ENZYMATIC PREPARATION OF [2,4,5,6,8-C-14, 5'-H-3]GUANOSINE FOR USE INBIOSYNTHETIC-STUDIES OF PTERINOID AND FLAVIN COMPOUNDS

To aid in investigations on the biosynthesis of pterinoid compounds, a procedure was developed for the preparation of double-labeled guanosi ne with H-3 in the ribose moiety and C-14 ill the guanine ring. Ribose , as ribose-1-phosphate, was removed from [2,8,5'-H-3]-adenosine in a reaction using purine nucleoside phosphorylase and was coupled to unla beled guanine using the same enzyme. The procedure was accomplished in one tube with no purification of intermediates necessary. The final p roduct, [5'-H-3]guanosine, was purified by affinity chromatography usi ng a boronate column. The overall yield of labeled guanosine is about 91%. HPLC analysis indicates the radiochemical purity at 98%. The puri ty of the guanosine makes it suitable for in vivo studies of flavins, pterins, and other guanosine-derived compounds. A doubly labeled prepa ration was achieved by the addition of [5'-H-3]guanosine to C-14-label ed guanosine in which only the guanine ring is labeled. The latter was formed in a manner similar to the one above. In this case, the labele d ribose moiety was enzymatically removed from [U-C-14]guanosine and r eplaced with unlabeled ribose. (C) 1994 Academic Press, Inc.