Catherine F. Silverio Maryam Azad Frank A. Gomez Department of Chemistry and Biochemistry, California State University, Los Angeles, CA, USA On-column derivatization of the antibiotics teicoplanin and ristocetin coupled to affinity capillary electrophoresis Binding constants between the glycopeptides teicoplanin (Teic) and ristocetin (Rist) and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column receptor synthesis coupled to partial-filling affinity capillary electrophoresis (PFACE) or affinity capillary electrophoresis (ACE). In these techniques, the column is first partially filled with increasing concentrations of D-Ala-D-Ala terminus peptides. This is followed by plugs of buffer, antibiotic and two noninteracting standards, and acetic and/or succinic anhydride (and buffer in the case of ACE). The order of the reagent plugs containing the antibiotic and 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 anti- biotic and the plug of D-Ala-D-Ala peptide. Analysis of the change in the relative migra- tion time ratio (RMTR) of the new glycopeptide relative to the standards, as a function of the concentration of the D-Ala-D-Ala ligand yields a value for the binding constant K b . The techniques described here can be used to assess how the derivatization of drugs alters their affinities for target molecules. Keywords: Affinity capillary electrophoresis / Ristocetin / Teicoplanin EL 5336 1 Introduction Glycopeptide antibiotics inhibit the growth of Gram-posi- tive bacteria by hindering cell wall peptidoglycan bio- synthesis [1–7]. These drugs bind to the D-Ala-D-Ala por- tion of peptidoglycan intermediates, thereby, inhibiting the transglycosylation reaction required for cross-linking of the cell wall. Vancomycin (Van) from Streptomyces orientalis is frequently called the antibiotic of last resort because it has been effective in treating infections caused by bacteria resistant to other antibiotics such as methi- cillin-resistant Staphylococcus aureus. However, bacteria have also conferred resistance to Van through the substi- tution of the D-Ala-D-Ala terminus of the peptidoglycan precursor by D-Ala-D-Lac [4, 8]. This has spurred interest in the design and synthesis of modified glycopeptides that may bind to other ligands on a bacteria cell wall. Over the past decade, advances in molecular biology have elucidated a myriad of molecular interactions includ- ing protein-peptide, protein-DNA, and antibody-antigen. These interactions coupled to the great numbers of potential drug targets readily synthesized using combi- natorial techniques have made the development of new analytical techniques for high-throughput screening of utmost importance. The ability to accurately determine the extent of interaction between a receptor and ligand is integral in rational drug design and development. Affinity capillary electrophoresis (ACE) is a versatile method that has been used to characterize a number of bimolecular noncovalent interactions [9–27]. For example, Progent et al. [9] used ACE to determine binding constants between anionic polydispersed polymers and peptides. Ding et al. [10] have examined the binding between porphyrins to human serum albumin using ACE. Finally, De Lorenzi et al. [11] used ACE to characterize the interaction between drugs and transthyretin. This technique uses the resolving power of CE to distinguish between the free and bound forms of a receptor as a function of the concentration of free ligand. Unlike other forms of binding assays, ACE does not require that free and bound receptor/ligand be known, hence, ACE simplifies the estimation of K b . Recently, we showed that on-column derivatization could be coupled to ACE [12]. In these studies, separate plugs of glycopeptide, D-Ala-D-Ala terminus peptides, fluorenyl- Correspondence: Professor Frank A. Gomez, Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032- 8202, USA E-mail: fgomez2@calstatela.edu Fax: + 323-343-6490 Abbreviations: ACE, affinity capillary electrophoresis; Fmoc, fluorenylmethoxycarbonyl; MO, mesityl oxide; PFACE, partial- filling affinity capillary electrophoresis; Rist, ristocetin; RMTR, relative migration time ratio; Teic, teicoplanin 808 Electrophoresis 2003, 24, 808–815  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/0503–808 $17.501.50/0