Abstract
Binding constants of the glycopeptide antibiotics teicoplanin (Teic), ristocetin (Rist), and vancomycin (Van), and their derivatives to D-Ala–D-Ala terminus peptides were determined by on-column ligand and receptor synthesis coupled to affinity capillary electrophoresis (ACE) or partial filling ACE (PFACE). In the first technique, 9-fluorenylmethoxycarbonyl (Fmoc)–amino acid–D-Ala–D-Ala species are first synthesized using on-column techniques. The initial sample plug contains a D-Ala–D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc–amino acid– N-hydroxysuccinimide (NHS) ester and buffer, respectively. Upon electrophoresis, the initial D-Ala–D-Ala peptide reacts with the Fmoc–amino acid NHS ester yielding the Fmoc–amino acid D-Ala–D-Ala peptide. Continued electrophoresis results in the overlap of the glycopeptide in the running buffer and the plug of Fmoc–amino acid–D-Ala–D-Ala peptide and non-interacting markers. Subsequent analysis of the change in the electrophoretic mobility ( μ) or relative migration time ratio (RMTR) of the peptide relative to the non-interacting standards, as a function of the concentration of the antibiotic, yields a value for the binding constant. In the second technique, derivatives of the glycopeptides Teic and Rist are first synthesized on-column before analysis by ACE or PFACE. After the column has been partially filled with increasing concentrations of D-Ala–D-Ala terminus peptides, a plug of buffer followed by two separate plugs of reagents are injected. The order of the reagent plugs containing the antibiotic and two non-interacting standards and the anhydride varies with the charge of the glycopeptide. Upon electrophoresis, the antibiotic reacts with the anhydride yielding a derivative of Teic or Rist. Continued electrophoresis results in the overlap of the derivatized antibiotic and the plug of D-Ala–D-Ala peptide. Analysis of the change in RMTR of the new glycopeptide relative to the non-interacting standards, as a function of the concentration of the D-Ala–D-Ala ligand yields a value for the binding constant.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.