HPLC and mass spectrometry analysis of the enzymatic hydrolysis of anti-HIV pronucleotide diastereomers.
In one current strategy to develop membrane-soluble pronucleotides, the phosphoramidate derivatives of the approved anti-HIV nucleosides 2',3'-didehydro-3'-deoxythymidine (d4T), 3'-azido-3'-deoxythymidine (AZT), (-)-beta-L-2',3'-dideoxy-3'- thiacytidine (3TC), and 2',3'-dideoxyadenosine (ddA) exhibit promising antiviral activity. However, the non-stereoselective synthetic route results in a mixture of diastereoisomers, which differ in the configuration of the phosphorus chiral center. Since it is believed that enzymatic ester hydrolysis is the first step in the intracellular activation of these prodrugs and that this process could be dependent on the stereochemistry at the phosphorus center, analytical methods must be developed. In the present work, in vitro evaluation of the selectivity of pig liver esterase (PLE) towards each diastereomer of d4T, AZT, 3TC, and ddA prodrugs has been investigated, applying our recently published HPLC-MS procedure using a polysaccharide-type chiral stationary phase. This method has been used to analyze the products of the PLE-catalyzed hydrolysis of the pronucleotides. It was found that both diastereomers of the four prodrugs were substrates for PLE.
Mesplet
Nathalie
N
Institut de Chimie Organique et Analytique, UMR CNRS 6005, Université d'Orléans, 45067 Orléans Cedex 2, France.
Saito
Yoshio
Y
Morin
Philippe
P
Agrofoglio
Luigi A
LA
D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D019380:Anti-HIV Agents / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004950:Esterases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D009705:Nucleosides / Q000737:chemistry* / Q000378:metabolism*; D010756:Phosphoric Acids / Q000737:chemistry / Q000378:metabolism; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors
D000577:Amides; D019380:Anti-HIV Agents; D009705:Nucleosides; D010756:Phosphoric Acids; D011355:Prodrugs; C011067:phosphoramidic acid; D004950:Esterases