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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Org Lett. Author manuscript; available in PMC 2010 May 21.
Published in final edited form as:
PMCID: PMC2688834
NIHMSID: NIHMS111031

Synthesis of Nucleoside Mono-, Di-, and Triphosphoramidates from Solid-Phase CycloSaligenyl Phosphitylating Reagents

Abstract

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Chloromethyl polystyrene resin was reacted with 5-hydroxysalicylaldehyde in the presence of potassium carbonate to afford polymer-bound 2-hydroxybenzaldehyde. Subsequent reduction with borane solution produced polymer-bound 2-hydroxybenzyl alcohol. The reaction of immobilized 2-hydroxybenzyl alcohol with appropriate phosphitylating reagents yielded solid-phase cycloSaligenyl mono-, di-, and triphosphitylating reagents, which were reacted with unprotected nucleosides, followed by iodine oxidation, deprotection of cyanoethoxy groups, and the basic cleavage, respectively, to afford 5′-O-nucleoside mono-, di-, and triphosphoramidates in 52-73% overall yield.

Antiviral and antitumor nucleoside analogs undergo three phosphorylation steps by cellular kinases to generate nucleoside 5′-triphosphates that act as competitive inhibitors of DNA polymerases or incorporate into DNA and cause chain termination.1 The first phosphorylation step is often the rate-limiting step. Thus several nucleoside phosphoramidate derivatives have been synthesized as prodrugs with the aim of delivering the corresponding 5′-mononucleotide intracellularly and bypassing the initial phosphorylation step.2-6 A number of phosphoramidate derivatives of antiviral and antitumor nucleosides have demonstrated to have enhanced activity and reduced cytotoxicity when compared with their corresponding parent nucleosides.3,6-8 Furthermore, oligonucleotide phosphoramidates have attracted considerable attention as potential antisense agents because of their stability toward nucleases and being able to form a duplex with complementary DNA or RNA sequences with higher affinity.9,10 Catalysis of many hydrolases and nucleases also occur through nucleoside phosphoramidate intermediates.11,12 Therefore, the synthesis of nucleoside phosphoramidates and phosphoramidate-based pronucleotides and oligonucleotides are subjects of considerable interest in nucleic acid research. The facile synthesis of larger quantities of phosphoramidate derivatives is essential for studying their biological properties.

The reported solution-phase methods for the synthesis of nucleoside 5′-phosphoramidates include the reaction of nucleoside diphosphates, triphosphates, chlorophosphates, H-phosphonates, or trimethaphosphates, with amines13-16 in the presence of a base and/or a coupling reagent (e.g., N-carbodiimide derivatives13,17,18 or trimethylsilyl chloride15,19). Alternatively, highly reactive phosphoramidate precursors (e.g., phosphoryldichloride derivatives or bis(benzotriazolyl)phosphoramidates) have been used in reaction with nucleosides for the synthesis nucleoside phosphoramidates.6 These methods have one or more disadvantages, such as the requirement for the synthesis of precursor nucleoside phosphates or phosphoramidates, the poor solubility of precursors in organic solvents, tedious purification of final products from intermediates and starting reagents, and low or moderate overall yields. We have previously reported the solid-phase synthesis of nucleoside mono-, di-, and triphosphates with high regioselectivity using polymer-bound linkers of p-hydroxybenzyl alcohol or p-acetoxybenzyl alcohol.20-4

CycloSaligenyl (cycloSal)-phosphate triesters of several nucleoside analogs have been designed as a pH-driven nucleotide delivery system.25-28 As part of our ongoing efforts to synthesize organophosphorus compounds,29 we report the synthesis of immobilized cycloSal phosphitylating reagents and their application for the synthesis of nucleoside mono-, di-, and triphosphoramidates to circumvent one or more of the problems associated with the solution-phase methods. To the best of our knowledge, this is the first paper on the synthesis of polymer-bound cycloSal phosphitylating reagents. Mono-, di-, and triphosphitylating reagents were first immobilized on polystyrene resin-bound linker of 2-hydroxybenzyl alcohol. Coupling reaction of unprotected nucleosides with the immobilized reagent followed by iodine oxidation, deprotection, and basic cleavage afforded nucleoside mono-, di-, and triphosphoramidates.

The advantages of this solid-phase strategy included: (i) The immobilization of hindered phosphitylating reagents on a rigid polymer-bound linker allowed for the regioselective reaction with the most reactive hydroxyl group in the presence of an excess of unprotected nucleosides to afford monosubstituted final products; (ii) Unprotected nucleosides were used instead of precursor nucleoside phosphate derivatives; (iii) Excess of nucleosides and unreacted reagents were removed in each step by washing the resins. Furthermore, the modified linker remained trapped on the resins. This facilitated isolation and purification of monosubstituted final products; and (iv) This strategy allowed the synthesis of nucleoside 5′-O-mono-, di-, and triphosphoramidates from the same polymer-bound linker.

Scheme 1 illustrates the synthesis of diphosphitylating and triphosphitylating reagents (4 and 7). Phosphorus trichloride was subjected to reaction with 3-hydroxypropionitrile (1 equiv) in the presence of 2,6-lutidine in anhydrous THF to yield 2-cyanoethyl phosphorodichloridate (1). The subsequent reaction of 1 with diisopropylamine (1 equiv) in the presence of 2,6-lutidine afforded 2-cyanoethyl diisopropylchlorophosphoramidite 2. Addition of water (1 equiv) and 2,6-lutidine gave the intermediate 3 that was reacted with phosphorus trichloride (1 equiv) in the presence of 2,6-lutidine to afford the diphosphitylating reagent (4, 93%)

Scheme 1
Synthesis of Diphosphitylating and Triphosphitylating Reagents (4 and 7).

In a separate reaction, 2-cyanoethyl phosphorodichloridate (1) was reacted with the intermediate 3 (1 equiv) in the presence of 2,6-lutidine in anhydrous THF to yield 5. Compound 5 was immediately treated with water (1 equiv) and phosphorus trichloride (1 equiv), respectively, in the presence of 2,6-lutidine to yield the triphosphitylating reagent (7, 87%).

The diphosphitylating and triphosphitylating reagents (4 and 7) were used immediately in coupling reactions with the polymer-bound 2-hydroxybenzyl alcohol. Compounds 4 and 7 were reacted with water and the chemical structures of their dihydroxy forms were confirmed by high-resolution time-of-flight electrospray mass spectrometry.

Scheme 2 shows the synthesis of nucleoside mono-, di, and triphosphoroamidates from polymer-bound 2-hydroxybenzylalcohol (10). Chloromethyl polystyrene resin (8) was reacted with 5-hydroxysalicylaldehyde in the presence of sodium iodide and potassium carbonate to afford polymer-bound 2-hydroxybenzaldehyde (9). Reduction of the aldehyde group in 9 in the presence of borane solution (1M) produced polymer-bound 2-hydroxybenzyl alcohol (10), which was reacted with phosphorus trichloride or N,N-diisopropyl phosphoramidous dichloride in the presence of 2,6-lutidine to produce the corresponding polymer-bound cycloSal monophosphitylating reagents, 11 and 12, respectively.

Scheme 2
Synthesis of Nucleoside Monophosphoramidates 28ah, Diphosphoramidates, and Triphosphoramidates 29-30a–b on the Solid Phase Using Polymer-Bound Linker 10.

Similarly, the reaction of 10 with diphosphitylating and triphosphitylating reagents, 4 and 7, in the presence of 2,6-lutidine produced polymer-bound cycloSal diphosphitylating and triphosphitylating reagents (13 and 14), respectively. The treatment of 11 or 12 with excess of unprotected nucleosides (e.g., 3′-azido-3′-deoxythymidine (a), adenosine (b), 3′-fluoro-3′-deoxythymidine (c), 2′,3′-didehydro-2′,3′-dideoxythymidine (d), thymidine (e), 2′-deoxyadenosine (f), 2′-deoxycytidine (g), and 3′-deoxyguanidine (h)) in the presence of pyridine or 5-(ethylthio)-1H-tetrazole, respectively, gave 15a–h. Similarly, reaction of 13 and 14 with excess of 3′-azido-3′-deoxythymidine (a) and adenosine (b) in the presence of 5-(ethylthio)-1H-tetrazole afforded 16–17a,b. The most reactive hydroxyl group of unprotected nucleosides reacted selectively with hindered polymer-bound reagents (1114) when an excess of nucleoside was used in coupling reaction.

Iodine oxidation of 15a–h and 16–17a,b yielded the corresponding polymer-bound nucleosides 5′-O-monophosphate (18a–h), diphosphate (19a,b), and triphosphate triester derivatives (20a,b). The removal of the cyanoethoxy group with DBU in 1920a,b afforded the corresponding polymer-bound nucleosides 21–22a,b.

The cleavage of polymer-bound compounds 18a–h and 21–22a,b was carried out under basic conditions (NH4OH). The intramolecular cleavage mechanism of final products from (23a–h and 24–25a,b) is shown in Scheme 2. The cleavage relies on a nucleophilic attack on the phosphate triester by ammonia and a subsequent hydrolysis pathway to yield the nucleoside phosphoramidate derivatives. The reaction of ammonium hydroxide on resin 26 at the same time produced the linker-trapped resin (27), which was separated from the final products by filtration. The crude products had a purity of 68-92% and were purified on the C18 Sep-Pak cartridges to afford 5′-O-nucleoside monophosphoramidates, diphosphoramidates, and triphosphoramidates (28ah, 2930a,b, Scheme 2) in 52-73% overall yield (calculated from 11–14, Table S1, see Supporting Information). The products were characterized by 1H NMR, 13C NMR, 31P NMR, and high-resolution mass spectrometry (ESI-TOF).

This is the first report of the synthesis of solid-phase cycloSal phosphitylating reagents and their application for the preparation of nucleoside 5′-O-mono- di-, and triphosphoramidates. The solid-phase strategy allowed facile synthesis and purification of nucleoside 5′-phosphoramidate derivatives from unprotected nucleosides by removing the unreacted reagents by washing in each step.

As a typical procedure (Scheme 2), 3′-azido-3′-deoxythymidine (a, 1 mmol, 4 equiv) and 5-(ethylthio)-1H-tetrazole (4 equiv) were dissolved in dry DMSO (3 mL) and were added to swollen 13 (229 mg, 0.25 mmol, 1.09 mmol/g) in THF (5 mL). The mixture was shaken for 28 h at room temperature. The resin was collected by filtration and washed with DMSO (2 × 10 mL) and THF (2 × 10 mL), respectively, and dried under vacuum to give 16a (267 mg). Iodine solution in pyridine/water (98:2 v/v) (1.5 equiv, 1.5 mL, 0.5 M) was added to swollen resin 16a in THF (5 mL). After 15 min shaking at room temperature, the resin was collected by filtration and washed with pyridine (2 × 10 mL), THF (2 × 10 mL), and DCM (2 × 10 mL), respectively, and was dried overnight at room temperature under vacuum to give 19a (273 mg). DBU (2 mmol) was added to swollen resin 19a in THF (5 mL). After 48 h shaking of the mixture at room temperature, the resin was collected by filtration and washed with THF (3 × 15 mL) and DCM (3 × 15 mL), respectively, and dried overnight at room temperature under vacuum to give 21a (244 mg). NH4OH (30%, 3 mL) was added to swollen resin 21a in THF (3 mL). After 75 min shaking of the mixture at room temperature, the resin was collected by filtration and washed with MeOH (2 × 10 mL). The solvents of filtrate solution were immediately evaporated at room temperature. The residue was mixed with Rexyn® 101 (H) (hydrogen form, 500 mg, 5.72 meq/g) in water:dioxane (75:25 v/v, 3 mL) for 15 min. After filtration, the solvents were removed using lyophilization and the crude products were purified on C18 Sep-Pak using appropriate solvents. The solvents were evaporated and the residues were dried under vacuum at −20 °C to yield 29a.

Supplementary Material

1_si_001

Acknowledgment

We acknowledge the financial support from National Science Foundation, Grant Number CHE 0748555. We acknowledge National Center for Research Resources, NIH, Grant Number 1 P20 RR16457 for sponsoring the core facility.

Footnotes

Supporting Information Available. Experimental procedures and characterization of resins with IR and final compounds with NMR and high-resolution mass spectrometry. This material is available free of charge via the Internet at http://pubs.acs.org.

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