Synthesis of acyclothymidine triphosphate and α-P-boranotriphosphate and their substrate properties with retroviral reverse transcriptase

Article abstract of DOI:10.1021/ol034538w

(Matrix presented) The first example of an acyclonucleoside α-P-boranotriphosphate has been synthesized via a phosphoramidite approach in a one-pot reaction with good yield. The presence of the α-P-BH₃ in 5b results in a 9-fold increase in efficiency of incorporation by MMLV retroviral reverse transcriptase relative to non-boronated 5a in pre-steady-state conditions. The preliminary results indicate that acyclonucleoside α-P-boranotriphosphates may have promising applications as a probe of enzyme mechanisms and in the design of new antiviral drugs.

Full text of DOI:10.1021/ol034538w

ORGANIC
LETTERS
2003
Vol. 5, No. 14
2401-2403
Synthesis of Acyclothymidine
Triphosphate and
r-P-Boranotriphosphate and Their
Substrate Properties with Retroviral
Reverse Transcriptase
Ping Li, Mikhail Dobrikov, Hongyan Liu, and Barbara Ramsay Shaw*
Department of Chemistry, Box 90346, Duke UniVersity,
Durham, North Carolina 27708-0346
brshaw@chem.duke.edu
Received March 27, 2003
ABSTRACT
The first example of an acyclonucleoside r-P-boranotriphosphate has been synthesized via a phosphoramidite approach in a one-pot reaction
with good yield. The presence of the r-P-BH in 5b results in a 9-fold increase in efficiency of incorporation by MMLV retroviral reverse
3
transcriptase relative to non-boronated 5a in pre-steady-state conditions. The preliminary results indicate that acyclonucleoside r-P-
boranotriphosphates may have promising applications as a probe of enzyme mechanisms and in the design of new antiviral drugs.
At the forefront of antiviral therapeutics has been the design
of new classes of nucleosides and nucleotides.1 Among these,
one type of nucleoside modification is an acyclic nucleoside
analogue,2,3 in which the pentafuranosyl sugar ring in the
natural nucleoside has been replaced with an acyclic moiety.
Such analogues have shown potent antiviral activity. For
example, 9-(2-hydroxyethoxymethyl)guanine (acyclovir,
ACV)2 is one of the most effective drugs against the
varicella-zoster virus (VZV),3 Epstein-Barr virus (EBV),3d
and herpes simplex viruses (HSV-1 and HSV-2).3b Most
acyclic nucleoside analogues become active after a series of
intracellular conversions to the corresponding triphosphates,
which are then incorporated into viral DNA and subsequently
cause chain termination.2b,c Numerous methods,4a including
pre-steady-state kinetic analyses4b of incorporation by HIV-1
reverse transcriptase (RT) and mitochondrial DNA poly-
merase γ, indicate that acyclonucleoside triphosphates (acy-
cloNTP, Figure 1) may serve as effective antiviral drugs.
(1) (a) DeClercq, E. Nature ReV., Drug DiscoVery 2002, 1, 13. (b)
Kramata, P.; Downey, K. M.; Paborsky, L. R. J. Biol. Chem. 1998, 273,
21966. (c) Sanghvi, Y. S.; Cook, P. D. In Nucleosides and Nucleotides as
Antitumor and AntiViral Agents; Chu, C. K., Baker, D. C., Eds.; Plenum
Press: New York, 1993; p 311. (d) DeMesmaeker, A.; Haener, R.; Martin,
P.; Moser, H. E. Acc. Chem. Res. 1995, 28, 366.
(2) (a) Schaeffer, H. J.; Beauchamp, L.; DeMiranda, P.; Elion, G. B.;
Bauer, D. J.; Collins, P. Nature 1978, 272, 583. (b) Elion, G. B.; Furman,
P. A.; DeMiranda, P.; Beauchamp, L.; Schaeffer, H. J. Proc. Natl. Acad.
Sci. U.S.A. 1977, 74, 5716. (c) Fyfe, J. A.; Keller, P. M.; Furman, P. A.;
Miller, R. L.; Elion, G. B. J. Biol. Chem. 1978, 253, 8721. (d) Emmert, D.
H. Am. Fam. Physician 2000, 61, 1697. (e) Chen, H. M.; Hosmane, R. S.
Nucleosides, Nucleotides & Nucleic Acids 2001, 20, 1599.
(3) (a) Snoeck, R.; Andrei, G.; DeClercq, E. Drugs 1999, 57, 187. (b)
Hamuy, R.; Berman, B. Eur. J. Derm. 1998, 8, 310. (c) Wutzler, P.
InterVirology 1997, 40, 343. (d) Pfleger, A.; Eber, E.; Popper, H.; Zach,
M. S. Eur. Respir. J. 2000, 15, 803.
(4) (a) Wutzler, P.; Thust, R. AntiVir. Res. 2001, 49, 55. (b) Johnson, A.
A.; Ray, A. S.; Hanes, J.; Suo, Z.; Colacino, J. M.; Anderson, K. S.; Johnson,
K. A. J. Biol. Chem. 2001, 276, 40847.
10.1021/ol034538w CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/18/2003

Scheme 1a
Figure 1. Structures of nucleoside triphosphate (NTP) and 5′-(R-
P-borano)triphosphate (NTP-R-BH3); acyclonucleoside triphosphate
(acycloNTP) and R-P-boranotriphosphate (acycloNTP-R-BH3).
They exhibit low mitochondrial toxicity as well as an absence
of HIV-1 RT mutations leading to drug resistance.4b
Nucleoside 5′-(R-P-borano)triphosphate5 (NTP-R-BH3,
Figure 1) is a new type of nucleotide modification, in which
a borane group (BH3) substitutes for one of the nonbridging
R-phosphate oxygens in nucleoside 5′-triphosphate (NTP).
The presence of the BH3 group at the R-phosphate of
triphosphates of clinically relevant dideoxy compounds, such
as3′-azido-3′-deoxythymidine(AZT),6a 2′,3′-didehydrodideoxy-
thymidine (D4T),6a and 2′,3′-dideoxyadenosine (ddA),6b,c
improves both phosphorylation by nucleotide diphosphate
kinase and incorporation by wild-type6a and mutant HIV-1
RTs.6b,c Moreover, after an R-P-borane group is incorporated
into DNA, repair of the blocked DNA chains by pyrophos-
phorolysis is reduced significantly with mutant RT enzymes
from drug-resistant viruses.6a
a Reagents and conditions: (i) [(iPr)2N]2PCl, DIPEA, DMAP,
CH3CN, 15 min; (ii) (HBu3N+)2P2O72-, 1H-tetrazole, 15 min; (iii)
I2/pyridine/H2O, total yield 48% from 1; (iv) 2 M BH3:SMe2 in
THF, 30 min; (v) H2O/Et3N, 5 h, total yield 53% from 1.
Because of the powerful antiviral activity of acyclonucleo-
sides and the advantages granted by the presence of an R-P-
borane group in triphosphates, we set out to synthesize an
acyclonucleoside R-P-boranotriphosphate (acycloNTP-R-
BH3, Figure 1) and determine whether it could be a substrate
for a viral RT. Specifically, the incorporation of acyclothy-
midine R-P-boranotriphosphate (acycloTTP-R-BH3, 5b) into
viral DNA by moloney murine leukemia virus (MMLV) RT
was investigated by using pre-steady-state kinetics.
Although the initial synthesis of NTP-R-BH3 used a
phosphoramidite approach,5a certain limitations, such as
isolation of one intermediate compound and two ion-
exchange column chromatography steps, resulted in a low
overall yield. However, we thought that with some alterations
the phosphoramidite approach would be a viable and efficient
way to synthesize R-P-boranotriphosphates. Here we dem-
onstrate that the sugar-substituted and R-phosphate-modified
triphosphate, e.g., acycloTTP-R-BH3 5b, can be synthesized
in a one-pot reaction via a phosphoramidite approach
(Scheme 1).
Formation of a triphosphate usually requires the use of a
phosphitylating reagent. Salicyl phosphochloridite, which has
been used extensively in the synthesis of NTP7 and NTP-
R-BH3,8a-c is difficult to handle because of its high reactivity
and hygroscopicity. As an alternate phosphitylating reagent,
we chose a reasonably reactive phosphorus compound, bis-
(diisopropylamino)chlorophosphine ([(iPr)2N]2PCl). Acy-
clothymidine 19 was first phosphorylated by [(iPr)2N]2PCl
dissolved in dry chloroform to form phosphoramidite 2 in
the presence of 4 equiv of diisopropylethylamine (DIPEA)
and 0.2 equiv of 1,4-(dimethylamino)pyridine (DMAP). This
step is completed in 15 min, and intermediate 2 was identified
by the appearance of one signal at δ 127.72, observed in
the 31P NMR spectra of the reaction mixture. A large excess
of the base, DIPEA, is required for the quick completion of
the reaction. Rather than carrying out the boronation step
(5) (a) Tomasz, J.; Shaw, B. R.; Porter, K.; Spielvogel, B. F. Angew.
Chem. Int. Ed. Engl. 1992, 31, 1373. (b) Sood, A.; Shaw, B. R.; Spielvogel,
B. F. J. Am. Chem. Soc. 1990, 112, 9000. (c) Shaw, B. R.; Sergueev, D. S.;
He, K.; Porter, K.; Summers, J. S.; Sergueeva, Z. A.; Rait, V. Method
Enzymol. 2000, 313, 226. (d) Summers, J. S.; Shaw, B. R. Curr. Med. Chem.
2001, 8, 1147. (e) Shaw, B. R.; Madison, J.; Sood, A.; Spielvogel, B. F.
Methods Mol. Biol. 1993, 20, 225.
(6) (a) Meyer, P.; Schneider, B.; Sarfati, S.; Deville-Bonne, D.; Guerreiro,
C.; Boretto, J.; Janin, J.; Veron, M.; Canard, B. EMBO J. 2000, 19, 3520.
(b) Selmi, B.; Boretto, J.; Sarfati, S. R.; Guerreiro, C.; Canard, B. J. Biol.
Chem. 2001, 276, 48466. (c) Deval, J.; Selmi, B.; Boretto, J.; Egloff, M.
P.; Guerreiro, C.; Sarfati, S.; Canard, B. J. Biol. Chem. 2002, 277, 42097.
(7) (a) Ludwig, J.; Eckstein, F. J. Org. Chem. 1991, 56, 1777. (b) Burgess,
K.; Cook, D. Chem. ReV. 2000, 100, 2047.
(8) (a) He, K.; Hasan, A.; Krzyzanowska, B. K.; Shaw, B. R. J. Org.
Chem. 1998, 63, 5769. (b) Krzyzanowska, B. K.; He, K.; Hasan, A.; Shaw,
B. R. Tetrahedron 1998, 54, 5119. (c) He, K.; Porter, K.; Hasan, A.; Briley,
D. J.; Shaw, B. R. Nucleic Acids Res. 1999, 27, 1788. (d) Li, P.; Shaw, B.
R. Org. Lett. 2002, 4, 2009. (e) Li, P.; Shaw, B. R. Chem. Commun. 2002,
2890.
(9) Rosowsky, A.; Kim, S. H.; Wick, M. J. Med. Chem. 1981, 24, 1177.
2402
Org. Lett., Vol. 5, No. 14, 2003

after the phosphitylation, as in the previously reported
phosphoramidite approach,5a compound 2 was treated directly
with the solution of pyrophosphate in DMF to form a cyclic
intermediate P1-acyclothymidinyl-P2,P3-dioxo-cyclotriphos-
phite 3. Formation of intermediate 3 was monitored by 31P
NMR, in which the singlet at δ 127.72 for phosphoramidite
2 was transformed to a triplet at δ 105.73 (P1, J ) 43.55
Hz) for cyclic intermediate 3 along with the appearance of
a doublet at δ -20.73 (P2,P3, J ) 43.39 Hz). Without the
addition of 1H-tetrazole, the formation of cyclotriphosphite
3 could be sluggish.10 However, the displacement reaction
by pyrophosphate was finished in 15 min when 4 equiv of
1H-tetrazole was added.
Cyclotriphosphite 3 was oxidized with iodine/pyridine/
water to yield the normal acyclothymidine triphosphate
(acycloTTP) 5a. Alternatively, an in situ boronation of
cyclotriphosphite 3 resulted in P1-acyclothymidinyl-P1-
borano-P2,P3-dioxo-cyclotriphosphate 4. The presence of the
PfB bond in cycloboranophosphate 4 was confirmed by 31
P
Figure 2. Concentration dependence of kinetic rate constants for
pre-steady-state incorporation of acycloTTP (b) and acycloTTP-
R-BH3 (9) by MMLV RT.
NMR spectra, which showed a broad peak centered at δ
90.30 for P1, characteristic of a boranophosphate group.5a,8
A slight upfield shift of the doublet at δ 24.47 (J ) 45.82
Hz) for P2 and P3 peaks in cyclic compound 4 was also
observed.7,8a-c Of several borane complexes tried for bor-
onation, a 2 M solution of borane-dimethyl sulfide in THF
gave the best results. Cycloboranophosphate 4 was finally
treated with water/triethylamine to give the ring-opened
product acycloTTP-R-BH3 5b. The addition of triethylamine
greatly reduced the time for the hydrolysis step.8 The final
products, triphosphates 5a11 and 5b, were purified by ion
exchange and HPLC with overall yields of 48% and 53%,
respectively.
Single nucleotide incorporation of acycloTTP 5a or its
analogue, acycloTTP-R-BH3 5b,12 into a 5′-HEX-modified
19-mer DNA primer was performed with MMLV RT
with use of a 27-mer DNA template. The initial and
elongated primers were separated by denaturing polyacry-
lamide gel electrophoresis and analyzed by fluorescent
imaging. Hyperbolic fitting4b of the data to the equation kobs
) kpol[acycloNTP]/(Kd + [acycloNTP]) was used to deter-
mine values of kinetic constants kpol (rate constant of
polymerization) and Kd (equilibrium constant of dissociation).
The R-BH3 substitution in acycloTTP increased the efficiency
for incorporation (kpol/Kd) of acycloTTP-R-BH3 by 9-fold in
pre-steady-state conditions with viral reverse transcriptase
(Figure 2). This difference in pre-steady-state incorporation
of acycloTTP-R-BH3 compared with acycloTTP by MMLV
RT involves an approximate 2.5-fold decrease in dissociation
constant Kd (from 90 to 36 µM) and a 3.5-fold increase in
rate constant kpol (from 1.3 × 10-3 s-1 to 4.6 × 10-3 s-1).
In conclusion, we have successfully synthesized acy-
clothymidine triphosphate 5a and acyclothymidine R-P-
boranotriphosphate 5b using a phosphoramidite approach in
good yield after isolation. The R-P-borane substitution in
acyclothymidine triphosphate results in a 9-fold increase in
the incorporation efficiency compared with the non-boronated
triphosphate in pre-steady-state conditions with viral reverse
transcriptase. These preliminary results, and the increase in
the lipophilicity imparted by the P-borane group,5e indicate
that acyclonucleoside R-P-boranotriphosphates may have
promising applications as a probe of enzyme mechanisms
and in the design of new antiviral drugs.
Acknowledgment. This work was supported by NIH
grant R01-AI-52061 to B.R.S. We thank Dr. Dmitri Sergueev
for providing the DNA template and primer.
(10) Nahum, V.; Zu¨ndorf, G.; Le´vesque, S. A.; Beaudoin, A. R.; Reiser,
G.; Fischer, B. J. Med. Chem. 2002, 45, 5384.
Supporting Information Available: Spectral data for
compounds 5a and 5b. This material is available free of
charge via the Internet at http://pubs.acs.org.
(11) (a) Nakayama, K.; Ruth, J. L.; Cheng, Y. C. J. Virol. 1982, 43,
325. (b) Compound 5a was first reported in ref 11a. No NMR data are
available in the literature, so we provide these in the Supporting Information.
(12) For preliminary investigations, acycloTTP-R-BH3 5b was used as
a mixture of R-P-diastereomers.
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