Synthesis of isotopically labeled puromycin derivatives for kinetic isotope effect analysis of ribosome catalyzed peptide bond formation

Abstract

The mechanism by which the ribosome catalyze peptide bond formation remains controversial. Here we describe the synthesis of dinucleotides that can be used in kinetic isotope effect experiments to assess the transition state of ribosome catalyzed peptide bond formation. These substrates are the isotopically labeled dinucleotide cytidylyl-(3′-5′)-3′-amino-3′-deoxy-3′- l-phenylalanyl- N 6, N 6-dimethyladenosine (Cm 6A NPhe-NH 2) and cytidylyl-(3′-5′)-3′-amino-3′-deoxy-3′-( l-2-hydroxy-3-phenylpropionyl)- N 6, N 6-dimethyladenosine (Cm 6A NPhe-OH). These substrates are active in peptide bond formation and can be used to measure kinetic isotope effects in ribosome catalyzed protein synthesis.