RESUMEN
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)
Asunto(s)
Péptidos/química , Secuencia de Aminoácidos , Estudios de Evaluación como Asunto , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , FosforilaciónRESUMEN
The model peptides glycylglycyltyrosylalanine (Gly-Gly-Tyr-Ala), glycylglycylthreonylalanine (Gly-Gly-Thr-Ala) and glycylglycylserylalanine (Gly-Gly-Ser-Ala) were phosphorylated at the hydroxyl groups of their tyrosyl, threonyl and seryl residues, respectively, and characterized by 31P and 1H NMR spectroscopy. The pKa-value of the phosphoryl group in the tyrosine-containing peptide determined from the pH dependence of chemical shifts is 5.9, the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -3.8 and 0.2 ppm, respectively. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the tyrosine residue; the amide resonance is shifted -0.02 ppm, the H alpha resonance 0.06 ppm, the H beta resonances 0.10 and -0.04 ppm, the H delta resonances 0.02 ppm and the H epsilon resonances 0.26 ppm. The pKa-value of the phosphoryl group in the threonine peptide determined from the pH dependence of chemical shifts is 6.1; the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -0.1 and 4.8 ppm, respectively. The corresponding values for the serine peptide are 6.1 (pKa), 0.6 ppm and 4.9 ppm. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the threonine and serine residues. In the threonine residue the amide resonance is shifted 0.25 ppm, the H alpha-resonance -0.43 ppm, the H beta-resonance 0.03 ppm and the H gamma-resonance 0.09 ppm. In the serine residue the amide resonance is shifted 0.21 ppm, the H alpha-resonance -0.17 ppm, and the H beta-resonances 0.17 ppm.