Synthesis and high-performance liquid chromatographic purification of tritiated thyrotrophin-releasing hormone-like peptides.

A simple method is presented for the synthesis and RP-HPLC purification of tritium-labelled thyrotrophin-releasing hormone (TRH)-like tripeptides. These peptides differ from TRH (pGlu-His-Pro-amide) in that they possess a neutral or acidic residue in place of the histidine of TRH. The method involves the preparation of the appropriate dipeptide by a solid-phase peptide synthesis procedure using 9-fluorenylmethoxycarbonyl (Fmoc) protection. Very small amounts of tritiated glutamine are then converted into tritiated pyroglutamic acid, and coupling to the dipeptide is effected using a mixed anhydride derived from Fmoc-phenylalanine and the tritiated pyroglutamic acid. The required labelled product is then separated from unlabelled material by reversed-phase HPLC, as the hydrophobicity of the phenylalanine-containing product ensures that it is strongly retained. The availability of a series of tritium-labelled markers prepared by this method has permitted the unequivocal identification of certain naturally occurring TRH-like peptides.