ENZYMATIC-SYNTHESIS OF PHOSPHOTYROSINE-CONTAINING PEPTIDES VIA ADENYLYLATED INTERMEDIATES

A novel two-part strategy has been developed for the specific O-phosphorylation of tyrosine residues in peptides. The first part involves the enzymatic transfer of an AMP moiety to the 0-tyrosyl side chains of the peptide substrate to produce a stable adenylylated intermediate. This step is mediated by Escherichia coli glutamine synthetase adenylyltransferase, whose native function is the specific adenylylation of tyrosine-397 in glutamine synthetase. The second step consists of either the enzymatic or chemical degradation of the adenylylated intermediate to produce the corresponding phosphotyrosine-containing peptide. Both an enzymatic procedure using micrococcal nuclease and an oxidative degradation procedure using sodium m-periodate were used in this latter step. Several peptides, including [Tyr5]bradykinin, angiotensin II, [Val5]angiotensin II, neurotensin, Leu-enkephalin, and a 17-residue peptide encompassing the tyrosine adenylylation site in glutamine synthetase, were subjected to this synthetic procedure with overall yields ranging from 3 to 40%. High-performance liquid chromatography, liquid secondary ion mass spectrometry, tandem mass spectrometry, and amino acid analysis were used to isolate and characterize the adenylylated intermediates and phosphotyrosine-containing products. These analyses showed that both the micrococcal nuclease and NaIO4 degradative methods produced a nearly quantitative yield of phosphorylated peptide from the adenylylated intermediate. © 1990, American Chemical Society. All rights reserved.