolution time-of-flight
In conclusion, nucleoside 5′-O-â-triphosphates were synthe-
sized by using a polymer-bound â-triphosphitylating reagent.
Only one type of monosubstituted product was formed using
the solid-phase strategy, probably due to the reaction of the
sterically rigid polymer-bound reagent with the most exposed
and reactive hydroxyl groups. The products were easily isolated
from the polymer-bound trapped linker. To the best of our
knowledge, this is the first paper on designing a polymer-bound
â-triphosphitylating reagent and its application in the synthesis
of nucleoside 5′-O-â-triphosphates. These compounds can have
diverse applications in nucleic acid research and studying and/
or inhibiting enzymes involved in the synthesis of nucleoside
triphosphates.
electrospray mass spectrometry. Stability studies using these
spectroscopic methods showed that the compound remained
stable even after 2 months storage at -20 °C and 10 days at
room temperature in DMSO solution.
A p-hydroxy benzyl alcohol linker was previously designed
for the solid-phase synthesis of nucleoside and carbohydrate
monophosphates and monomethyl phosphates.18,19 Bradley and
colleagues developed a polymer-bound N-Boc p-acetoxybenzyl
alcohol (aminomethyl polystyrene resin linked through a reduced
amide bond with p-acetoxybenzyl alcohol) as a safety-catch
2
0
linker for solid-phase synthesis of a squalamine analogue.
Polymer-bound N-Boc p-acetoxybenzyl alcohol with a reduced
2
0
amide bond and other safety-catch linkers, such as polymer-
21
bound p-acetoxybenzyl alcohol containing an amide bond and
Experimental Section
polymer-bound oxathiophospholane,22 were used for the syn-
thesis of carbohydrate and nucleoside monophosphates and
As a representative example, thymidine (2.0 mmol) and 1H-
tetrazole (71 mg, 1.0 mmol) were added to 9 (790 mg, 0.51 mmol/
g) in anhydrous THF (2 mL) and DMSO (3 mL). The mixture was
shaken for 24 h at room temperature. The resin was collected by
filtration and washed with DMSO, THF, and MeOH, respectively,
and dried under vacuum to give 10d. t-Butyl hydroperoxide in
decane (5-6 M, 6.0 mmol) was added to resin 10d in THF. After
monothiophosphates,2
1,22
diphosphates, diphosphodithioates,
23
triphosphates, or triphosphotrithioates. These studies revealed
that the p-acetoxybenzyl alcohol is an appropriate linker for
attachment to solid-phase resins and can be used in a variety of
reactions. The aminomethyl polystyrene resin linked through a
reduced amide bond with p-acetoxybenzyl alcohol (8) was
synthesized from aminomethyl polystyrene resin in multiple-
1
h shaking at room temperature, the resin was collected by filtration
and washed with THF and MeOH, respectively, and was dried
overnight at room temperature under vacuum to give 11d. To
swelled resin 11d in THF was added DBU (4.0 mmol). After 48 h
shaking of the mixture at room temperature, the resin was collected
by filtration and washed with THF and MeOH, respectively, and
dried overnight at room temperature under vacuum to give 12d.
To swelled resin 12d in anhydrous DCM was added DCM/TFA/
water (74:24:2 v/v, 4 mL). After the mixture was shaken for 30
min at room temperature, the resin was collected by filtration and
washed with DCM, THF, and MeOH, respectively. The solvents
of the filtrate solution were immediately evaporated at -20 °C.
The residue was mixed with Amberlite AG-50W-X8 (100-200
mesh, hydrogen form, 1.0 g) in water/dioxane (75:25 v/v, 5 mL)
for 15 min. After filtration, the solvents were removed using
lyophilization, and the crude product was purified using a C18 Sep-
20,21
step reactions according to the previously reported procedure.
Scheme 2 shows the synthesis of nucleoside 5′-O-â-triphos-
phates (14a-f). The aminomethyl polystyrene resin-bound
linker (8, 3.75 g, 0.72 mmol/g) was subjected to reaction with
the â-triphosphitylating reagent (7, 10 mmol) in the presence
of triethylamine (10 mmol) to produce the corresponding
polymer-bound â-triphosphitylating reagent (9). Unprotected
nucleosides (e.g., adenosine (a), uridine (b), 3′-azido-3′-deoxy-
thymidine (c), thymidine (d), inosine (e), and cytidine (f)) were
(
18) Parang, K.; Fournier, E. J.-L.; Hindsgaul, O. Org. Lett. 2001, 3,
3
6
07-309.
(
(
19) Parang, K. Bioorg. Med. Chem. Lett. 2002, 12, 1863-1866.
20) Chitkul, B.; Atrash, B.; Bradley, M. Tetrahedron Lett. 2001, 42,
1
Pak to yield thymidine-5′-O-â-triphosphate (14d). H NMR (DMSO-
211-6214.
d , 400 MHz, δ ppm): 1.74 (d, J5-CH ,6 ) 1.1 Hz, 3H), 2.10-2.11
6
(21) Ahmadibeni, Y.; Parang, K. J. Org. Chem. 2005, 70, 1100-1103.
(22) Ahmadibeni, Y.; Parang, K. Org. Lett. 2005, 7, 1955-1958.
(23) Ahmadibeni, Y.; Parang, K. Org. Lett. 2005, 7, 5589-5592.
3
(m, 2H), 3.49-3.65 (m, 2H), 3.72-3.80 (m, 1H), 4.19-4.28 (m,
1H), 4.95-5.05 (m, 1H), 5.15-5.25 (m, 1H), 6.17 (t, J1′,2′ and J1′,2′′
5
838 J. Org. Chem., Vol. 71, No. 15, 2006

SCHEME 2. Synthesis of Polymer-Bound â-Phosphitylating Reagent (9) and Nucleosides 5′-O-â-Triphosphates (14a-f) using
Polymer-Bound Linker 8
TABLE 1. Overall Isolated Yields and Purity of Crude Products
6 3 4
d and H PO 85% in water as external standard, 162 MHz, δ
for Nucleoside 5′-O-â-Triphosphates (14a-f)
ppm): -17.83 (t, Jâ,R ) 11 Hz, â-P, 1P), 1.66 (d, JR,â ) 11 Hz,
R-P, 2P); HR-MS (ESI-TOF) (m/z): calcd, 481.9893; found,
overall yield (%)
purity of
+
no.
calcd from 9
crude products
481.6183 [M] ; Anal. Calcd, P 19.27%; found 19.52%.
1
4a
4b
4c
4d
4e
4f
79
65
87
78
63
81
90
88
96
89
87
92
Acknowledgment. We acknowledge the financial support
from the National Center for Research Resources, NIH, Grant
P20 RR16457.
1
1
1
1
1
1
Supporting Information Available: Experimental procedures
and characterization of resins with FT-IR and final compounds with
1
13
31
H NMR, C NMR, P NMR, and high-resolution mass spec-
trometry. This material is available free of charge via the Internet
at http://pubs.acs.org.
)
8.0 Hz, 1H), 7.69 (d, J6,5-CH3 ) 1.1 Hz, 1H), 11.05-11.15 (br s,
H); 13C NMR (DMSO-d
, 100 MHz, δ ppm): 13.2, 40.8, 62.3,
1.5, 84.8, 88.2, 110.5, 137.2, 151.6, 164.9; P NMR (in DMSO-
1
7
6
31
JO0610107
J. Org. Chem, Vol. 71, No. 15, 2006 5839