A GENERAL AND EFFICIENT ROUTE FOR CHEMICAL AMINOACYLATION OF TRANSFER-RNAS

A general and expedient procedure for the synthesis of aminoacyl tRNA has been developed. The preparation of aminoacyl pdCpA in one step and in high yield by reaction of the cyanomethyl active esters of N-protected alpha-amino acids with pdCpA is detailed. The preparation and photodeprotection of aminoacyl pdCpA derivatives containing nitroveratryl (NVOC) N-protected amino acids is also studied. The utility of the above methods for preparing aminoacyl tRNA was confirmed by enzymatically ligating NVOC-phenylalanyl pdCpA to tRNA-C(OH) followed by photolysis to provide unprotected phenylalanyl tRNA. The phenylalanyl tRNA was shown to be competent in an in vitro protein biosynthesis system. These protocols greatly simplify the use of chemically misacylated tRNAs in the synthesis of proteins containing unnatural amino acids, as well as in studies of protein biosynthesis.