Wojciech Bocian

NMR and molecular modeling study, as complementary techniques to capillary electrophoresis method to elucidate the separation mechanism of linezolid enantiomers.

NMR study and molecular modeling were performed to improve the level of understanding of the chiral recognition process occurring between linezolid and anionic single-isomer cyclodextrin-heptakis-(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD). NMR spectrometry allowed to estimate the stoichiometry of the complexes between HDAS-beta-CD and S- or R-linezolid and to determine the binding constants. The 1:1 complex stoichiometry was detected in millimolar concentrations and the mode of binding was proposed. The binding constants Ka of the complexes were of the order of 30-80 M(-1). Molecular dynamic simulations of 40ns for four complexes and calculations of binding free energies were performed. These calculations allowed determining the mode of binding of linezolid to HDAS-beta-CD and explaining the binding enantioselectivity.

Assessment of Oversulfated Chondroitin Sulfate in Low Molecular Weight and Unfractioned Heparins Diffusion Ordered Nuclear Magnetic Resonance Spectroscopy Method

Heparins can be contaminated with oversulfated chondroitin sulfate, OSCS, the impurity being linked to adverse clinical events that certain lots of heparins have had on humans. Here, we propose labeling of the N-acetyl peaks in (1)H NMR spectra of heparins with the parameter D(t), describing the translational diffusion coefficient available from DOSY NMR. We show how DOSY can be applied as a routine method for screening the lots of heparins for obtaining the impurity profile when using (1)H NMR.

Direct insight into insulin aggregation by 2D NMR complemented by PFGSE NMR

The aggregation of Zn(II)‐bound and zinc‐free human insulin was studied in solution using the Hβ∼CH3 crosspeaks of threonine residues in 2D COSY, TOCSY, and NOESY NMR spectra which allow viewing of the oligomers in equilibrium. This is complemented by PFGSE measurements of the translational diffusion coefficient, Di, used for monitoring the changes in equilibrium composition of aggregates on dilution of both insulins in physiological medium. The back calculation of the dilution isotherm allows establishing the association constants for oligomeric equilibria in solution and discussion of the models of association. Proteins 2008.

Interaction of flavonoid topoisomerase I and II inhibitors with DNA oligomers

The binding affinities of flavonoids, genistein and quercetin, to DNA oligomers have been established by means of studying by NMR the diffusion coefficients of these compounds with and without the presence of DNA. Genistein was found to bind very weakly, Ka = 1.54 × 102 M−1, as compared to quercetin, Ka = 5.75 × 103 M−1 and luteolin, Ka = 2.17 × 104 M−1 (as reported in the literature). In the case of genistein a hydrogen bond between the NHF,B cytidine protons of the edge base pair and a genistein hydroxyl is proposed, based on the shape of the DOSY spectrum. MP2 and DFT calculations of genistein show a 9 kcal mol−1 excess energy of the planar conformation as compared with a twisted one. For this reason intercalation of the planar genistein into the base pairs is rather unlikely.

NMR structure of biosynthetic engineered human insulin monomer B31(Lys)-B32(Arg) in water/acetonitrile solution. Comparison with the solution structure of native human insulin monomer.

A solution NMR-derived structure of a new long -acting, B31(Lys)-B32(Arg) (LysArg), engineered human insulin monomer, in H(2)O/CD(3)CN, 65/35 vol %, pH 3.6, is presented and compared with the available X-ray structure of a monomer that forms part of a hexamer (Smith, et al., Acta Crystallogr D 2003, 59, 474) and with NMR structure of human insulin in the same solvent (Bocian, et al., J Biomol NMR 2008, 40, 55-64). Detailed analysis using PFGSE NMR (Pulsed Field Gradient Spin Echo NMR) in dilution experiments and CSI analysis prove that the structure is monomeric in the concentration range 0.1-3 mM. The presence of long-range interstrand NOEs in a studied structure, relevant to the distances found in the crystal structure of the monomer, provides the evidence for conservation of the tertiary structure. Therefore the results suggest that this solvent system is a suitable medium for studying the native conformation of the protein, especially in situations (as found for insulins) in which extensive aggregation renders structure elucidations in water difficult or impossible. Starting from the structures calculated by the program CYANA, two different molecular dynamics (MD) simulated annealing refinement protocols were applied, either using the program AMBER in vacuum (AMBER_VC), or including a generalized Born solvent model (AMBER_GB). Here we present another independent evidence to the one presented recently by us (Bocian et al., J Biomol NMR 2008, 40, 55-64), that in water/acetonitrile solvent detailed structural and dynamic information can be obtained for important proteins that are naturally present as oligomers under native conditions.

Solution and solid state 13C NMR and X-ray studies of genistein complexes with amines. Potential biological function of the C-7, C-5, and C-4′-OH groups

Parent genistein and its new amine complexes with morpholine and piperazine were studied comparatively in the solid and liquid states by X-ray crystallography and 13C and 15N NMR spectroscopy. Biochanine A and its complexes were used as reference. Secondary deuterium isotope effects on 13C chemical shifts in solution were studied in parent isoflavones and their morpholine and piperazine complexes to aid in evaluation of the electronic distribution in both systems. In addition, to quantify the extent of proton transfer as well as to establish strong hydrogen bonding of the 7-OH group in a morpholine complex, proton transfer from the 7-OH group to the piperazine nitrogen atom was also confirmed by 13C NMR in the solid state and by X-ray studies. The effect of 7-OH deprotonation yields a high frequency shift of 7–8 ppm on the C-7 carbon atom of the piperazine complex whereas it is as large as 12 ppm in the morpholine complex in the solid. The former trend is confirmed from solution state concentration studies which also show that the isoflavones have a strong tendency to form complexes with bases. Depending on the pKa difference between the isoflavones and the base this leads either to proton transfer and ion-pair formation or, in the case of a larger pKa difference, to a hydrogen bonded ion pair. The concentration studies show formation of a 1 : 1 genistein–piperazine complex in DMSO. Addition of water leads to formation of solvent separated ions. The C-5 OH group is involved in strong intramolecular hydrogen bonding leading to a pseudo aromatic ring extending the aromatic part of the drug pharmacophore. The analysis also suggests the way that both the C-7 and C-4′ hydroxyl group of genistein may participate in stabilising the ternary inhibitor complexes of tyrosine-specific kinases or DNA topoisomerase II.

Binding of Topotecan to a Nicked DNA Oligomer in Solution

Topotecan (TPT) is in clinical use as an antitumor agent. It acts by binding to the covalent complex formed between nicked DNA and topoisomerase I, and inserts itself into the single-strand nick, thereby inhibiting the religation of the nick and acting as a poison. A crystal structure analysis of the ternary complex has shown how the drug binds (B. L. Staker, K. Hjerrild, M. D. Feese, C. A. Behnke, A. B. Burgin, L. Stewart, Proc. Natl. Acad. Sci. U.S.A., 2002, 99, 15 387-15 392), but has left a number of unanswered questions. Herein, we use NMR spectroscopy and molecular modeling to show that the solution structure of a complex of TPT with nicked natural DNA is similar, but not identical to the crystal conformation, and that other geometries are of very low population. We also show that the lactone form of TPT binds approximately 40 times more strongly than the ring-opened carboxylate.

Complexation of steroid hormones: prednisolone, ethinyloestradiol and estriol with β-cyclodextrin. An aqueous 1H NMR study

The inclusion complexes of β-cyclodextrin (β-CD) with prednisolone 1, ethinyloestradiol 2 and estriol 3 in aqueous solution were investigated using 1H NMR and molecular modelling. The NMR spectra of the steroids studied in the presence of β-CD are fully assigned and interpreted by means of 2D GCOSY and NOESY spectra. The parallel interpretation of β-CD chemical shift changes and dipolar contacts allows the mode of binding to be established. On the basis of ROESY data, the “low resolution” β-cyclodextrin complexes of 1, 2 and 3 have been determined by multistep restrained molecular dynamics calculations. Calculated structures of β-cyclodextrin complexes with 1, 2 and 3 fully agree with experimental data. Combined approaches allow the distinction of weak nonspecific binding for 1 as compared to stronger, “through cavity”, inclusion established for 2 and 3.

An assessment of polydispersed species in unfractionated and low molecular weight heparins by diffusion ordered nuclear magnetic resonance spectroscopy method

The primary goal of this project is to extend a (1)H NMR based method, which combines elements of separation on the basis of molecular size with the information specific to (1)H-1D NMR, to the assessment of the heparin contaminant oversulfated chondroitin sulfate (OSCS) and process related impurity dermatan sulfate (DS), and their polydisperse degradation products in samples of unfractionated heparins (UFHs) and low molecular weight heparins (LMWHs) used as the active pharmaceutical ingredients (APIs) in finished pharmaceutical products. The method has been briefly introduced by us in a recent contribution (vide infra). We propose a labelling of the N-acetyl peaks in the (1)H NMR spectra of the UFHs and LMWHs with the parameter D(i), the translational diffusion coefficient available from DOSY NMR. It is shown how DOSY can be applied for screening lots of unfractionated and depolymerised heparins for obtaining molecular size information for heparins and any impurities when using (1)H NMR. The evidence has been presented that title method can be applied as a routine means for assessment of the OSCS and DS contaminants and the polydisperse chemical entities present in the UFHs and LMWHs used as the APIs in heparin pharmaceuticals.

Insights into the Tautomerism in meso‐Substituted Corroles: A Variable‐Temperature 1H, 13C, 15N, and 19F NMR Spectroscopy Study

Tris(pentafluorophenyl)corrole and its (15)N-enriched isotopomer were studied in [D8]toluene solution by 1D and 2D variable-temperature NMR techniques to establish the mechanisms of tautomerization of the NH protons inside the interior of the corrole macrocycle. Three such rate processes could be identified of which two modulate the spectral line shapes at temperatures above 205 K and the third is NMR-inaccessible as it is very fast. The latter involves the proton engaged in an unsymmetrical proton sponge unit formed by two pyrrole nitrogen atoms. Temperature and concentration dependences of the two remaining processes were determined. One of them is purely intramolecular and the other is intermolecular at low temperatures, with growing contribution of an intramolecular mechanism at elevated temperatures. The proposed microscopic mechanisms of all these processes are semi-quantitatively confirmed by quantum chemical calculations using density functional theory.