Synthesis of Stereoisomers and Isoforms of a Tryptic Heptapeptide Fragment of Human Growth Hormone and Analysis by Reverse‐Phase HPLC and Capillary Electrophoresis

Abstract

The amino acid sequence Asn-Gly has at pH 7 a tendency to induce deamidation of asparagine to aspartic acid via the formation of a cyclic imide. This imide opens up to yield Asp-Gly or the isoaspartic acid (isoAsp) form, isoAsp-Gly. Both isomers may be found in their l-form or d-form. Like Asn-Gly, the sequence Asp-Gly has a tendency for isomerization and racemization via the formation of a cyclic imide intermediate. When human growth hormone is digested with trypsin, one of the fragments is a heptapeptide (amino acid residues 128–134) containing the amino acid sequence Asp-Gly (amino acid residues 130 and 131). This heptapeptide, as well as stereoisomers and isoforms where l-Asp was replaced by d-Asp, l-isoAsp, d-isoAsp or the l-cyclic imide, respectively, has been synthesized and used as a standard to achieve separation of the five forms by capillary electrophoresis and by reverse-phase HPLC. Capillary electrophoresis analysis was performed in uncoated capillaries by the use of aspartic acid/cyclodextrin buffers at low pH. The elution order of the aspartic-acid-containing heptapeptides was d-Asp, asp, l-isoAsp, d-isoAsp and l-cyclic imide. Reverse-phase HPLC analysis was performed on a C₁₈ column by the use of a shallow acetonitrile gradient in trifluoroacetic acid/water. The elution order was d-isoasp, l-isoAsp, l-asp, d-Asp and l-cyclic imide. Human growth hormone samples were degraded by incubation at high temperature and analyzed for their potential content of isomerization and racemization products. Only l-forms of aspartic acid and isoaspartic acid of the heptapeptide fragment were found.