Preparative-scale synthesis and reversed-phase purification of a gonadotropin-releasing hormone antagonist

Abstract

The preparation of “Nal—Glu” antagonist (Ac— d-Nal— d-Cpa— d-Pal—Ser—Arg— d-2-amino-5-oxo-5-(4-methoxyphenyl)pentanoic acid—Leu—Arg—Pro— d-Ala—NH 2) was accomplished in two steps: (i) preparation of [Ac— d-Nal 1, d-Cpa 2, d-Pal 3, Arg 5, d-Glu 6, d-Ala 10]-GnRH via standard solid-phase synthetic techniques and (ii) acylation of anisole (Friedel—Crafts) by the glutamic acid residue in position 6. The preparative-scale, reversed-phase high-performance liquid chromatographic (HPLC) purification of the crude “Nal—Glu” antagonist employs first a triethylammonium phosphate (TEAP) (Ph 2.25)—acetonitrile solvent system, followed by an HPLC-based desalting procedure, yielding the acetate salt of the peptide. This repetitive process of purification in TEAP—acetonitrile, followed by counter-ion exchange with 0.5% acetic acid—acetonitrile is highly reproducible and allows large amounts of a given peptide to be purified efficiently in a batchwise fashion. The procedure described for the synthesis, purification and characterization of the “Nal—Glu” antagonist is presented as a model for the multi-gram synthesis and purification of peptides to be used in clinical investigations.