Protein synthesis with conformationally constrained cyclic dipeptides

Abstract

We have synthesized several conformationally constrained dipeptide analogues as possible substrates for incorporation into proteins. These have included three cyclic dipeptides formed from Boc derivatives of 2,4-diaminobutyric acid, ornithine and lysine, having 5-, 6-, and 7-membered lactam rings, respectively. These dipeptides were used to activate a suppressor tRNA transcript, the latter of which had been prepared by in vitro transcription. Using modified E. coli ribosomes described previously, these activated suppressor tRNAs enabled the incorporation of the three cyclic dipeptides into dihydrofolate reductase (DHFR) at positions 18 and 49. The suppression yields increased with increasing lactam ring size and were found to proceed in suppression yields ranging from 3.4 to 8.9% at two different protein sites for the 5-, 6- and 7-membered lactam dipeptides. The greater facility of incorporation of the 7-membered lactam prompted us to prepare two 7-membered cyclic acylhydrazides (4 and 5) by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)-mediated cyclization of amino acids having selectively protected hydrazine functional groups in their side chains. In common with the lactam dipeptides, acylhydrazide dipeptides 4 and 5 could be used to activate the same suppressor tRNA transcript and to incorporate the cyclic dipeptides into DHFR. They were incorporated into the same two DHFR sites in suppression yields ranging from 8.3 to 11.2%.

Keywords: Conformational constraint; Dipeptides; Lactam and acyl hydrazide cassettes; Non-proteinogenic; Protein modification.