Hoffmann, R. and Wachs, W. O. and Berger, R. G. and Kalbitzer, Hans Robert and Waidelich, D. and Bayer, E. and Wagner-Redeker, W. and Zeppezauer, M. (1995) Chemical phosphorylation of the peptides GGXA (X = S; T; Y): an evaluation of different chemical approaches. International journal of peptide and protein research 45 (1), pp. 26-34.
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An evaluation was made of the two methods most commonly used for phosphorylation of hydroxyamino acids in peptides, i.e. the tetrazole-catalysed phosphitylation by di-tert-butyl-N,N-diethylphosphoramidite followed by oxidation and the phosphorylation by dibenzylphosphochloridate. As model system the sequence GGXA (X = S, T, Y) was used which represents a random-coil sequence avoiding the influence on the reaction kinetics of secondary structure formation. In the case of serine- and threonine-containing peptides, both synthetic methods gave comparable yields of the desired phosphopeptides. The phosphorylation of tyrosine was achieved more favorably via the phosphoramidite method. However, phosphotyrosine peptides are most easily obtained by peptide synthesis using Fmoc-Tyr(PO3Me2)OH as building block. The dibenzylphosphochloridate method yields the expected phosphopeptides as the only peptide derivative and in addition, a great number of unidentified by-products which can be removed by ion-exchange chromatography. The phosphoramidite method consistently resulted in three peptide derivatives, i.e. the desired phosphopeptide, the phosphitylated peptide and a bridged derivative with two GGXA fragments linked through a phosphodiester bridge. The derivatives were characterised by RP and ion-exchange chromatography, 31P- and 1H-NMR spectroscopy, and ion-spray and electrospray mass 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 phosphorylation sites in peptides. Both the phosphorylated serine and threonine peptide lost phosphoric acid (98 mass units), the tyrosine peptide lost phenyl phosphate (174 mass units).(ABSTRACT TRUNCATED AT 250 WORDS)
|Additional information (public):||The journal of peptide research = online-Ausgabe|
|Institutions:||Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Dr. Hans Robert Kalbitzer|
|Subjects:||500 Science > 570 Life sciences|
|Created at the University of Regensburg:||Unknown|
|Deposited On:||08 Sep 2010 08:28|
|Last Modified:||08 Sep 2010 08:28|