ABSTRACT
The synthesis of N4-benzoyl-5'-O-dimethoxytrityl-2',3'-dideoxy-3'-thiocytidine and its phosphorothioamidite is described for the first time, together with a shortened procedure for the preparation of 5'-O-dimethoxytrityl-3'-deoxy-3'-thiothymidine and its corresponding phosphorothioamidite. The first fully automated coupling procedure for the incorporation of a phosphorothioamidite into a synthetic oligodeoxynucleotide has been developed, which conveniently uses routine activators and reagents. Coupling yields using this protocol were in the range of 85-90% and good yields of singularly modified oligonucleotides were obtained. Coupling yields were also equally good when performed on either a 0.2 or 1 micro mol reaction column, thus facilitating large scale syntheses required for mechanistic studies.
Subject(s)
Dideoxynucleosides/chemistry , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Phosphates/chemistry , Thymidine/analogs & derivatives , Thymidine/chemistry , Zalcitabine/analogs & derivatives , Zalcitabine/chemistry , Automation/methods , Chromatography, High Pressure Liquid , Dideoxynucleosides/chemical synthesis , Dideoxynucleotides , Oligodeoxyribonucleotides/isolation & purification , Organothiophosphorus Compounds/chemical synthesis , Organothiophosphorus Compounds/chemistry , Organothiophosphorus Compounds/isolation & purification , Thionucleosides/chemical synthesis , Thionucleosides/chemistry , Thymidine/chemical synthesis , Thymidine/isolation & purification , Zalcitabine/chemical synthesis , Zalcitabine/isolation & purificationABSTRACT
A liquid chromatographic method capable of separating and quantitating the stereoisomers of zalcitabine has been developed and validated. The separation was achieved with an Astec Cyclobond I--RSP column and a mobile phase of 0.25% triethylamine in water adjusted to a pH of 6.5 with glacial acetic acid. All enantiomers were found to exhibit a linear response in the range of 0.1-10% in the presence of 100% zalcitabine. Precision of analysis was found to be less than 1.5% at a level of 1% relative to zalcitabine. The limit of detection for two of the three enantiomeric impurities was determined to be 0.05% relative to zalcitabine. The detection limit for the third was found to be 0.1%. This method was successfully applied to the analysis of reference standards and several production scale batches. All of these materials were found to be stereochemically pure to a level of 99.8% or better.
Subject(s)
Zalcitabine/isolation & purification , Chromatography, High Pressure Liquid , Cyclodextrins , Hydrogen-Ion Concentration , Solvents , StereoisomerismABSTRACT
The experimental anti-AIDS glycerophosphatidic acid: nucleoside (sn-1/sn-2 diacylglycerol:dideoxynucleotide) drugs 3'-azido-3'-deoxythymidine monophosphate diglyceride (AZT-MP-DG) and 2',3'-dideoxycytidine monophosphate diglyceride (ddC-MP-DG) were isolated and purified by reversed-phase high-performance liquid chromatography (HPLC). The chromatographic separation was based on the glycerophospholipid moiety of the drugs and detection of the nucleoside component. The separations were optimized on method development columns packed with the stationary phase to be used in the micro-preparative column and monitored by a UV detector. Fractions were collected and analyzed for purity by analytical-scale HPLC and by thin-layer chromatography (TLC). The purity of the recovered drugs based on UV and light-scattering detection and on TLC was greater than 99%. The purified compounds were isolated for studies on structure confirmation, physical, biophysical and formulation properties and anti-HIV efficacy in culture.
