Enzymatic protecting group techniques for glyco- and phosphopeptide chemistry: Synthesis of a glycophosphopeptide from human serum response factor

The covalent modification of proteins by phosphorylation and by glycosylati on with GlcNAc residues are important regulatory processes which mediate bi ological signal transduction. For the study of such biological phenomena in molecular detail characteristic peptides which embody both types of modifi cation may serve as efficient tools. However, their synthesis is complicate d by their pronounced acid and base lability as well as their multifunction ality. For this purpose the enzyme labile choline eater was developed. The choline ester can be removed selectively and in high yields from various Gl cNAc-glycopeptides and phosphopeptides at pH 6.5 and 37 degrees C. The cond itions under which the enzymatic deprotections proceed are so mild that no undesirable side reactions are observed (i.e., no cleavage or anomerization of the glycosidic bonds and no beta-elimination of the phosphate or the ca rbohydrate occur). The specificity of the biocatalyst guarantees that neith er the peptide bonds nor the other protecting groups present are being atta cked. When this enzymatic protecting group technique was combined with the enzyme-labile 4-(phenylacetoxy)benzyloxycarbonyl (PhAcOZ) urethane protecti ng group a complex glycophosphopeptide could be built up. The glycopeptide is equipped with a biotin label by which it can be traced in biological sys tems. This peptide represents a characteristic partial structure of a glyco sylated and phosphorylated sequence from the transactivation domain of seru m response factor (SRF), a widely occuring human transcription factor.