Use of ¹³C‐n.m.r. for the characterisation of linkages in acidic peptides.

A method involving the measurement of 13 C‐n.m.r. spectra in the presence and absence of lanthanides has been found to be useful for distinguishing α‐linked and β‐linked aspartyl residues in peptides. Lanthanides were found to induce shifts of 2–4 p.p.m. in the resonances of β‐carbons of α‐linked aspartyl residues, or α‐carbons of β‐linked aspartyl residues, whilst no shifts were observed in the resonances of carbons positioned further from ionisable carboxyl groups. These shifts were more marked than the corresponding shifts induced by pH‐ionisation, and thus are more useful for distinguishing α from β peptides linkages. The method has been applied to a comparison between solution and solid‐phase synthesis of aspartyl‐rich peptides. The two activation peptides of human trypsinogen, Asp‐Asp‐Asp‐Asp‐Lys and Ala‐Pro‐Phe‐Asp‐Asp‐Asp‐Asp‐Lys were synthesised by solution and solid‐phase methods, respectively, the latter employing Fmoc‐protection of amino groups during synthesis. The purified products were found to contain aspartyl residues that were exclusively α‐linked.