PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Org Biomol Chem. Author manuscript; available in PMC 2010 August 26.
Published in final edited form as:
PMCID: PMC2928660
NIHMSID: NIHMS225754

Synthesis of nucleoside 5′-O-α,β-methylene-β-triphosphates and evaluation of their potency towards inhibition of HIV-1 reverse transcriptase

Abstract

A polymer-bound α, β-methylene-β-triphosphitylating reagent was synthesized and subjected to reactions with unprotected nucleosides, followed by oxidation, deprotection of cyanoethoxy groups, and acidic cleavage to afford nucleoside 5′-O-α, β-methylene-β-triphosphates. Among all the compounds, cytidine 5′-O-α, β-methylene-β-triphosphate inhibited RNase H activity of HIV-1 reverse transcriptase with a Ki value of 225 μM.

Modified nucleoside triphosphates have received much attention as mimics of naturally occurring deoxyribo- and ribonucleoside triphosphates, as probes in several biochemical pathways involving DNA and RNA synthesis, and as potential diagnostic and therapeutic agents.1,2 The structural similarity of modified nucleotides to natural nucleoside triphosphates make them useful reagents as substrates or inhibitors for DNA or RNA polymerases.3,4 Although most natural polymerase enzymes incorporate natural nucleoside triphosphates into nucleic acids, there are certain polymerases that are capable of incorporating unnatural nucleoside triphosphates into nucleic acids.57

A number of approaches have been focused on modifications and/or substitution on the base,89 carbohydrate,1013 and linear triphosphate moieties1417 to design modified nucleotides for diverse applications in nucleic acid and antiviral research.

Early in the life cycle of human immunodeficiency virus type 1 (HIV-1), viral RNA is reverse transcribed into double stranded DNA for integration into the genome of the infected cell.18 This process is catalyzed by reverse transcriptase (RT), a virus encoded heterodimeric enzyme composed of 66 and 51 kD subunits (p66 and p51), possessing DNA polymerase and ribonuclease H (RNase H) activities.19 DNA polymerase activity is required for the synthesis of RNA: DNA heteroduplex from the single stranded viral RNA. Whereas, RNase H activity is responsible for hydrolyzing the RNA strand of RNA: DNA heteroduplex molecules that are generated during reverse transcription and create the primer for plus strand DNA synthesis.20 Both DNA polymerase and RNase H activities of HIV-1 RT have been considered as potential targets for antiretroviral therapy. 3′-Substituted modified nucleoside triphosphate analogues (e.g., 3′-azidodeoxythymidine triphosphate) are incorporated into DNA, behave as chain terminators, and inhibit DNA synthesis by HIV-1 RT. On the other hand, the discovery of potent and selective inhibitors of HIV-1 RNase H has been challenging, and inhibitors of this enzyme function have yet to reach clinical development stage.18 Although there are more than a dozen FDA approved drugs that inhibit the polymerase function of HIV-1 RT, there is not a single RNase H inhibitor in clinics. Therefore, a potential RNase H inhibitor can serve as a mechanistic probe and/or a lead compound against HIV-1 RT. Nucleotide analogues have been described as inhibitors of HIV-1 RNase H activity.21,22

Less attention has been given to the synthesis of modified nucleotides containing branched triphosphate groups that can have diverse applications in studying the enzymes (e.g., RNase H) that use natural nucleoside triphosphates. Previously, we reported the synthesis of 5′-O-nucleoside β-triphosphates using a solid-phase β-triphosphitylating reagent.23 Herein, we report the solid-phase synthesis of 5′-O-nucleoside β-triphosphates containing α,β-methylene triphosphate bridge by using a novel solid-phase phosphitylating reagent. The unusual branched triphosphate in nucleoside triphosphate analogues may be useful in elucidation of enzymatic functions and mechanism. Furthermore, the presence of an α, β-methylene group in the modified nucleotide may provide more stability to cleavage by cellular hydrolytic enzymes. We determined the potency of the modified analogues towards HIV-1 RT polymerase and RNase H activity.

α,β-Methylene-β-triphosphitylating reagent was first immobilized on polystyrene resin-bound linker of p-acetoxybenzyl alcohol. Coupling reactions of unprotected nucleosides with the immobilized reagent followed by oxidation, deprotection, and cleavage afforded nucleoside 5′-O-α,β-methylene-β-triphosphates. The advantages of this solid-phase strategy included major 5′-O-monophosphitylation, the facile isolation and purification of products, removal of unreacted reagents and starting materials in each step, and trapping of the linker on the resin in the final cleavage reaction.

Scheme 1 illustrates the synthesis of α,β-methylene β-triphosphitylating reagent 6. The reaction of bis(dichloro-phosphino)methane (1) with diisopropylamine (1 equiv) in the presence of 2,6-lutidine (1 equiv) in anhydrous THF afforded 2. Because of the bulkiness of the N,N-diisopropylamine group, the replacement of −Cl groups in the less hindered phosphorous group in 2 is significantly more desirable. Reaction of 2 with 3-hydroxypropionitrile (2 equiv) in the presence of 2,6-lutidine (2 equiv) afforded 3, which was subjected to reaction with water (1 equiv) and 2,6-lutidine (1 equiv) to produce the intermediate 4.

Scheme 1
Synthesis of α,β-methylene-β-triphosphitylating reagent 6.

In a separate reaction, phosphorus trichloride was reacted with 3-hydroxypropionitrile (1 equiv) in the presence of 2,6-lutidine (1 equiv) in anhydrous THF to yield 2-cyanoethoxyphosphorodichloridite (5), which was reacted with 4 (1 equiv) to yield α,β-methylene-β-triphosphitylating reagent 6, which was immediately used in the coupling reaction with polymer-bound p-acetoxybenzyl alcohol 7. The chemical structure of 6 was confirmed by high-resolution time-of-flight electrospray mass spectrometry (ESI-TOF), but obtaining NMR for this compound was not feasible because of its rapid decomposition. Compound 6 was immediately used for the next coupling reaction with polymer-bound p-acetoxybenzyl alcohol 7.

We have previously reported the synthesis of aminomethylpolystyrene resin-bound p-acetoxybenzyl alcohol 7 (Scheme 2).24 Our earlier studies revealed the application of polymer-bound p-acetoxybenzyl alcohol linkers in the synthesis of a diverse number of compounds.25 Polymer-bound linker 7 was selected for immobilization of reagent 6.

Scheme 2
Synthesis of nucleoside 5′-O-α,β-methylene-β-triphosphates 15a–f using polymer-bound linker 7.

Scheme 2 shows the synthesis of nucleoside 5′-O-α,β-methylene-β-triphosphates. Aminomethyl polystyrene resin-bound linker of p-acetoxybenzyl alcohol 7 was reacted with reagent 6 in the presence of 2,6-lutidine to produce the corresponding polymer-bound α,β-methylene-β-triphosphitylating reagent (8). The treatment of excess of unprotected nucleosides (adenosine (a), 3′-azido-3′-deoxythymidine (b), 3′-fluoro-3′-deoxythymidine (c), 2′,3′-didehydrothymidine (d), cytidine (e), and inosine (f)) in the presence of 5-(ethylthio)-1H-tetrazole gave 9a–f. Oxidation with t-butyl hydroperoxide followed by removal of the cyanoethoxy group with DBU, afforded the corresponding polymer-bound nucleosides 5′-O-α,β-methylene-β-triphosphosphate diester derivatives, 11a–f. The cleavage of polymer-bound compounds was carried out under acidic conditions, DCM/TFA/water/EDT (72.5: 23: 2.5: 2 v/v/v/v). The intramolecular cleavage mechanism of final products from 12a–f is shown in Scheme 2. The linker-trapped resin (13) was separated from the final products by filtration and was converted to 14 by reaction with potassium hydroxide. The crude products had a purity of 68–83% and were purified on the C18 Sep-Pak cartridges to afford 5′-O-nucleoside α,β-methylene-β-triphosphates (15a–f, Scheme 2) in 47–73% overall yield (calculated from 8, 212–344 mg, Table S1, see the Supporting Information).

Nucleoside 5′-O-α,β-methylene-β-triphosphates were synthesized with high selectivity as a result of this sequence because of the presence of the phosphitylating reagent on the solid support having a hindered structure, thereby allowing for the regioselective reaction for unprotected nucleosides (e.g., adenosine, inosine, and cytidine). The most reactive hydroxyl group of unprotected nucleosides reacted selectively with hindered polymer-bound reagents when an excess of nucleoside was used. There was no need to protect the free amino group in adenosine and cytidine. The compounds were characterized by 1H NMR, 13C NMR, 31P NMR, ESI-TOF, and phosphorus elemental analysis. 31P NMR indicated the presence of only one diastereoisomer, suggesting that one of the diastereoisomers of each compound was purified from the crude as the major product. On the other hand, there is a slight possibility that diastereoisomers may not be distinguishable by 31P NMR because of overlapping peaks.

Purification of HIV-1 RT and enzyme assays was performed according to the previously reported procedures.2628 Initially, these compounds were screened towards the polymerase and RNase H activity of HIV-1 RT at a fixed concentration of the compound (1 mM). In comparison to the wild type enzyme, the polymerase activity was not affected in the presence of these compounds 15a–f, however a few compounds exhibited inhibitory activity against RNase H function. The results of the polymerase assay are presented in Figure S1 (see Supporting Information).

The results of the RNase H analysis are presented in Fig. 1A. Lane W represents the RNase H cleavage products representative of the primary and secondary cleavages in the absence of any inhibitor. Lanes a–f represent the RNase H cleavage products in the presence of the compounds 15a–f, respectively. Although compounds 15a–d and 15f did not show any inhibitory activity, the RNase H cleavage of HIV-1 RT was inhibited by 5′-O-cytidine α,β-methylene-β-triphosphate (15e) (Fig. 1A, Lane e). To further evaluate the inhibitory potency of 15e, RNase H assay was performed at increasing concentrations of 15e. Since RNase H inhibition was not observed up to 100 μM concentration, the results from 100 μM–1mM are presented in Fig. 1B.

Fig. 1
A. RNase H analysis of HIV-1 RT in the presence of compounds 15a–e. Lane w represents no inhibitor, lane a (15a), lane b (15b), lane c (15c), lane d (15d), lane e (15e), and lane f (15f), at 1 mM concentrations. B. RNase H activity as a function ...

To further characterize the nature of inhibition, we performed Dixon analysis for the compound 15e.29a The results of this analysis indicated a competitive mode of inhibition with a Ki value of 225 μM (Fig. 2). Subsequently, the IC50 value was calculated to be 585 μM for compound 15e by the Cheng–Prusoff equation29b for competitive inhibition [i.e., K i = IC50/(1+ [S]/Km)] towards the RNase H activity of HIV-1 RT.

Fig. 2
Inhibition kinetics analyses of compound 15e with RNase H of HIV-1 RT. The enzyme activity was estimated using the substrate at 200 nM (▲) and 400 nM (■) at increasing concentrations of 15e. Rate of reaction represents rate of RNase H ...

To the best of our knowledge, this is the first report of the synthesis of nucleoside 5′-O-α,β-methylene-β-triphosphates. This solid-phase methodology using novel polymer-bound phosphitylating reagent (8) allowed for the expeditious development of these analogues in a short synthetic route without the need for the nucleoside phosphate precursors or protected nucleosides. The solid-phase strategy offered the advantages of monosubstitution, high selectivity, and facile isolation and purification of products. Further exploring and optimization of 5′-O-cytidine α,β-methylene-β-triphosphate is required to design cell-permeable compounds that can potentially inhibit the RNase H activity of HIV-1 RT at lower concentrations.

Supplementary Material

Supplemental data

Acknowledgments

We acknowledge the financial support from National Science Foundation, Grant Number CHE 0748555. The work at NCI was supported in part by the Intramural Research Program of the NIH, NCI and Center for Cancer Research.

Footnotes

Electronic supplementary information (ESI) available: Experimental procedures, characterization of resins with IR and final compounds with NMR, high-resolution mass spectrometry, and quantitative phosphorus analysis, DNA polymerase assay results.

Notes and references

1. Spelta V, Mekhalfia A, Rejman D, Thompson M, Blackburn GM, North RA. Br J Pharmacol. 2003;140:1027. [PMC free article] [PubMed]
2. Wang G, Boyle N, Chen F, Rajappan V, Fagan P, Brooks JL, Hurd T, Leeds JM, Rajwanshi VK, Jin Y, Prhavc M, Bruice TW, Cook PD. J Med Chem. 2004;47:6902. [PubMed]
3. Arabshahi L, Khan NN, Butler M, Noonan T, Brown NC, Wright GE. Biochemistry. 1990;29:6820. [PubMed]
4. Ono K, Nakane H, Herdewijn P, Balzarini J, De Clercq E. Mol Pharmacol. 1989;35:578. [PubMed]
5. Joyce CM. Proc Natl Acad Sci U S A. 1997;94:1619. [PubMed]
6. Porter KW, Tomasz J, Huang F, Sood A, Shaw BR. Biochemistry. 1995;34:11963. [PubMed]
7. Huang Y, Eckstein F, Padilla R, Sousa R. Biochemistry. 1997;36:8231. [PubMed]
8. Kawate T, Allerson CR, Wolfe JL. Org Lett. 2005;7:3865. [PubMed]
9. Hoffmann C, Genieser HG, Veron M, Jastorff B. Bioorg Med Chem Lett. 1996;6:2571.
10. Wu W, Meyers CLF, Borch RF. Org Lett. 2004;6:2257. [PubMed]
11. Chong Y, Gumina G, Mathew JS, Schinazi RF, Chu CK. J Med Chem. 2003;46:3245. [PubMed]
12. von Janta-Lipinski M, Costisella B, Ochs H, Hubscher U, Hafkemeyer P, Matthes E. J Med Chem. 1998;41:2040. [PubMed]
13. Anastasi C, Quelever G, Burlet S, Garino C, Souard F, Kraus JL. Curr Med Chem. 2003;10:1825. [PubMed]
14. Okruszek A, Olesiak M, Balzarini J. J Med Chem. 1994;37:3850. [PubMed]
15. Ludwig J, Eckstein F. Nucleosides, Nucleotides Nucleic Acids. 1991;10:663.
16. Ludwig J, Eckstein F. J Org Chem. 1991;56:5860.
17. Ma QF, Bathurst IC, Barr PJ, Kenyon GL. J Med Chem. 1992;35:1938. [PubMed]
18. Telesnitsky A, Goff SP. In: Retroviruses. Coffin JM, Hughes SH, Varmus HE, editors. Cold Spring Harbor Laboratory Press; Plainview, New York: 1997. p. 121.
19. Le Grice SF. In: Reverse transcriptase. Skalka AM, Goff S, editors. Cold Spring Harbor Laboratory Press; Plainview, New York: 1993. p. 163.
20. Klumpp K, Mirzadegan T. Curr Pharm Des. 2006;12:1909. [PubMed]
21. Allen SJW, Krawczyk SH, Mcgee LR, Bischofberger N, Mulato AS, Cherrington JM. Antivir Chem Chemother. 1996;7:37.
22. Tan CK, Civil R, Milan AM, So AG, Downey KM. Biochemistry. 1991;30:4831. [PubMed]
23. Ahmadibeni Y, Parang K. J Org Chem. 2006;71:5837. [PubMed]
24. Kumar A, Ye G, Ahmadibeni Y, Parang K. J Org Chem. 2006;71:7915. [PMC free article] [PubMed]
25. (a) Parang K, Fournier EJL, Hindsgaul O. Org Lett. 2001;3:307. [PubMed] (b) Parang K. Bioorg Med Chem Lett. 2002;12:1863. [PubMed] (c) Ahmadibeni Y, Parang K. J Org Chem. 2005;70:1100. [PubMed] (d) Ahmadibeni Y, Parang K. Org Lett. 2005;7:5589. [PubMed] (e) Ahmadibeni Y, Parang K. Angew Chem, Int Ed. 2007;46:4739. [PubMed] (f) Ahmadibeni Y, Parang K. Org Lett. 2007;9:4483. [PubMed] (g) Ahmadibeni Y, Parang K. Curr Protoc Nucleic Acid Chem. 2008;Chapter 13(Unit 13.8) [PubMed] (h) Ahmadibeni Y, Parang K. Curr Protoc Nucleic Acid Chem. 2009;Chapter 13(Unit 13.9) [PubMed] (i) Ahamadibeni Y, Tiwari R, Sun G, Parang K. Org Lett. 2009;11:2157. [PubMed]
26. Le Grice SF, Cameron CE, Benkovic SJ. Methods Enzymol. 1995;262:130. [PubMed]
27. Dash C, Rausch JW, Le Grice SFJ. Nucleic Acids Res. 2004;32:1539. [PMC free article] [PubMed]
28. Dash C, Fisher TR, Prasad VR, Le Grice S. J Biol Chem. 2006;281:27873. [PubMed]
29. (a) Dixon M. Biochem J. 1953;55:170–171. [PubMed] (b) Cheng Y, Prusoff WH. Biochem Pharmacol. 1973;22:3099. [PubMed]