Synthesis of Isotopically Labeled P-Site Substrates for the Ribosomal Peptidyl Transferase Reaction

Abstract

Isotopomers of the ribosomal P-site substrate, the trinucleotide peptide conjugate CCA-pcb (Zhong, M.; Strobel, S. A. Org. Lett. 2006, 8, 55-58), have been designed and synthesized in 26-35 steps. These include individual isotopic substitution at the alpha-hydrogen, carbonyl carbon, and carbonyl oxygen of the amino acid, the O2' and O3' of the adenosine, and a remote label in the N3 and N4 of both cytidines. These isotopomers were synthesized by coupling cytidylyl-(3',5')-cytidine phosphoramidite isotopomers as the common synthetic intermediates, with isotopically substituted A-Phe-cap-biotin (A-pcb). The isotopic enrichment is higher than 99% for 1-13C (Phe), 2-2H (Phe), and 3,4-15N2 (cytidine), 93% for 2'/3'-18 O (adenosine), and 64% for 1-18 O (Phe). A new synthesis of highly enriched [1-18 O2]phenylalanine has been developed. The synthesis of [3'-18 O]adenosine was improved by Lewis acid aided regioselective ring opening of the epoxide and by an economical SN2-SN2 method with high isotopic enrichment (93%). Such substrates are valuable for studies of the ribosomal peptidyl transferase reaction by complete kinetic isotope effect analysis and of other biological processes catalyzed by nucleic acid related enzymes, including polymerases, reverse transcriptases, ligases, nucleases, and ribozymes.