Histidine racemization in the synthesis of an analog of the luteinizing hormone releasing factor. Investigation by HPLC and enzymatic methods.
To investigate histidine racemization in the synthesis of a LHRH analog, (D-Trp)6-LHRH2-10 was built up by stepwise elongation of the sequence 3-10 using the solid phase technique on a 1% cross-linked chloromethyl polystyrene. For the whole synthesis the tert.-butyloxycarbonyl (BOC) group was used for temporary N-terminal protection. To protect the pi-nitrogen in histidine the benzyl-oxymethyl (BOM) group was utilized. The condensation position in the (D-Trp)6-LHRH analog was chosen so as to be able to investigate the racemization of histidine. We coupled BOC-His(BOM) with the (D-Trp)6-LHRH3-10 fragment using three different activating agents, mixed anhydride, carbonyldiimidazole (1 equiv.)/1-hydroxybenzotriazole (2 equiv.) and dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The racemization was investigated by enzymatic digestion and by HPLC. For HPLC, (D-His(BOM)2-(D-Trp)6-LHRH2-10 was also synthesized. It could be proved that practically no racemization occurs during the actual peptide synthesis. The small amount (1%) of D-histidine found is due to racemization in the synthesis of BOC-His(BOM).
D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D007987:Gonadotropin-Releasing Hormone / Q000138:chemical synthesis* / Q000031:analogs & derivatives; D006639:Histidine; D007202:Indicators and Reagents; D050830:L-Amino Acid Oxidase; D010446:Peptide Fragments / Q000138:chemical synthesis*; D013237:Stereoisomerism
D007202:Indicators and Reagents; D010446:Peptide Fragments; C049698:LHRH (2-10), Trp(6)-; D007987:Gonadotropin-Releasing Hormone; D006639:Histidine; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase