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 of Polyethylene Glycol Vancomycin Derivatives to Peptide Ligands Using Affinity Capillary Electrophoresis

Vancomycin (Van) from Streptomyces orientalis has been derivatized with polyethylene glycol [PEG; PEG-550 (1), 750 (2), 1,100 (3), 2,000 (4), 5,000 (5), and 8,000 (6) g mol⁻¹] at the N-terminus of the glycopeptide backbone and their binding to d-Ala-d-Ala terminus peptides assessed using affinity capillary electrophoresis (ACE). Utilizing ACE, a plug of Van-PEG and non-interacting standards are injected and electrophoresed. Analysis of the change in the relative migration time ratio of the Van-PEG species, relative to the non-interacting standards, as a function of the concentration of peptide, yields a value for the binding constant (K _b). Values of K _b for N-acetyl-d-Ala-d-Ala, 7 to the Van-PEG derivatives are weaker than those for N _α,N _ε-diacetyl-Lys-d-Ala-d-Ala, 8 (for example, values of K _b for 7-1 and 8-1 are 1.8 and 47.7 × 10³ M⁻¹, respectively). These results demonstrate that derivatization of Van with PEG has little effect on the affinity of d-Ala-d-Ala peptide ligands to it. The findings further prove the versatility of ACE and its ability to estimate binding parameters of ligands to antibiotics.     

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.     

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.     

Magnetic microsphere-based methods to study the interaction of teicoplanin with peptides and bacteria

Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal d-Ala-d-Ala intermediates in the peptidoglycan of the cell wall of Gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two d-Ala-d-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled d-Ala-d-Ala-d-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 × 10⁻⁶ and 3.0 × 10⁻⁶ mol L⁻¹, respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of Gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide–antibiotic interaction.     

Characterization of structural variations in the peptidoglycan of vancomycin-susceptible <Emphasis Type="Italic">Enterococcus faecium</Emphasis>: Understanding glycopeptide-antibiotic binding sites using mass spectrometry

Enterococcus faecium, an opportunistic pathogen that causes a significant number of hospital-acquired infections each year, presents a serious clinical challenge because an increasing number of infections are resistant to the so-called antibiotic of last resort, vancomycin. Vancomycin and other new glycopeptide derivatives target the bacterial cell wall, thereby perturbing its biosynthesis. To help determine the modes of action of glycopeptide antibiotics, we have developed a bottom-up mass spectrometry approach complemented by solid-state nuclear magnetic resonance (NMR) to elucidate important structural characteristics of vancomycin-susceptible E. faecium peptidoglycan. Using accurate-mass measurements and integrating ion-current chromatographic peaks of digested peptidoglycan, we identified individual muropeptide species and approximated the relative amount of each. Even though the organism investigated is susceptible to vancomycin, only 3% of the digested peptidoglycan has the well-known d-Ala-d-Ala vancomycin-binding site. The data are consistent with a previously proposed template model of cell-wall biosynthesis where d-Ala-d-Ala stems that are not cross-linked are cleaved in mature peptidoglycan. Additionally, our mass-spectrometry approach allowed differentiation and quantification of muropeptide species seen as unresolved chromatographic peaks. Our method provides an estimate of the extent of muropeptides containing O-acetylation, amidation, hydroxylation, and the number of species forming cyclic imides. The varieties of muropeptides on which the modifications are detected suggest that significant processing occurs in mature peptidoglycan where several enzymes are active in editing cell-wall structure.     

The evidence for complex formation between N-acetyl-l-tyrosine methyl ester and N-acetylprocainamide hydrochloride using NMR spectroscopy

In order to reveal the possible mechanism of the recognition of antiarrhythmic agents class I and class III by the amino acid residues, which are responsible for drug binding to the selectivity filters either in the sodium or potassium ion channels, co-crystallizations of procainamide hydrochloride and N-acetylprocainamide hydrochloride with N-acetyl-l-tyrosine methyl ester and N-acetyl-l-phenylalanine methyl ester were performed using various conditions. Because the crystallization of the complexes failed, the intermolecular interactions between the components were evidenced using NMR spectroscopy. Exclusively, in the case of N-acetylprocainamide hydrochloride and N-acetyl-l-tyrosine methyl ester, two-dimensional NMR experiments and Job Plot analysis indicated the formation of the 1:1 complex in DMSO-d ₆  solution (with the association constant of 16 M⁻¹), whereas for the mixture of procainamide hydrochloride with N-acetyl-l-tyrosine methyl ester, the complex formation was not confirmed. The NMR results were discussed using crystal structure data obtained for N-acetylprocainamide hydrochloride, procainamide hydrochloride, as well as procainamide dihydrochloride, and were compared with the known pharmacological activity of the antiarrhythmic agents.     

Glycoconjugates of amines: alkylation of primary and secondary amines withN-chloroacetyl-β-glycopyranosylamines

Efficient monoalkylation of a series of primary and secondary amines was demonstrated with the use ofN-chloroacetylglycosylamines derived fromd-glucose,d-galactose,d-mannose,N-acetyl-d-glucosamine, and lactose. The reaction was shown to be useful for incorporation of carbohydrate residues into physiologically active compounds. Glycoconjugates of some derivatives of piperazine, 2-phenylethylamine, tryptamine, and important biogenic amines (norephedrine, octopamine, dopamine) were prepared. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya. No. 6, pp. 1244–1247, June, 1998.     

N-glycyl-β-glycopyranosylamines, derivatives of mono- and disaccharides, and their use for the preparation of carboxylic acid glycoconjugates

A convenient preparative procedure was developed for the synthesis ofN-glycyl-β-glycopyranosylamines, derivatives of monosaccharides (d-galactose,d-mannose,l-fucose, andN-acetyl-d-glucosamine) and disaccharides (lactose, melibiose, cellobiose, and maltose). These compounds were demonstrated to be useful for the preparation of glycoconjugates of biologically active compounds containing the carboxy group (nicotinic, orotic, kynurenic, and indoleacetic acids). Synthetic pathways were developed for conversions ofN-glycyl-β-glycopyranosylamines into derivatives containing the carboxy group with the use of malonic andl-tartaric acid derivatives. Published inIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1461–1466, August, 2000.     

Mechanistic studies on the conjugation of methyl isocyanate with N-acetyl-cysteine in rats

In order to investigate the utility of selected thiols as scavengers of MIC, we first assessed the chemical stability of SMG, AMCC and SMC by measuring their rates of reaction in vitro with thiophan. The inital rates of carbamoylation of thiorphan (0.5 mM) by the above conjugates (0.5 mM) in aqueous buffer at pH 7.4 and 37°C were 2.51, 0.76 and 8.47 mol L^–1 min^–1, respectively, indicating that the mercapturate AMCC was the most stable of the three MIC conjugates.In light of these results, studies were conducted to examine the effect of pretreatment withN-acetyl-l-cysteine (l-NAC; 500 mg kg^–1, i.p.) on the urinary elimination of AMCC in rats dosed with MIC (15 mg kg^–1, i.p.). In separate experiments, groups of rats were pretreated with eitherN-acetyl-d-cysteine (d-NAC) orN-trideuteroacetyl-l-cysteine (d₃-l-NAC) in order to explore the mechanism by which MIC undergoes conjugation to AMCCin vivo. The results indicated that exogenous NAC effectively enhances the urinary excretion of MIC in the form of AMCC, and that it does so largely by direct conjugation with the isocyanate, rather thanvia biosynthesis to GSH.     

Purification and Characterization of a Novel Thermostable Mycelial Lectin from <Emphasis Type="Italic">Aspergillus terricola</Emphasis>

Lectin has been isolated from mycelia of Aspergillus terricola by single step purification on porcine stomach mucin-Sepharose 4B affinity column. Lectin could be effectively purified with 75% recovery and 4.47-fold increase in specific activity. Lectin migrated as a single band on SDS-PAGE with an apparent molecular mass of 32.5 kDa. Sugar inhibition assay revealed that the lectin did not strongly interact with most carbohydrates and their derivatives tested while strong binding affinity to d-glucose, d-sucrose, N-acetyl-d-galactosamine, asialofetuin, porcine stomach mucin, and bovine submaxillary mucin was indicated. Neuraminidase and protease treatment to erythrocytes enhanced lectin titre. Lectin activity was stable within the pH range of 7.0–10.5. A. terricola lectin displayed remarkable thermostability and remained unaffected upon incubation at 70 °C for 2.5 h. Lectin did not require metal ions for its activity. Incubation with denaturants (urea, thiourea, and guanidine–HCl) substantially reduced lectin activity. Carbohydrate analysis revealed that it is a glycoprotein with 9.76% total sugars.     

Synthesis of β-<Emphasis Type="SmallCaps">d</Emphasis>-galactopyranosylamine derivatives with the terminal amino group in the spacer as mono-, di-, and trivalent ligands of galectins

Glycoclusters were obtained by N-alkylation of N-glycyl-β-d-galactopyranosylamine with N-chloroacetyl derivatives of β-d-galactopyranosylamine and N,N″-iminodiacetyl-di-β-d-galactopyranosylamine. The glycoclusters with two and three galactopyranosylamine residues and the monovalent ligand N-diglycyl-β-d-galactopyranosylamine with an amino group in the spacer are suitable for subsequent conjugation with carboxyl-containing physiologically active compounds.     

Mixed-ligand complexes of nickel(II) involving sulphur-containing ligands and diaminocarboxylic acids

Summary Stability constants for mixed-ligand complexes of the types [NiABH₂], [NiABH] and [NiAB] formed by Ni^II with l-cysteine (cys), d-penicillamine (pen) or l-cysteic acid (cya) as ligand A and dl-2,3-diaminopropionic acid (dapa), dl-2,4-diaminobutyric acid (daba) or dl-ornithine (orn) as ligand B have been determined by the computerbased analysis of pH titration data obtained at 37 °C and I = 0.15 mol dm^–3 (NaClO₄). In the [NiABH] species, for all three secondary ligands (B), when A = pen or cya the labile proton appears to be attached to the terminal amino group of ligand B, whereas when A = cys it is not clear where the proton is located. In all the systems in the [NiABH₂] species, one proton resides with the primary ligand (A) and the other with the secondary ligand (B). In the [NiAB]-type complexes, cys and pen chelate through the amino and thiolato groups, while cya binds in a glycine-like mode and the secondary ligands (B) coordinate in a terdentate manner.Author to whom all correspondence should be directed.     

Enzymatic preparation of optically active silicon-containing amino acids and their application

Optically active 3-trimethyl silylalanine (TMS-Ala) was prepared by hydrolysis of N-acetyl-dl-TMS-Ala catalyzed by acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC3.5.1.14). Acylase I from porcine kidney (PKA) was found to be more effective than that from Aspergillus melleus in the preparation of l-TMS-Ala. Under the optimized conditions, optically pure l-TMS-Ala (>99% enantiomeric excess, ee) was obtained with a 72% yield. Furthermore, a highly optically pure d-TMS-Ala (96% ee) could also be obtained with a 76% yield by chemical hydrolysis of the residual substrate. Enzymatic synthesis of peptides containing TMS-Ala was also attempted in ethyl acetate. Benzyloxycarbonyl (Z)-l-TMS-Ala served as the substrate for thermolysin, whereas l-TMS-Ala-OMe was inactive as the amino component. In the case of inhibitory activity of dipeptides toward thermolysin, l-Leu-(l-TMS-Ala) was found to be a more potent inhibitor than l-Leu-l-Leu, which is known to be one of the most effective inhibitors of thermolysin among the dipeptides consisting of natural aminoacids.     

Micro-Scale Frontal Affinity Chromatography with Mass Spectrometric Detection: A New Method for the Screening of Compound Libraries

In one run the binding constants K_d for all the active components of a ligand library at sub-microgram quantities can be determined. A mixture of ligands is continuously infused through a column of immobilized receptor, and the eluent analyzed by electrospray mass spectrometry. From the affinity chromatogram produced (see picture) the breakthrough volume of a single compound and hence its K_d value can be determined.     

Synthesis and assessment of molecular recognizability by RP-HPLC of an N-alkyl-β-Ala-L-Phe-derived organic phase with self-assembling ability

An N-alky-β-Ala-L-Phe derivative, N'-octadecyl-N ^α -[(N-acryloyl)-β-alanyl]-L-phenylalanineamide (1), with a polymerizable head group has been synthesized and telomerized with the silane coupling agent 3-mercaptopropyltrimethoxysilane (MPS). SEM and DSC observations indicated that both 1 and its telomer (T-1) could self-assemble into fibrillar forms with highly ordered structures in organic media such as benzene through complementary hydrogen bonding between the amide moieties. T-1 was grafted onto porous silica gels through the terminal trimethoxysilyl group and then packed into a stainless steel column. RP-HPLC results for polycyclic aromatic hydrocarbons (PAHs) demonstrated that significantly higher molecular shape recognition could be achieved by silica-supported T-1 (Sil-T-1). In this paper, the mechanism of the selectivity enhancement in HPLC by Sil-T-1 is discussed on the basis of comparing with the corresponding L-Phe derivative N'-octadecyl-N ^α -(acryloyl)-L-phenylalanineamide (2) without β -Ala and the stationary phase (Sil-T-2) obtained from it. The HPLC column materials Sil-T-1 and Sil-T-2 were characterized by DSC, TGA, DRIFT-IR, and ¹³C and ²⁹Si CP-MAS NMR spectroscopic measurements. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dimericd- anddl-dysprosium(iii) tartrates: paramagnetic birefringence,molecular mechanics,stereoselectivity

The molar constants of paramagnetic birefringence (PBR) for the dimeric dysprosium(iii)d- anddl-tartrates, Dy₂(d-Tart)(l-Tart)^2– (1) and Dy₂(d-Tart)₂ ^2– (2) have been determined by means of pH-metric and PBR measurements. The simulation of the structure of the ligand and solvate environment has been carried out using the method of molecular mechanics (Dashevsky-Plyamovaty model, the MIND program). In addition to the four oxygen atoms from the ligand, each Dy^III ion coordinates four molecules of water and a Na⁺ ion.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1029–1032, June, 1994.     

Tris{2‐[N‐(diethylaminothiocarbonyl)benz(‐amidino;‐imidoxy;‐imidothio)‐N′‐yl]ethyl}amine – neue tripodale Liganden. Darstellung,Komplexstabilität und Extraktionsverhalten ihrer Silber(I)‐Komplexe

Tris{2‐[ N ‐(diethylaminothiocarbonyl)benz(‐amidino; imidoxy; ‐imidothio)‐ N ′‐yl]ethyl}amines – New Tripodal Ligands. Synthesis, Complex Stability, and Extraction Behaviour of their Silver(I) Complexes N‐(Thiocarbamoyl)‐benzimidoylchlorides react with trivalent nucleophiles to give four novel tripodal ligands. Two of them have been characterized by X‐ray methods. The ligands form with silver(I) cationic mononuclear complexes in which the three arms of the ligand are coordinated monodentately via sulfur. The results of FAB and ESI mass spectrometry as well as ESCA and NMR investigations verify this binding mode. The protonation constants of the ligands and the stability constants of silver(I) complexes have been determined potentiometrically. The novel tripodal compounds behave as powerful extractands for silver(I).     

CCR4 expressing cells cultured adherently on a capillary wall and formaldehyde fixed as the stationary phase for ligand screening by ACE

We have developed an on‐line screening method for CC chemokine receptor 4 (CCR4) ligands, in which the whole cells expressed with CCR4 were cultured adherently and immobilized on the inner wall of the capillary as the stationary phase for the first time. Moreover, in this method it is unnecessary to isolate and purify the target receptors from cell membranes. Therefore, it is possible to almost completely preserve the native conformation of the target receptors. The binding activities of the immobilized CCR4 did not change. A known antagonist of CCR4, compound A, was employed to validate the bioactivity of the cell layer and stability of this method. The intraday, interday, and batch‐to‐batch reproducibilities were investigated (RSD ≤ 13.9%). Nonlinear chromatography was used to calculate the binding constant between the compound A and CCR4 (6.4 × 10⁴/M, RSD = 4.96%). Using this method, the qualitative and quantitative characterizations of 23 computer‐aided drug design compounds were achieved and the kinetic parameters (K, k_a, k_d, and k′) were obtained by nonlinear chromatography. Three active compounds were screened out, which also showed activity in chemotaxis inhibition assay. The experimental results show that this method is simple, sensitive, and efficient for drug screening. Moreover, it offered a novel way to detect the nonspecific interactions between ligands and cell membrane.