On-column synthesis coupled to affinity capillary electrophoresis for the determination of binding constants of peptides to glycopeptide antibiotics

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 (mu) 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.     

Estimation of binding constants between ristocetin and teicoplanin and peptides using on-column ligand derivatization coupled to affinity capillary electrophoresis

This work utilizes on-column ligand synthesis and affinity capillary electrophoresis (ACE) to determine binding constants (K_b) of 9-flourenylmethyloxy carbonyl (Fmoc)-amino acid derivatives to the glycopeptide antibiotics ristocetin (Rist) and teicoplanin (Teic). In this technique, two separate plugs of sample are injected on to the capillary column and electrophoresed. The initial sample plug contains a d-Ala-d-Ala terminus peptide and either one or two non-interacting standard(s). The second plug contains a Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester. The electrophoresis is then carried out with an increasing concentration of Rist or Teic in the running buffer. Upon electrophoresis the initial d-Ala-d-Ala peptide reacts with the Fmoc-amino acid yielding a new Fmoc-amino acid-d-Ala-d-Ala peptide derivative. Continued electrophoresis results in the binding of Rist or Teic to the Fmoc-amino acid-d-Ala-d-Ala peptide derivatives. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility () of the Fmoc-amino acid-d-Ala-d-Ala peptide derivatives relative to the non-interacting standards, as a function of the concentration of Rist and Teic, yields a value for K_b. These findings demonstrate the advantage of coupling on-column ligand synthesis to ACE for estimating binding parameters between antibiotics and ligands.Abbreviations Rist Ristocetin - Teic Teicoplanin - ACE Affinity capillary electrophoresis - RMTR Relative migration time ratio     

Determination of binding constants between the antibiotic ristocetin A and D-Ala-D-Ala terminus peptides by affinity capillary electrophoresis

Summary Binding constants between the antibiotic ristocetin A (Rist A) and D-Ala-D-Ala terminus peptides were determined using affinity capillary electrophoresis (ACE). In these experiments two techniques are used to obtain binding constants. In the first, a plug of Rist A and non-interacting standards are injected and electrophoresed. Analysis of the change in the relative migration time ratio (RMTR) of Rist, relative to the non-interacting standards, as a function of the concentration of peptide, yields a value for the binding constant (Kb). In the second, samples of peptide and standards are injected and electrophoresed in increasing concentrations of Rist A in the running buffer. Analysis using theRMTR yields aK _b. The findings described here demonstrate the advantage of using ACE for estimating binding parameters between antibiotics and ligands.     

Partial filling multiple injection affinity capillary electrophoresis (PFMIACE) to estimate binding constants of receptors to ligands

Partial filling multiple injection affinity capillary electrophoresis (PFMIACE) is used to determine binding constants between vancomycin (Van) from Streptomyces orientalis, teicoplanin (Teic) from Actinoplanes teicomyceticus and ristocetin (Rist) from Nocardia lurida to d-Ala-d-Ala terminus peptides and carbonic anhydrase B (CAB, E.C.4.2.1.1) to arylsulfonamides. Two variations of PFMIACE are described herein. In the first technique, the capillary is partially filled with ligand at increasing concentrations, a non-interacting standard, three or four separate plugs of receptor each separated by small plugs of buffer, a plug containing a second non-interacting standard, and then electrophoresed in buffer. Upon continued electrophoresis, equilibrium is established between the ligand and receptors causing a shift in the migration time of the receptors with respect to the non-interacting standards. This change in migration time is utilized for estimating multiple binding constants (K_b) for the same interaction. In the second technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column. The capillary is partially filled with a series of buffers containing an antibiotic at increasing concentrations and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities since their charge-to-mass ratios are approximately the same but remain as distinct zones due to the buffer plug between peptides. Upon electrophoresis, the plug of antibiotic flows into the peptide plugs affecting a shift in the migration time of the peptides with respect to the non-interacting standards occurs due to formation of the of the antibiotic-peptide complex. The shift in the migration time of the peptides upon binding to the antibiotic is used for the Scatchard analysis and measurement of a K_b. The PFMIACE technique expands the functionality and potential of ACE as an analytical tool to examine receptor-ligand interactions. In PFMIACE, a smaller amount of sample is required in the assay compared to both conventional ACE and MIACE. Furthermore, a wide array of data is obtained from a single experiment, thus, expediting the assay of biological species.     

On-column ligand synthesis coupled to partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to a receptor

This paper describes a two-step procedure whereby on-column ligand synthesis and partial-filling affinity capillary electrophoresis (PFACE) are sequentially coupled to each other to determine the binding constants of 9-fluorenylmethoxy carbonyl (Fmoc)-amino acid-D-Ala-D-Ala species to vancomycin (Van) from Streptomyces orientalis. In this technique four separate plugs of sample are injected onto the capillary column and electrophoresed. 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 running buffer, respectively. The fourth sample plug contains an increasing concentration of Van partially-filled onto the capillary column. 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 plugs of Van and Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Analysis of the change in the relative migration time ratio of the Fmoc-amino acid-D-Ala-D-Ala peptide relative to the non-interacting standards, as a function of the concentration of Van, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques.     

Partial-filling affinity capillary electrophoresis

Partial-filling affinity capillary electrophoresis (PFACE) is used to examine the binding interactions between two model biological systems: D-Ala-D-Ala terminus peptides to the glycopeptide antibiotic vancomycin (Van) from Streptomyces orientalis, and arylsulfonamides to carbonic anhydrase B (CAB, EC 4.2.1.1, bovine erythrocytes). Using these two systems, modifications in the PFACE technique are demonstrated including flow-through PFACE (FTPFACE), competitive flow-through PFACE (CFTPFACE), on-column ligand synthesis PFACE (OCLSPFACE), and multiple-step ligand injection PFACE (MSLIPFACE). In PFACE small plugs of sample are injected into the capillary column and an equilibrium is established between receptor and ligand during electrophoresis. Binding constants are then obtained by Scatchard analysis using changes in the migration time of the receptor/ligand on changing the concentration of the ligand/receptor. Data demonstrating the quantitative potential of these methods are presented. This review focuses on the unique capabilities of the different PFACE techniques as applied to two model biological systems.     

Frontal analysis microchip capillary electrophoresis to study the binding of ligands to receptors derivatized on magnetic beads

The model binding of the glycopeptide antibiotic teicoplanin (Teic) from Actinoplanes teichomyceticus, immobilized on magnetic microspheres, to d-Ala-d-Ala terminus peptides was assessed using microchip capillary electrophoresis (MCE) with continuous frontal analysis (FA). Teic is closely related to vancomycin (Van), historically, the drug of last resort used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to the d-Ala-d-Ala terminus on the cell wall of Gram-positive bacteria via hydrogen bonds, thereby preventing the enzyme-mediated cross-linking of peptidoglycan and eventual cell death. In this work direct and competitive bead-based assays in a microfluidic chip are demonstrated. The binding constants obtained using the technique are comparable with values reported in the literature.     

Molecular interactions of glycopeptide antibiotics investigated by affinity capillary electrophoresis and bioaffinity electrospray ionization-mass spectrometry

Many analytical approaches are available to evaluate (bio)molecular interactions, all of which have their particular advantages and disadvantages. In recent years, two relatively new techniques have emerged that may be used by the bioanalytical community to evaluate such interactions, namely affinity capillary electrophoresis (ACE) and bioaffinity electrospray ionization-mass spectrometry (ESI-MS). In this paper, we describe and evaluate the use of both these techniques for the investigation of the interactions of glycopeptide antibiotics with peptides that mimic the bacterial cell wall binding site. We focus particularly on the effect of the sugar moieties attached to the antibiotic peptide backbone and on the noncovalent dimerization of these glycopeptide antibiotics.     

Multiple-injection affinity capillary electrophoresis to examine binding constants between glycopeptide antibiotics and peptides

Multiple-injection affinity capillary electrophoresis (MIACE) was used to determine binding constants (K(b)) between vancomycin, ristocetin, and teicoplanin from Streptomyces orientalis, Nocardia lurida, and Actinoplanes teichomyceticus, respectively, and fluorenylmethoxycarbonyl (Fmoc)-(Gly, Ala, Val, and Phe)-D-Ala-D-Ala peptides. In this technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities but remain as distinct zones due to the buffer plug between peptides. The electrophoresis is then carried out in an increasing concentration of antibiotic in the running buffer. Continued electrophoresis results in a shift in the migration time of the peptides upon binding to the antibiotic. Analysis of the change in the relative migration time ratio (RMTR) of the resultant complexes relative to the non-interacting standards, as a function of the concentration of antibiotic yields a value for K(b). MIACE is a versatile technique that can be used to measure affinity constants between ligands of similar relative molecular mass and charge without the need of separate binding experiments. The findings described, herein, demonstrate the advantages of using MIACE to estimate binding parameters between ligands and receptors.     

Use of a Dual-Marker Form of Analysis to Estimate Binding Constants Between Receptors and Ligands by Affinity Capillary Electrophoresis

This work describes the use of a dual-standard analysis approach termed the time-average ratio (TAR) in affinity capillary electrophoresis (ACE) to estimate binding constants of receptors to ligands. In this form of analysis the TAR is the migration time of the receptor divided by the average of the sum of the migration times of two non-interacting standards. This change in TAR as a function of the concentration of ligand yields a value for the binding constant. This concept is demonstrated using three model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, vancomycin (Van) and ristocetin (Rist) from Streptomyces orientalis and Nocardia lurida, respectively, and d-Ala- d-Ala terminus peptides. Three ACE techniques are used to examine the three systems: standard ACE, flow-through partial-filling ACE (FTPFACE), and on-column derivatization coupled to ACE. The findings described here demonstrate that ACE data analyzed using the TAR form of analysis yield binding constants between receptors and ligands comparable to those estimated using other ACE forms of analysis. A comparison to three other forms of analysis is described.     

Multiple-step ligand injection affinity capillary electrophoresis for determining binding constants of ligands to receptors

This work demonstrates the use of multiple-step ligand injection affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique a sample plug of receptor and non-interacting standards is injected by pressure and electrophoresed in a buffer containing a given concentration of ligand. The sequence is repeated for all concentrations of ligand generating a single electropherogram containing a series of individual sample plugs superimposed on environments of buffer containing increasing concentrations of ligand. Analysis of the change in the relative migration time ratio, RMTR, relative to the non-interacting standards, as a function of the concentration of the ligand, yields a value for the binding constant. A competitive assay using the technique is also demonstrated using neutral ligands for CAB. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method are presented.     

Multiple-injection affinity capillary electrophoresis to estimate binding constants of receptors to ligands

Multiple-injection affinity capillary electrophoresis (MIACE) is used to determine binding constants (K _b) between receptors and ligands using as model systems vancomycin and teicoplanin from Streptomyces orientalis and Actinoplanes teichomyceticus, respectively, and their binding to D-Ala-D-Ala peptides and carbonic anhydrase B (CAB. EC 4.2.1.1) and the binding of the latter to arylsulfonamides. A sample plug containing a non-interacting standard is first injected followed by multiple plugs of sample containing the receptor and then a final injection of sample containing a second standard. Between each injection of sample, a small plug of buffer is injected which contains an increasing concentration of ligand to effect separation between the multiple injections of sample. Electrophoresis is then carried out in an increasing concentration of ligand in the running buffer. Continued electrophoresis results in a shift in the migration time of the receptor in the sample plugs upon binding to their respective ligand. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility (μ) of the resultant receptor–ligand complex relative to the non-interacting standards, as a function of the concentration of ligand yields a value for K _b. The MIACE technique is a modification in the ACE method that allows for the estimation of binding affinities between biological interactions on a timescale faster than that found for standard ACE. In addition sample volume requirements for the technique are reduced compared to traditional ACE assays. These findings demonstrate the advantage of using MIACE to estimate binding parameters between receptors and ligands.     

On-column derivatization and analysis of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis

This work demonstrates the use of an in-capillary procedure for derivatization of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis (CE). Migrating in an uncoated fused-silica capillary, plugs of substrate and anhydride are injected separately and electrophoresed. Differential transport velocities permit the separate zones to penetrate each other under an applied field, thereby facilitating reaction. In initial experiments the extent of reaction between tryptophan and acetic anhydride was examined and product amounts quantitated by CE. In separate experiments a series of amino acids and peptides were injected into the capillary and reacted with phthalic anhydride on-column to yield the phthalic derivatized species. Finally, on-column derivatization of alkylamines with phthalic anhydride was investigated and electrophoretic mobility related to molecular weight of the derivatized amines. These procedures illustrate the use of the capillary as a microreactor in the facile synthesis of derivatized molecules and ease of quantitation of reaction products under conditions of electrophoresis.     

Binding of glycopeptide antibiotics to a model of a vancomycin-resistant bacterium

BACKGROUND: The vancomycin group of glycopeptide antibiotics is active against a wide range of gram-positive bacteria. The increasing resistance to vancomycin is the result of a change of an amide linkage (D-Ala-D-Ala) to an ester linkage (D-Ala-D-Lactate) in the bacterial cell-wall precursors. RESULTS: We have used a peptide terminating in the sequence -Lys-D-Ala-D-Lactate linked by its amino terminus to a docosanoyl (C22) acyl chain and anchored in a supported lipid monolayer to mimic the surface of vancomycin-resistant enterococci. Surface plasmon resonance analysis was then used to investigate the binding of glycopeptide group antibiotics to this surface. Vancomycin, which dimerises weakly, bound with low affinity, whereas strongly dimerising antibiotics, such as chloroeremomycin, bound with higher affinities. Antibiotics that have attached hydrophobic groups, such as teicoplanin and biphenylchloroeremomycin (LY307599), bound to the lipid monolayer. This resulted in an enhanced affinity for the lipid-anchored peptide at the surface relative to affinities for an analogous non-anchored peptide in solution. CONCLUSIONS: We have shown that the affinities of glycopeptide antibiotics for a model of the surface of a vancomycin-resistant bacterium are enhanced relative to affinities determined in free solution. We have also shown that antibiotics that have membrane anchors bind tightly to the model surface and that this feature is an important determinant of the ability of an antibiotic to kill vancomycin-resistant enterococci.     

Conformational studies of resin-bound vancomycin and the complex of vancomycin and Ac2-L-Lys-D-Ala-D-Ala

The molecular target of vancomycin, a commonly used glycopeptide antibiotic, is the D-Ala-D-Ala dipeptide subunit on the bacterial cell wall. The molecular basis of interaction between vancomycin and D-Ala-D-Ala in solution is well-known. However, there is no structural data on vancomycin, and its interaction with D-Ala-D-Ala when the drug is tethered to a solid support. In this Article, vancomycin was directly coupled onto TentaGel or PEGA resin through its C terminus. High-resolution magic angle spinning NMR studies indicated that conformation of PEGA bead-bound vancomycin is identical to that of the free drug. Broadening and shifts of the same proton resonances were observed in solution-phase vancomycin or PEGA-bound vancomycin when complexed with Ac(2)-L-Lys-D-Ala-D-Ala. This study demonstrates that bead-bound molecules can behave the same as solution-phase molecules in terms of molecular interaction with its target molecule, thus validating the on-bead screening approach of the "one-bead-one-compound" combinatorial library method.     

Affinity capillary electrophoresis on microchips

The present study shows that the application of the method of affinity capillary electrophoresis (ACE) to investigate interactions between ligands and their substrates can be realized on microchips. With ACE it is possible to characterize non-covalent molecular interactions (complexation and partition equilibria). Binding constants (K(B)) provide a measured value of the affinity of a ligand molecule to a substrate, which is basic information for the understanding of hormones, drugs and their targets, e.g. receptors in the human body. A microchip electrophoresis instrument equipped with a UV-detector and a home-built chip-station with electrochemical detection were used. ACE could be achieved with model solutions of neurotransmitters using sulfated beta-cyclodextrin (sCD) as substrate in a background buffer. This paper describes the advantages of microchip-ACE (MC-ACE) to traditional affinity capillary electrophoresis on a capillary. The results show that MC-ACE has great potential as a tool for fast scanning of interactions and to calculate binding constants of ligands with their substrates.     

Recent advances in capillary electrophoresis/electrospray-mass spectrometry

In this review, the progress in hyphenation of capillary electrophoresis (CE) with electrospray ionization-mass spectrometry (ESI-MS) since the article of Banks (Banks, J. F., Electrophoresis 1997, 18, 2255-2266) is reported. In all capillary-based electromigration techniques, such as capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), capillary isoelectric focussing (CIEF), micellar electrokinetic chromatography (MEKC), affinity capillary electrophoresis (ACE), as well as in the hybrid techniques capillary electrochromatography (CEC), and pressurized capillary electrochromatography (pCEC) progress has been made in experimental setups, and for many groups of analytes, such as peptides, proteins, nucleotides, saccharides, drugs and their metabolites, CE/ESI-MS has been successfully applied. Electromigration is further miniaturized. New preconcentration methods allow the investigation of compounds, which are not sensitively detected with ESI-MS. Coordination ion spray (CIS) MS is another method for sensitivity enhancement by on-line formation of charged coordination compounds.     

Total synthesis and evaluation of [Psi[CH2NH]Tpg4]vancomycin aglycon: reengineering vancomycin for dual D-Ala-D-Ala and D-Ala-D-Lac binding

An effective synthesis of [Psi[CH(2)NH]Tpg(4)]vancomycin aglycon (5) is detailed in which the residue 4 amide carbonyl of vancomycin aglycon has been replaced with a methylene. This removal of a single atom was conducted to enhance binding to D-Ala-D-Lac, countering resistance endowed to bacteria that remodel their D-Ala-D-Ala peptidoglycan cell wall precursor by a similar single atom change (ester O for amide NH). Key elements of the approach include a synthesis of the modified vancomycin ABCD ring system featuring a reductive amination coupling of residues 4 and 5 for installation of the deep-seated amide modification, the first of two diaryl ether closures for formation of the modified CD ring system (76%, 2.5-3:1 kinetic atropodiastereoselectivity), a Suzuki coupling for installation of the hindered AB biaryl bond (90%) on which the atropisomer stereochemistry could be thermally adjusted, and a macrolactamization closure of the AB ring system (70%). Subsequent DE ring system introduction enlisted a room-temperature aromatic nucleophilic substitution reaction for formation of the remaining diaryl ether (86%, 6-7:1 kinetic atropodiastereoselectivity), completing the carbon skeleton of 5. Consistent with expectations and relative to the vancomycin aglycon, 5 exhibited a 40-fold increase in affinity for D-Ala-D-Lac (K(a) = 5.2 x 10(3) M(-1)) and a 35-fold reduction in affinity for D-Ala-D-Ala (K(a) = 4.8 x 10(3) M(-1)), providing a glycopeptide analogue with balanced, dual binding characteristics. Beautifully, 5 exhibited antimicrobial activity (MIC = 31 microg/mL) against a VanA-resistant organism that remodels its D-Ala-D-Ala cell wall precursor to d-Ala-d-Lac upon glycopeptide antibiotic challenge, displaying a potency that reflects these binding characteristics.     

毛细管电泳中的毛细管区段灌注及其相关技术

毛细管区段灌注是一种随手性毛细管电泳、亲和毛细管电泳和胶束电动力色谱-质谱发展而产生的实验技术。由于它能消除采用以上方法时遇到的缓冲溶液对检测的影响。因此引起人们的关注。本文对区段灌注技术的原理,区段灌注技术在手性毛细管电泳、亲和毛细管电泳和胶束电动力色谱中的应用,以及目前区段灌注技术存在的一些问题进行了评述。本文还对由区段灌注技术发展而来的逆流毛细管电泳、液相预柱毛细管电泳、多步配体进样等实验技术进行了简介。     

Use of a partial-filling technique in affinity capillary electrophoresis for determining binding constants of ligands to receptors

This work evaluates the concept of a partial-filling technique in affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique the capillary is first partially-filled with ligand followed by a sample of receptor and non-interacting standard and electrophoresed. Analysis of the change in the mobility ratio, M, of the receptor, relative to the non-interacting standard, as a function of the concentration of the ligand, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.