Improved Proton Decoupling in NMR Spectroscopy of Crystalline Solids Using the SPINAL-64 Sequence

Summary.  The performance of three different spin decoupling schemes, CW, TPPM, and SPINAL-64 is compared, by recording proton decoupled ¹³C NMR spectra of a crystalline glycine sample, with 20% isotopic labelling. At a magnetic field of B ₀ = 14.1 T, the two phase modulated pulse schemes, TPPM and SPINAL-64, are shown to give decisively better results than CW decoupling, and the SPINAL-64 sequence is found to perform better than TPPM. It is suggested that in NMR of crystalline solids, SPINAL-64 offers a viable and competitive alternative to the well established TPPM decoupling technique, especially at higher magnetic fields. Corresponding author. E-mail: thomas.braeuniger@durham.ac.uk Received May 6, 2002; accepted May 22, 2002     

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.     

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.     

Low-temperature heat capacity of heptacopper(II) complex [Cu7(μ3-Cl)2(μ3-OH)6-(d-pen-disulfide)3]

A single crystal calorimetry of a heptacopper(II) complex of [Cu₇(μ₃-Cl)₂(μ₃-OH)₆-(d-pen-disulfide)₃] which has a double-cubane structure supported by d-penicillaminedisulfides has been performed at low-temperature region below 8 K. This compound is a metal complex which contains seven Cu(II)s in a cluster unit. These Cu(II)s are magnetically coupled each other by strong intra-complex interactions. The heat capacities under magnetic fields exhibit Schottky type anomalies explained by the Zeeman splitting of the doublet ground state of the complex. The g-value of the ground state is evaluated as 1.86 from the systematic analysis of the Schottky peak under magnetic fields. The first excited state of the cluster seems to be separated at least by several Kelvins, which is consistent with the theoretical calculations and magnetic susceptibility results.     

Mixed valency in the high-temperature phases of transition metal molybdates, A MoO4 ( A =Fe, Co, Ni)

Transition metal molybdates of the formulaAMoO₄ whereA=Fe, Co or Ni exhibit a first-order phase transition between 670K–970K. An investigation of the lowtemperature (lt) and high-temperature (ht) phases by x-ray photoelectron spectroscopy, x-ray absorption spectroscopy, magnetic susceptibility and other physical methods shows that the phase transition is associated with a valence change of the typeA ²⁺+Mo⁶⁺αA ³⁺+Mo⁵⁺ in the cases of iron and cobalt molybdates. Contribution No. 311 from the Solid State and Structural Chemistry Unit.     

Chromium(III) complexes of naturally occurring amino acids

Summary Amino acid complexes of CrCl₃Py₃ have been prepared and studied by elemental analysis, magnetic susceptibility, vibrational (i.r.), electronic and circular dichroism spectroscopy. Two peaks in the visible spectra are assigned to a d-d transition in pseudo octahedral symmetry. The spectrochemical parameters (Dq, B and ₃₅) for the complexes were calculated which confirm that pyridine nitrogen and/or chlorine are not removed. Prolonged heating or bubbling of air through the solution of CrCl₃-Py₃ containing l-(-)-histidine or l-(-)-threonine for several hours enhanced formation of chromium (VI).     

Reactions of heteroaromatic chromophores with lanthanide complexes of p-sulfonatothiacalix[4]arene

The conditions for the formation of heteroleptic complexes of a lanthanide(iii) ion (Ln = La, Gd, and Tb) with p-sulfonatothiacalix[4]arene and a heteroaromatic chromophore in water were found using X-ray diffraction analysis, pH-metry, ¹H NMR and UV—Vis spectroscopy, and nuclear magnetic relaxation. In the resulting complexes, the heteroaromatic chromophore is in the calix[4]arene cavity and the lanthanide ion is coordinated by the electron-donating groups of the upper or lower calix[4]arene rim. Emission spectroscopic studies revealed changed emission properties of Tb^III ions in the terbium(iii)—p-sulfonatothiacalix[4]arene—bipy complex. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1871–1878, September, 2008.     

Synthesis and characterization of a functionalized amphiphilic diblock copolymer: MePEG-b-poly(DL-lactide-co-RS-β-malic acid)

A class of novel amphiphilic diblock copolymer of MePEG-b-poly(DL-lactide-co-RS-β-malic acid) has been synthesized via the hydrogenation over palladium on charcoal of MePEG-b-poly(DL-lactide-co-RS-β-benzyl malolactonate), which was prepared by ring-opening copolymerization of DL-lactide and RS-β-benzyl malolactonate (MABz) using methyl-polyethylene glycol (MePEG) as the initiator and stannous octoate as the catalyst. The influence of copolymerization temperature, reaction time, macro-initiator (MePEG-5000) proportion and monomer ratio was studied. Gel permeation chromatography measurements revealed that the molecular weight decreased with increasing MABz feeding dose. The configurational structures of the protected and de-protected copolymers were determined by ¹³C nuclear magnetic resonance (NMR), ¹H NMR and Fourier transform infrared. A water-swollen core of the nanospheres formed from the de-protected copolymer was discovered by transmission electron microscopy measurement. Additionally, the degradation experiments indicated that more hydrophilic malic acid content led to higher degradation rate.     

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.     

Structure-function relationships of a catalytically efficient β-<Emphasis Type="SmallCaps">D</Emphasis>-xylosidase

β-d-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k _cat, k _cat/K _m) for promoting hydrolysis of 1,4-β-d-xylooligosaccharides. ¹H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 1,4-β-d-xylobiose (X2). Progress curves of 4-nitrophenyl-β-d-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k _cat and k _cat/K _m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites −1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites bey ond−1 are blocked by protein and subsites bey ond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k _cat and k _cat/K _m by <10³ and to asimilar extent for substrates 4NPX and 4-nitrophenyl-α-l-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs k _cat/K _m with pK _a values of 5.0 and 7.0 assigned to D14 and E186, respectively. k _cat (4NPX) has a pK _a value of 7.0 and k _cat (4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, “dianionic” enzyme form (D14-E187-) binds 4NPX but not 4NPA. The mention of firm names or trade products does not imply that they are end orsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Cloning of two genes (LAT1,2) encoding specific L: -arabinose transporters of the L: -arabinose fermenting yeast Ambrosiozyma monospora

We identified and characterized two genes, LAT1 and LAT2, which encode specific l-arabinose transporters. The genes were identified in the l-arabinose fermenting yeast Ambrosiozyma monospora. The yeast Saccharomyces cerevisiae had only very low l-arabinose transport activity; however, when LAT1 or LAT2 was expressed, l-arabinose transport was facilitated. When the LAT1 or LAT2 were expressed in an S. cerevisiae mutant where the main hexose transporters were deleted, the l-arabinose transporters could not restore growth on d-glucose, d-fructose, d-mannose or d-galactose. This indicates that these sugars are not transported and suggests that the transporters are specific for l-arabinose.     

Characterization and complementation of a Pichia stipitis mutant unable to grow on d-xylose or l-arabinose

Pichia stipitis CBS 6054 will grow on d-xylose, d-arabinose, and l-arabinose. d-Xylose and l-arabinose are abundant in seed hulls of maize, and their utilization is important in processing grain residues. To elucidate the degradation pathway for l-arabinose, we obtained a mutant, FPL-MY30, that was unable to grow on d-xylose and l-arabinose but that could grow on d-arabinitol. Activity assays of oxidoreductase and pentulokinase enzymes involved in d-xylose, d-arabinose, and l-arabinose pathways indicated that FPL-MY30 is deficient in d-xylitol dehydrogenase (D-XDH), d- and l-arabinitol dehydrogenases, and d-ribitol dehydrogenase. Transforming FPL-MY30 with a gene for xylitol dehydrogenase (PsXYL2), which was cloned from CBS 6054 (Gen Bank AF127801), restored the D-XDH activity and the capacity for FPL-MY30 to grow on l-arabinose. This suggested that FPL-MY30 is critically deficient in XYL2 and that the d-xylose and l-arabinose metabolic pathways have xylitolas a common intermediate. The capacity for FPL-MY30 to grow on d-arabinitol could proceed through d-ribulose.     

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.     

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     

Micellar structure of an oligopeptide surfactant “trimeric N -dodecanoyl-L-proline potassium salt” in aqueous solution – small-angle neutron scattering study

Structures of the micelles which are formed by the chiral oligopeptide surfactant N-dodecanoyl-L-proline tripeptide anions have been examined using small-angle neutron scattering spectral analysis. Results show that the chiral N-dodecanoyl-L-proline trimeric anions may form a spherical micelle with an aggregation number of 36 and that the oligopeptide portions with a poly-L-proline I-type helical structure are saturated with water. Received: 21 March 2001 Accepted: 5 April 2001     

Synthesis and properties of fused tetracyclic derivatives of 1,4-naphthoquinone thioglycosides

A series of 1,4-naphthoquinone O-acetylthioglycosides have been synthesized by the condensation of fully O-acetylated derivatives of 1-thio-d-xylose, 1-thio-L-arabinose, 1-thio-d-galactose, 1-thio-d-mannose, and 1-thiomaltose with 3-chloro-2-methoxy-1,4-naphtho-quinone. Their deacetylation with MeONa/MeOH proceeded with simultaneous heterocyclization to yield linear carbohydrate-containing tetracyclic quinones. Tetracycles with trans junction of the carbohydrate and quinone rings are poorly soluble in water and organic solvents.     

Enthalpies of Dilution and Enthalpies of Mixing of Amino Acids with Sucrose in Aqueous Solutions at 298.15 K

The enthalpies of mixing of aqueous solutions have been determined for sucrose with six different amino acids (glycine, l-alanine, l-serine, l-valine, l-proline and l-threonine) at 298.15 K, by using a LKB-2277 flow microcalorimetric system. These results, along with the enthalpies of dilution of these solutes for the initial solutions, were used to determine the enthalpic interaction coefficients (h _xy, h _xyy, h _xxy) of the McMillan–Mayer Theory. The pair-wise cross interaction coefficients of amino acids and sucrose are discussed from the viewpoint of solute–solute interactions.     

Coordination complexes of l-azetidine-2-carboxylic acid with platinum(II) and palladium(II)

Summary The complexes K[Pt(l-aze)Cl₂, [Pt(l-aze)₂] and [Pd(l-aze)₂] (l-aze = l-azetidine-2-carboxylate) were prepared. X-ray structures show that [Pt(l-aze)₂] and [Pd(l-aze)₂] are isomorphous, having a planar tetragonal geometry with a trans configuration around the Pt and Pd atoms. Slight puckerings of the MN(1)N(11)O(11) chelate ring (M = Pt or Pd) and the azetidine ring were observed. The circular dichroism (c.d.) spectra of the complexes in aqueous solution agree with the structures found in the solid state as far as the hexadecant rule is concerned, giving, for the trans configuration of [M(l-ia)₂] (where ia = imino acid), the profile of the c.d. signs for the three predominant d-d transitions as: +,-,-. I.r., conductivity and n.m.r. measurements are also reported and are in accord with the proposed structures.     

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.     

Temperature- and pressure-responsive properties of <Emphasis Type="SmallCaps">l</Emphasis>- and<Emphasis Type="SmallCaps"> dl</Emphasis>-forms of poly(<Emphasis Type="Italic">N</Emphasis>-(1-hydroxymethyl)propylmethacrylamide) in aqueous solutions

The structural transition of the l- and dl forms of poly(N-(1- hydroxymethyl)propylmethacrylamide (PHMPMA) in aqueous solution was studied by measuring the pressure dependence of the apparent scattering intensity, differential scanning calorimetry (DSC), and circular dichroism (CD). The thermodynamic implications of the results are discussed in relation to the chiral structure of the side chain, and differences in the thermal and barometric transitions. T-P diagrams of the transition showed characteristic ellipsoid features. Antagonism of the temperature and pressure effects was observed only for P(dl-HMPMA). For P(l-HMPMA), the transition temperature (T _tr) decreased with increasing pressure, and the highest T _tr was observed at atmospheric pressure (0.1 MPa). For both polymers, the highest P _trs were observed at the lowest temperatures. The l polymer showed a specific negative peak in its CD spectrum at around 220 nm in the lower temperature region and the temperature dependence was reproduced by a single-step transition, with the midpoint corresponding to the T _tr obtained from the scattering measurements. Coupled with the results from the DSC, the different behavior between the P(l-HMPMA) and P(dl-HMPMA) could be explained in terms of the chain states before and after the transition. The cooperative factors derived from the DSC measurement revealed that about 4 to 5 polymers of the present size were necessary to perform a thermal transition for P(l-HMPMA), and that P(dl-HMPMA) underwent its transition as an almost single molecular event.This revised version was published online in June 2005 with correction to the article category.