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

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-NH2) was accomplished in two steps: (i) preparation of [Ac-D-Nal1, D-Cpa2, D-Pal3, Arg5, D-Glu6, D-Ala10]-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.