The chemical step of protein synthesis, peptide bond formation, is catalyzed by the large subunit of the ribosome. Crystal structures have demonstrated that the active site for peptide bond formation is composed entirely of RNA1. Recent work has focused on how an RNA active site is able to catalyze this fundamental biological reaction at a suitable rate for protein synthesis. Based on the absence of important ribosomal functional groups2, lack of a dependence on pH3, and the dominant contribution of entropy to catalysis4, it has been suggested that the role of the ribosome is limited to bringing the substrates into close proximity. Alternatively, the importance of the 2′-hydroxyl of the peptidyl-tRNA5 and a Bronsted coefficient near zero6 were taken as evidence that the ribosome coordinates a proton-transfer network. Here we report the transition state of peptide bond formation based upon kinetic isotope effect analysis at five positions at the reaction center of a peptidyl-tRNA mimic. Our results indicate that in contrast to the uncatalyzed reaction, formation of the tetrahedral intermediate and proton-transfer from the nucleophilic nitrogen both occur in the rate-limiting step. Unlike previous proposals, the reaction is not fully concerted, instead breakdown of the tetrahedral intermediate occurs in a separate fast step. This suggests that in addition to substrate positioning, the ribosome is contributing to chemical catalysis by changing the rate-limiting transition state.

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Purine alkylations have been plagued with formation of mixtures of N9 (usually desired), N7, and other regioisomers. We have developed methods for synthesis of 6-(azolyl)purine derivatives whose X-ray crystal structures show essentially coplanar conformations of the linked azole-purine rings. Such ring orientations position the C-H of the azole above N7 of the purine, which results in protection of N7 from alkylating agents. Treatment of 6-(2-butylimidazol-1-yl)-2-chloropurine (9) with sodium hydride in DMF followed by addition of ethyl iodide resulted in exclusive formation of 6-(2-butylimidazol-1-yl)-2-chloro-9-ethylpurine (10), whereas identical treatment of 2-chloro-6-(4,5-diphenylimidazol-1-yl)purine (11) produced a regioisomeric mixture 12/13 (N9/N7, approximately 5:1). The linked imidazole and purine rings are coplanar in 9 (the butyl side chain is extended away from the purine ring and C-H is over N7) but are rotated approximately 57 degrees in 11, and the more bulky azole substituent in 11 did not prevent formation of the minor N7 regioisomer 13. Access to various regioisomerically pure 9-alkylpurines is now readily available.