Tripeptide discriminations using circular dichroism detection

Abstract

A general spectroscopic method is described that might be applied to validating amino acid sequences in peptides and protein fragments with a view to it becoming a routine procedure with which to characterize biotechnology drug products. The tripeptides are the L-enantiomers of GGA, GGH, GGI, GGL, GGF, GHG, LGG, and YGG. The simple procedure calls for their complexation with Cu(II) ion in strong aqueous base. Binding the first three residues in the sequence, beginning at the amine terminus, completes the coordination sphere of the Cu(II) ion, so duplication of the initial sequence from peptide to peptide could be an important limiting factor in determining the extent of differentiation that is possible. The analytical focus is the selectivity associated with the chirality properties of the peptides. Detection is by circular dichroism operating in the visible range. The eight analytes were chosen as representative of a series where the sequences are most similar and therefore potentially the most difficult to discriminate spectroscopically. All have just one chiral center. Using ellipticity data at all (n = 1500) wavelengths in the measured spectra, and two novel data reduction procedures, total discrimination among all eight analytes is achieved. The method has considerable potential for use in quality control of peptide and protein biotechnological drug forms, especially their enantiomeric purities.

A convenient assay method for the quality control of peptides and proteins

“…The SD values were 7.42 ( 0.07 mdeg and -214 ( 0.60 mdeg, respectively, within and between stocks. 2,7 Results and Discussion CD activity in the visible range for chiral Cu(II) complexes is a result of disymmetric perturbations of the ground and excited-state ligand field orbitals by the chiral ligands. 6 Three CD-active electronic transitions are reported to occur over the wavelength range of the visible absorbance band.…”

“…1,2 Spectra for Cu(II)-D-histidine and Cu(II)-(D-histidinepeptide) mixed complexes were the focus for di-and tripeptide ligands. 3,7 Evidence was provided that confirmed that the stoichiometries and the complex formation con- stants were similar in magnitude within each series.…”

“…1 The 3-D model, in contrast, is better suited for enantiomeric identification, and chemical purity determinations. 2,3 Only the former is considered in the context of this article.…”

“…This was resolved by complexing the Cu(II) ion with the auxiliary ligand Dhistidine which was made to undergo ligand exchange with the same peptide analytes under the same solution conditions. 2,3 The Cu(II)-D-histidine host complex in effect acts as a chiral derivatizing agent which produces a diastereoisomer, or mixed chiral complex, on partial ligand exchange with each peptide.…”

“…With D-histidine as the auxiliary ligand, the quantity of the analyte material can be decreased by a factor of 10-100 relative to the amounts used earlier. 2,3 This is a significant savings factor if amounts of the analytes are very small to begin with as they invariably are in the production of trial quantities of new drug forms.…”