ABSTRACT
Adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD), an NH analog of ADP-ribose, was chemically synthesized and shown to be a potent and specific inhibitor of poly-(ADP-ribose) glycohydrolase. The synthetic starting material was the protected pyrrolidine, (2R,3R,4S)-1-(benzyloxycarbonyl)-2-(hydroxymethyl)pyrrolidine-3,4-diol 3,4-O-isopropylidene acetal. This starting pyrrolidine was phosphorylated, coupled to adenosine 5'-monophosphate, and deprotected, yielding the title inhibitor ADP-HPD. ADP-HDP was shown to inhibit the activity of poly(ADP-ribose) glycohydrolase by 50% (IC50) at 0.12 microM, a value 1000-times lower than the IC50 of the product, ADP-ribose. The NAD glycohydrolase from Bungarus fasciatus venom was less sensitive to inhibition by ADP-HPD, exhibiting an IC50 of 260 microM. ADP-HPD did not inhibit either poly(ADP-ribose) polymerase or NAD:arginine mono(ADP-ribosyl)-transferase A at inhibitor concentrations up to 1 mM. At low ADP-HPD concentration, inhibition was therefore shown to be highly specific for poly(ADP-ribose) glycohydrolase, the hydrolytic enzyme in the metabolism of ADP-ribose polymers.
Subject(s)
Adenosine Diphosphate/analogs & derivatives , Enzyme Inhibitors , Glycoside Hydrolases/antagonists & inhibitors , Adenosine Diphosphate/pharmacology , Animals , Cattle , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Kinetics , NAD+ Nucleosidase/antagonists & inhibitors , Thymus Gland/enzymologyABSTRACT
(2R,3R,4S)-2-Hydroxymethylpyrrolidine-3,4-diol (1,4-dideoxy-1,4-imino-D-ribitol) was synthesized in five steps from N-protected (2S)-3,4-dehydroproline methyl esters. The stereoselective reaction of osmium tetraoxide with dehydroproline derivatives gave high yields of (2S,3R,4S)-3,4-dihydroxyprolines (2,3-trans-3,4-cis-3,4-dihydroxy-L-prolines) accompanied by small amounts (< 15%) of the diastereomeric (2S,3S,4R)-3,4-dihydroxyprolines (2,3-cis-3,4-cis-3,4-dihydroxy-L-prolines). The mixture of the diastereomeric glycols was converted into the isopropylidene acetals, and the isomers separated efficiently on a preparative scale. The resulting protected (2S,3R,4S)-3,4-dihydroxyproline methyl ester was reduced (LiBH4) to the 2-hydroxymethylpyrrolidine and deprotected, resulting in the production of (2R,3R,4S)-2-hydroxymethylpyrrolidine-3,4-diol in high yield and in high purity. The 1H and 13C NMR signals of the product have been unambiguously assigned using two-dimensional NMR techniques, and the identity of the title pyrrolidine confirmed by comparisons of its spectra with those reported for the authentic material.