2-cyanoethyl Phosphate Research Articles

Biotinylation of a propargylated cyclic (3'-5') diguanylic acid and of its mono-6-thioated analog under "click" conditions.

Commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(propargyl)guanosine is converted to its 3'-O-levulinyl ester in a yield of 91%. The reaction of commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine with N(2)-isobutyryl-2'-O-propargyl-3'-O-(levulinyl)guanosine provides, after P(III) oxidation, 3'-/5'-deprotection, and purification, the 2'-O-propargylated guanylyl(3'-5')guanosine 2-cyanoethyl phosphate triester in a yield of 88%. Phosphitylation of this dinucleoside phosphate triester with 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole, followed by an in situ intramolecular cyclization, gives the propargylated cyclic dinucleoside phosphate triester, which is isolated in a yield of 40% after P(III) oxidation and purification. Complete removal of the nucleobases, phosphates, and 2'-O-tert-butyldimethylsilyl protecting groups leads to the desired propargylated c-di-GMP diester. Cycloaddition of a biotinylated azide with the propargylated c-di-GMP diester under click conditions provides the biotinylated c-di-GMP conjugate in an isolated yield of 62%. Replacement of the 6-oxo function of N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-levulinyl-2'-O-(propargyl)guanosine with a 2-cyanoethylthio group is effected by treatment with 2,4,6-triisopropybenzenesulfonyl chloride and triethylamine to give a 6-(2,4,6-triisopropylbenzenesulfonic acid) ester intermediate. Reaction of this key intermediate with 3-mercaptoproprionitrile and triethylamine, followed by 5'-dedimethoxytritylation, affords the 6-(2-cyanoethylthio)guanosine derivative in a yield of 70%. The 5'-hydroxy function of this derivative is reacted with commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine. The reaction product is then converted to the mono-6-thioated c-di- GMP biotinylated conjugate under conditions highly similar to those described above for the preparation of the biotinylated c-di-GMP conjugate, and isolated in similar yields.

Convenient synthesis of a propargylated cyclic (3'-5') diguanylic acid and its "click" conjugation to a biotinylated azide.

The ribonucleoside building block, N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine, was prepared from commercial N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-propargyl guanosine in a yield of 91%. The propargylated guanylyl(3'-5')guanosine phosphotriester was synthesized from the reaction of N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine with N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl] guanosine and isolated in a yield of 88% after P(III) oxidation, 3'-/5'-deprotection, and purification. The propargylated guanylyl(3'-5')guanosine phosphotriester was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole and was followed by an in situ intramolecular cyclization to give a propargylated c-di-GMP triester, which was isolated in a yield of 40% after P(III) oxidation and purification. Complete N-deacylation of the guanine bases and removal of the 2-cyanoethyl phosphate protecting groups from the propargylated c-di-GMP triester were performed by treatment with aqueous ammonia at ambient temperature. The final 2'-desilylation reaction was effected by exposure to triethylammonium trihydrofluoride affording the desired propargylated c-di-GMP diester, the purity of which exceeded 95%. Biotinylation of the propargylated c-di-GMP diester was easily accomplished through its cycloaddition reaction with a biotinylated azide derivative under click conditions to produce the biotinylated c-di-GMP conjugate of interest in an isolated yield of 62%.

Nitromethane as a scavenger of acrylonitrile in the deprotection of synthetic oligonucleotides

A novel deprotection procedure for synthetic oligonucleotides was developed to prevent nucleobase alkylation. Acrylonitrile, a side product of the deprotection of a 2-cyanoethyl phosphate protecting group and which causes nucleobase alkylation, was found to be trapped by the addition of some acidic compounds, which generate a carbanion species under the conventional deprotection conditions using aqueous NH 3. The 2-cyanoethylation of thymidine was inhibited effectively in the presence of nitromethane.

The 4-[N-Methyl-N-(2,2,2-trifluoroacetyl)amino]butyl Group as an Alternative to the 2-Cyanoethyl Group for Phosphate Protection in the Synthesis of Oligodeoxyribonucleotides

The 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl group for phosphate protection in the synthesis of oligodeoxyribonucleotides has been developed to completely prevent nucleobase alkylation by acrylonitrile that could potentially occur upon deprotection of the traditional 2-cyanoethyl phosphate protecting group. The properties of this new phosphate protecting group were evaluated using the model phosphotriester 9. The mechanism of phosphate deprotection was studied by treating 9 with concentrated NH4OH. NMR analysis of the deprotection reaction demonstrated that cleavage of the N-trifluoroacetyl group is rate-limiting. The resulting phosphotriester intermediate 13 was also shown to undergo rapid cyclodeesterification to produce O,O-diethyl phosphate 15 and N-methylpyrrolidine 16 (Scheme 2). Given the facile removal of the 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl phosphate protecting group under mild basic conditions, its utilization in oligonucleotide synthesis began with the preparation of the...

Sequencing of oligonucleotide phosphorothioates based on solid-supported desulfurization.

We described a solid-supported desulfurization procedure allowing easy access to the sequence analysis of oligonucleotide phosphorothioates. The described method is based upon selective removal of the 2-cyanoethyl phosphate protecting groups, followed by iodine-promoted desulfurization of the resulting phosphorothioate diesters. Automatic oxidation of oligonucleotide phosphorothioates, anchored via an ester linkage to a standard solid support (LCAA/CPG), is combined with Maxam-Gilbert solid-support sequencing. The overall procedure allows rapid simultaneous sequence analysis of several oligonucleotide analogs.

A Convenient Synthesis of Cyclohexylammonium 2-Chlorophenyl 2-Cyanoethyl Phosphate

A Convenient Synthesis of Cyclohexylammonium 2-Chlorophenyl 2-Cyanoethyl Phosphate

Metronidazole Phosphate—A Water-Soluble Prodrug for Parenteral Solutions of Metronidazole

To develop a parenteral solution of relatively water-insoluble metronidazole (2-methyl-5-nitro-lH-imidazole-l-ethanol), its phosphate ester was synthesized via two routes. One route utilized 2-cyanoethyl phosphate and the other utilized pyrophosphoryl tetrachloride. The first method used dicyclohexylcarbodiimide as a coupling agent and the cy-anoethyl group was removed under mild alkaline conditions. The second method was a one-step procedure in which free acid of metronidazole phosphate was isolated as a crystalline solid. The solubility of metronidazole in various solvents was determined at 25°. From the pH-depen-dence of its aqueous solubility, the pKa of the conjugate acid of metronidazole was estimated to be 2.62, which agreed well with the pKa values of other nitroimidazoles. Metronidazole phosphate behaved as a zwit-terionic compound in an acidic medium with a minimum solubility at pH 2.0. At pH 7, its solubility was ~50 times that of metronidazole. The phosphate ester was so soluble at pH higher than 7 that it was difficult to measure the solubility accurately. In human serum, the hydrolysis of metronidazole phosphate followed zero-order kinetics at an initial concentration of 0.25 mg/ml or higher, presumably due to enzyme saturation (0.035 mg/ml/hr at 37°). A reversed-phase HPLC procedure was adopted to monitor the appearance of metronidazole and the disappearance of metronidazole phosphate. Subcutaneous administration of metronidazole phosphate to rats produced a blood level of bioactivity comparable to that observed after administration of metronidazole.

The synthesis of unlabeled and 32P-labeled phosphocitrate and analytical systems for its identification

A method for the synthesis of phosphocitrate is described using 2-cyanoethyl phosphate to phosphorylate triethyl citrate. Following alkaline hydrolysis of the coupled intermediate, phosphocitrate was purified by ion-exchange chromatography on an AG 1-X8 (HCO 3 −) column. The method was also used to prepare [ 32P]phosphocitrate. Phosphocitrate was characterized by 1H NMR, 31P NMR, and 13C NMR spectroscopy. In addition methods for thin-layer chromatography and enzyme assay are detailed for the detection of phosphocitrate.

A new chemical synthesis of 2-amino-( N- d-ribofuranosyl)acetamide 5′-phosphate

2-amino-( N- d-ribosyl)acetamide 5′-phosphate (GAR, 10) has been prepared in a readily scaled-up synthesis from 2,3- O-isopropylidene- d-ribofuranosylamine p-toluenesulfonate ( 3) by condensation with the mixed anhydride of N-(benzyloxy-carbonyl)glycine, followed by phosphorylation with 2-cyanoethyl phosphate and removal of the protecting groups. By varying the conditions, the α- 4 and the β- 5 anomers could be obtained and separated from each other. Anomerization occurs upon removing the O-isopropylidene group from either 7 or 8, so that the final compound ( 10) is an equimolar mixture of both anomers, of which only one is active toward GAR-transformylase.

Purines, pyrimidines, and imidazoles. Part XXXI. Some reactions of the allylic group in the plant cell-division promoting factor, zeatin, and its nucleoside and nucleotide derivatives

Attempted diphosphorylation of 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9-(2′,3′-O-isopropylidene-β-D-ribofuranosyl) purine with 2-cyanoethyl phosphate and dicyclohexylcarbodi-imide in pyridine followed by removal of protecting groups gave 6-(3-methyl-4-pyridiniobut-trans-2-enylamino)-9-β-D-ribofuranosylpurine 5′-phosphate, which was hydrolysed by an alkaline phosphatase to the riboside and by acid to the aglycone. The latter compound was synthesised from zeatin and methanesulphonyl chloride in pyridine, and also by reaction of 6-(4-chloro-3-methylbut-trans-2-enylamino)purine, (prepared from zeatin and methanesulphonyl chloride in NN-dimethylformamide) or, less readily, O-p-tolylsulphonylzeatin (from zeatin and toluene-p-sulphonyl chloride) and pyridine. Structures assigned to the compounds were confirmed by u.v. absorption, 1H n.m.r., and mass spectroscopic studies. The related 3-carbamoyl-1-[2-methyl-4-(purin-6-ylamino)but-trans-2-enyl]pyridinium chloride was obtained in a similar manner and the quaternisation of pyridine by allyl alcohols including zeatin in the presence of 2-cyanoethyl phosphate and dicyclohexylcarbodi-imide was shown to be general.

Synthesis of nucleotides. II. Selective phosphorylation of ribonucleosides with 2-cyanoethyl phosphate

2-Cyanoethyl phosphate employed as an efficient phosphorylating agent in the synthesis of nucleotide was found to be an excellent catalyst for the conversion of ribonucleosides to their 2', 3'-O-isopropylidene derivatives. This fact has led to the establishment of a novel, single-step conversion of ribonucleosides to 5'-ribonucleotides in a high yield using 2-cyanoethyl phosphate, acetone, pyridine, and dicyclohexylcarbodiimide without isolation of the 2', 3'-O-isopropylidene derivatives.

2-Cyanoethyl Phosphate and its Use in the Synthesis of Phosphate Esters1

2-Cyanoethyl Phosphate and its Use in the Synthesis of Phosphate Esters¹