David B. Davies

The effect of variation of substitution on the solution conformation of heparin: a spectroscopic and molecular modelling study

The effect of variations in substitution on the conformation of iduronate-containing sequences in heparin and heparan sulphate has been studied using a series of chemically-modified heparins in which substitution with O- and N-sulphate and N-acetyl substituents has been systematically altered. Monosaccharides corresponding to residues in these modified heparins have also been investigated. The conformations of the glycosidic linkages in O- and N-desulphated re-N-acetylated heparin, O-desulphated re-N-sulphated heparin, and 6-O-desulphated re-N-sulphated heparin have been compared with those of N-desulphated re-N-acetylated heparin and of heparin itself, which have been compared with those of N-desulphated re-N-acetylated heparin and of heparin itself, which have previously been reported [B. Mulloy, M.J. Forster, C. Jones, and D.B. Davies, Biochem. J., 293 (1993) 849-858]. The overall conformation of all the polysaccharides is shown to be similar, regardless of substitution pattern. The conformational equilibrium of the pyranose ring of iduronic acid residues in the polysaccharides has been monitored by the use of 13C NMR chemical shift temperature coefficients, and shown to be similar for all the modified heparins with the exception of N-desulphated re-N-acetylated heparin. Circular dichroism spectra of all the polysaccharides are reported, and their variations attributed to differences in the proportions of pyranose ring forms in the iduronate conformational equilibrium.

1 H NMR investigation of self-association of aromatic drug molecules in aqueous solution. Structural and thermodynamical analysis

Self-association of aromatic drug molecules, proflavine (PF), Acridine Orange (AO), ethidium bromide (EB) and actinomycin D (ActD), in aqueous salt solution has been studied by one- and two-dimensional 500 MHz 1H NMR spectroscopy. 2D-COSY and 2D-NOESY measurements were used for complete assignment of proton signals of EB and ActD in solution and for a qualitative determination of their self-association, i.e. the mutual arrangement of the drug molecules in the complexes. Concentration and temperature dependences of proton chemical shifts of the drugs have been measured. Experimental results have been analysed using indefinite non-cooperative and cooperative models of molecular self-association, enabling the determination of equilibrium reaction constants, parameters of cooperativity, the thermodynamical parameters (enthalpy and entropy) of the self-association reactions and the limiting values of drug proton chemical shifts in the complexes. The most favourable dimer structures of PF, AO, EB and ActD have been constructed using calculated values of induced chemical shifts of drug protons in conjunction with intermolecular NOEs. The results show that the drug chromophores have an antiparallel orientation in all the dimers studied.

1H NMR investigation of the hetero-association of aromatic molecules in aqueous solution: factors involved in the stabilization of complexes of daunomycin and acridine drugs

Hetero-association of the anthracycline drug, daunomycin (DAU), with typical mutagens, the acridine dyes proflavine (PF) and acridine orange (AO), has been studied by 500 MHz 1H NMR spectroscopy as a function of concentration and temperature in 0.1 mol dm−3 phosphate buffered aqueous solutions at pD = 7.1. The results have been analysed in terms of a statistical-thermodynamical model of hetero-association of aromatic molecules, described previously [Davies, D. B., Veselkov, D. A., and Veselkov, A. N., 1999, Molec. Phys., 97, 439], but generalized in this work, so that there is no limitation on the magnitudes of the self-association constants of the interacting molecules. Expressions suitable for the analysis of NMR parameters of both components in the mixed solution have been developed enabling both the structural and thermodynamic properties of hetero-association to be determined. The magnitude of the equilibrium constant for hetero-association of PF + DAU is found to be substantially higher than the self-association constants of these molecules, whereas that for hetero-association of AO + DAU is intermediate between the equilibrium constants of self-association of AO and DAU. Intermolecular cross-peaks observed in 2D-ROESY spectra of PF + DAU mixed solutions are consistent with formation of a hetero-association complex in which an intermolecular hydrogen bond can form between either of the 3,6-diamino groups of the PF chromophore and the 9-MeCO group of DAU, which is in contrast to AO + DAU hetero-association, where such hydrogen bonds are unable to form. Quantitative structural and thermodynamical analysis of PF + DAU complexation is consistent with an intermolecular hydrogen bond contributing to the stability of the hetero-complex in aqueous solution. The NMR results show that hydrophobic interactions play a substantial role in the stabilization of the AO-DAU complex, characterized by a relatively small entropy change on complexation, compared to the PF-DAU hetero-complex, which is mainly stabilized by hydrogen bond and dispersive van der Waals interactions.

NMR Investigation of the Complexation of Daunomycin with Deoxytetranucleotides of Different Base Sequence in Aqueous Solution

Abstract 500 MHz NMR spectroscopy has been used to investigate the complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary deoxytetranucleotides, 5′-d(CGCG), 5′-d(GCGC), 5′-d(TGCA), 5′-d(ACGT) and 5′-d(AGCT), of different base sequence in aqueous salt solution. 2D homonuclear 1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H—31P NMR spectroscopy (HMBC) have been used for complete assignment of the non-exchangeable protons and the phosphorus resonance signals, respectively, and for a qualitative determination of the preferred binding sites of the drug. Analysis shows that DAU intercalates preferentially into the terminal sites of each of the tetranucleotides and that the aminosugar of the antibiotic is situated in the minor groove of the tetramer duplex, partly eclipsing the third base pair. A quantitative determination of the complexation of DAU with the deoxytetranucleotides has been made using the experimental concentration and temperature dependences of the drug proton chemical shifts; these have been analysed in terms of the equilibrium reaction constants, limiting proton chemical shifts and thermodynamical parameters (enthalpies AH, entropies AS) of different drug-DNA complexes (1:1, 1:2, 2:1, 2:2) in aqueous solution. It is found that DAU interacts with sites containing three adjacent base pairs but does not show any significant sequence specificity of binding with either single or double-stranded tetranucleotides, in contrast with other intercalating drugs such as proflavine, ethidium bromide and actinomycin D. The most favourable structures of the 1:2 complexes have been derived from the induced limiting proton chemical shifts of the drug in the intercalated complexes with the tetranucleotide duplex, in conjunction with 2D NOE data. It has been found that the con- formational parameters of the double helix and the orientation of the DAU chromophore in the intercalated complexes depend on base sequence at the binding site of the tetramer duplexes in aqueous solution.

Intramolecular hydrogen bonding in 1'-sucrose derivatives determined by SIMPLE proton NMR spectroscopy

Intramolecular hydrogen bonding in a series of 1’-sucrose derivatives having different numbers of hydroxyl groups has been investigated in Me2SO-d6 solution by SIMPLE ‘H NMR measurements, Le., Secondary Isotope Multiplet NMR Spectroscopy of Partially Labeled Entities. Isotope effects, transmitted through intramolecular hydrogen bonds, are observed for the hydroxyl proton resonances; each separate hydrogen bond is manifested as a separate isotope shift. The existence of an intramolecular hydrogen bond in which OH3’ is the donor and OH2 is the acceptor hydroxyl group is revealed by isotope shift measurements of four 1’-chloro1’-deoxysucrose derivatives: namely, 1,6-dichloro1,6-dideoxy-~-~-fructofuranosyl 4chloro-4-deoxy-3-O-methyl-a-~-galactopyranoside (1 1; 1,6-dichloro1,6-dideoxy-~-~-fructofuranosyl 4-chloro-4-deoxy-cu-~galactopyranoside (2), 1,6-dichloro1,6-dideoxy-~-~-fructofuranosyl a-D-ghcopyranoside (3), and 1 -chloro1 -deoxy-P-Dfructofuranosyl a-D-glucopyranoside (4). It is found that the OH3’-02 interresidue hydrogen bond in 1’-chloro1’-deoxysucrose derivatives in solution is weaker than the analogous OH 1’.-02 hydrogen bond in 3’-substituted sucrose derivatives. ’H NMR measurements also show that the interresidue hydrogen bond stabilizes a weak hydrogen bond network between adjacent hydroxyl groups; the network extends to both sugar residues and the hydrogen bonds become progressively weaker at distances further from the relatively strong interresidue hydrogen bond, Le., OH6.OH4-OH3-OH2..-OH3’-.OH4’. For this series of molecules it is also shown that the interresidue hydrogen bonds become stronger as the hydrogen-bonding network extends to more of the molecule, Le., the process is cooperative. Hydrogen bonding is an important interaction involved in determining the secondary structures of biomolecules such as proteins, nucleic acids, and carbohydrates. Evidence about the existence of hydrogen bonds in these systems comes from crystal structure determinations and, in particular, from neutron diffraction studies which provide the location of hydrogen atoms in the lattice. Hydrogen bonding in carbohydrate crystals has been studied extensively by Jeffrey and co-workersId and by Saenger and c o w ~ r k e r s . ~ ~ ~ Many examples of hydrogen bonding in carbohydrate crystals occur as part of networks of intermolecular hydrogen bonds which may be chain-like’” or ~ i r c u l a r . ~ ~ ~ Under these circumstances, it is found that bond distances are shorter (and hence hydrogen bonds presumed stronger) for hydroxyl groups involved in both donor and acceptor interactions compared to those where the hydroxyl group is a donor only (cooperative effect on hydrogen bonding). Quantum mechanical c a l c ~ l a t i o n s ~ ~ ~ ~ confirm that chain-like hydrogen bonds in the crystal structure are energetically favored above individual ones. By comparison with crystallographic studies such detailed information about the strength and direction of hydrogen bonding is generally not available for molecules in solution, though a number of N M R methods have been used to provide information on the presence of both interand intramolecular hydrogen bonds, e.g., chemical shifts, solvent and temperature dependence of chemical shifts, solvent exchange studies, NOE effects, and magnitudes of appropriate spin coupling constants.” In an early study of intramolecular hydrogen bonding in solution Lemieux and Paviai2 showed that hydrogen bonding of a strong acceptor such as Me2S0 to a hydroxyl group increased the ability of that hydroxyl to participate in intramolecular hydrogen bonding. Although N M R methods are used to indicate the presence of hydrogen bonding in carbohydrates, they are usually unable to discriminate between the donor and acceptor hydroxyl groups or to provide a basis for comparison of the relative strengths of hydrogen bonds in these molecules. This is particularly difficult in solvents like Me2S0, which are strong hydrogen bond acceptors and where intermolecular (solvent) hydrogen bonding is predominant. On the other hand, recent ’H N M R studies of carbohydrates in Me2SO-d6 solution (sucrose,’3i5 cyclodextrin,’6 and 3,3’,4’,6’-tetra-0-acetylsucrose (5)17) have shown that, for mol’ Birkbeck College. ‘Tate & Lyle Group Research and Development, Chart I compno R’ R2 R 3 R4 no. of OHgroups

Determination of glycosidic bond conformations of pyrimidine nucleosides and nucleotides using vicinal carbon–proton coupling constants

Measurements have been made of 3J(C-6,H-1′) and 3J(C-2,H-1′) magnitudes of pyrimidine nucleosides and nucleotides in solution by natural abundance, 13C n.m.r. spectroscopy and the results have been interpreted in terms of the glycosidic bond conformations of the nucleic acid derivatives. Based on X-ray crystal structure results a four-state conformational model for the glycosidic bond is introduced consisting of an equilibrium between two syn and two anti conformations; each syn or anti conformation is symmetrically related to the C-1′–H-1′ bond direction and appropriate syn and anti conformations are symmetrically related by 180°. It is shown that a quantitative estimate of the glycosidic bond conformation may be determined from the sum of observed coupling constants [i.e., ΣJ=J(C-6,H-1′)+J(C-2,H-1′)] and that the equilibrium composition of syn and anti conformers may be determined from the difference in observed coupling constants, i.e., ΔJ=J(C-6,H-1′)–J(C-2,H-1′). Results for uridine and cytidine derivatives are discussed.

A general nuclear magnetic resonance analysis of hetero-association of aromatic molecules in aqueous solution

A general nuclear magnetic resonance analysis of a statistical-thermodynamical model of hetero-association of aromatic molecules in solution has been developed to take “edge effects” into consideration, i.e., the dependence of proton chemical shifts on the position of the molecule situated inside or at the edge of the aggregate. This generalized approach is compared with a previously published model, where an average contribution to proton shielding is considered irrespective of the position of the molecule in the stack. Association parameters have been determined from experimental concentration and temperature dependences of 500 MHz proton chemical shifts of the hetero-association of the acridine dye, proflavine, and the phenanthridinium dye, ethidium bromide, in aqueous solution. Differences in the parameters in the range 10%–30% calculated using the basic and generalized approaches have been found to depend substantially on the magnitude of the equilibrium hetero-association constant Khet—the larger th...

The structural and stereogenic properties of pentaerythritoxy-bridged cyclotriphosphazene derivatives: spiro-spiro, spiro-ansa and ansa-ansa isomers.

Reactions of pentaerythritol with hexachlorocyclotriphosphazene, N3P3Cl6, and gem-disubstituted cyclotriphosphazene derivatives, N3P3Cl4R2 [R = Ph, NHBu(t) or (OCH2CF2CF2CH2O)0.5] gave a series of pentaerythritol-bridged derivatives linked spiro-spiro, spiro-ansa and ansa-ansa. The structures and stereogenic properties of the products were characterised by X-ray crystallography and 31P NMR spectroscopy on addition of the chiral solvating agent, (S)-(+)-2,2,2-trifluoro-1-(9-anthryl)ethanol. Molecules with spiro-spiro and spiro-ansa bridged gem-disubstituted cyclophosphazenes [R = Ph, NHBu(t) or (OCH2CF2CF2CH2O)0.5] are found to be chiral and exist as racemates. Molecules with ansa-ansa bridged cyclophosphazenes [R = Cl or (OCH2CF2CF2CH2O)0.5] have been characterised for the first time and are shown to have meso configurations. Analysis of crystal structure data shows that the six-membered chair form of the spiro rings and the eight-membered boat-chair form of the ansa rings in the bridged compounds are similar to analogous spiro and ansa exocyclic ring conformations of 1,3-propanedioxy-derivatives of cyclophosphazenes.

Structural investigations of phosphorus–nitrogen compounds. 7. Relationships between physical properties, electron densities, reaction mechanisms and hydrogen-bonding motifs of N3P3Cl(6 − n)(NHBut)n derivatives

A series of compounds of the N3P3Cl(6 - n)(NHBu(t))n family (where n = 0, 1, 2, 4 and 6) are presented, and their molecular parameters are related to trends in physical properties, which provides insight into a potential reaction mechanism for nucleophilic substitution. The crystal structures of N3P3Cl5(NHBu(t)) and N3P3Cl2(NHBu(t))4 have been determined at 120 K, and those of N3P3Cl6 and N3P3Cl4(NHBu(t))2 have been redetermined at 120 K. These are compared with the known structure of N3P3(NHBu(t))6 studied at 150 K. Trends in molecular parameters [phosphazene ring, P-Cl and P-N(HBu(t)) distances, PCl2 angles, and endo- and exocyclic phosphazene ring parameters] across the series are observed. Hydrogen-bonding motifs are identified, characterized and compared. Both the molecular and the hydrogen-bonding parameters are related to the electron distribution in bonds and the derived basicities of the cyclophosphazene series of compounds. These findings provide evidence for a proposed mechanism for nucleophilic substitution at a phosphorus site bearing a PCl(NHBu(t)) group.

Location and quantitation of the sites of O-acetylation on the capsular polysaccharide from Streptococcus pneumoniae type 9V by 1H-n.m.r. spectroscopy: comparison with type 9A

The 1H-n.m.r. spectra of the Streptococcus pneumoniae type 9V (S68 in American nomenclature) capsular polysaccharide (PS) and its O-deacetylated derivative [which is structurally identical to the S. pneumoniae type 9A (S33) PS] were assigned using COSY, relayed-COSY, and 2D-NOESY experiments. The positions of the OAc groups in the alpha-GlcA, beta-ManNAc, and alpha-Glc residues of the native 9V PS were established using 2D-n.m.r. and chemical shift arguments, and the relative proportions of different O-acetylated species were estimated by integration of well-resolved 1H-n.m.r. signals. The locations of the OAc substituents differ from those previously reported. [formula: see text].