CHEMICAL PHOSPHORYLATION OF THE PEPTIDES GGXA (X=S, T, Y) - AN EVALUATION OF DIFFERENT CHEMICAL APPROACHES

An evaluation was made of the two methods most commonly used for phosp horylation of hydroxyamino acids in peptides, i.e. the tetrazole-catal ysed phosphitylation by di-tert-butyl-N,N-diethylphosphoramidite follo wed by oxidation and the phosphorylation by dibenzylphosphochloridate. As model system the sequence GGXA (X = S, T, Y) was used which repres ents a random-coil sequence avoiding the influence on the reaction kin etics of secondary structure formation. In the case of serine- and thr eonine-containing peptides, both synthetic methods gave comparable yie lds of the desired phosphopeptides. The phosphorylation of tyrosine wa s achieved more favorably via the phosphoramidite method. However, pho sphotyrosine peptides are most easily obtained by peptide synthesis us ing Fmoc-Tyr(PO(3)Me(2))OH as building block. The dibenzylphosphochlor idate method yields the expected phosphopeptides as the only peptide d erivative and in addition, a great number of unidentified by-products which can be removed by ion-exchange chromatography. The phosphoramidi te method consistently resulted in three peptide derivatives, i.e. the desired phosphopeptide, the phosphitylated peptide and a bridged deri vative with two GGXA fragments linked through a phosphodiester bridge. The derivatives were characterised by RP and ion-exchange chromatogra phy, P-31- and H-1-NMR spectroscopy, and ion-spray and electrospray ma ss spectrometry. Interestingly, even these mild ionisation techniques resulted in partial fragmentation, The observed fragmentation pathways seem to be a diagnostic tool for the identification of phosphorylatio n sites in peptides. Both the phosphorylated serine and threonine pept ide lost phosphoric acid (98 mass units), the tyrosine peptide lost ph enyl phosphate (174 mass units). Fragmentation of the dephosphorylated peptides in MS/MS experiments yielded fragments of the masses 69.1u a nd 83.1u, corresponding to dehydroalanine derived from phosphoserine a nd alpha-aminodehydrobutyric acid, derived from phosphothreonine. (C) Munksgaard 1995.