Inhibition of the peptidyltransferase acceptor site by 2′(3′)- O-cycloleucyl- and α-aminoisobutyryl derivatives of cytidylyl-(3′–5′)adenosine

Abstract

2′(3′)- O-( N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5′- O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2′- and 3′-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2′,5′-di- O-tetrahydropyranylcytidine 3′-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3′→5′)2′(3′)- O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3′→5′)2′(3′)- O-( α-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[ 14C]Phe-tRNA·poly(U)·70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10 −5 M puromycin) was achieved at 9.5·10 −6 M of compound 1c and 9·10 −5 M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[ 14C]Phe to the A site of peptidyltransferase in a system containing tRNA Phe·poly(U)·70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of α-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the α-substituent.