Epitope mapping of ligand-receptor interactions by diffusion NMR

A novel method based on diffusion NMR for the epitope mapping of ligand binding is presented. The intermolecular NOE builds up during a long diffusion period and creates a deviation from the linearity. The ligand proton nearest the protein generates the strongest NOE from protein during the diffusion period and has the largest deviation. Therefore, this diffusion artifact can be used to characterize the ligand binding epitope. The concept was investigated using dihydrofolate reductase (DHFR) and its ligand trimethoprim (TMP), and the epitope map of TMP on DHFR generated with this method is in excellent agreement with the structural and dynamic studies by crystallography and NMR, as well as the medicinal chemistry results.     

NMR analysis of aromatic interactions in designed peptide beta-hairpins

Designed octapeptide beta-hairpins containing a central (D)Pro-Gly segment have been used as a scaffold to place the aromatic residues Tyr and Trp at various positions on the antiparallel beta-strands. Using a set of five peptide hairpins, aromatic interactions have been probed across antiparallel beta-sheets, in the non-hydrogen bonding position (Ac-L-Y-V-(D)P-G-L-Y/W-V-OMe: peptides 1 and 2), diagonally across the strands (Boc-Y/W-L-V-(D)P-G-W-L-V-OMe: peptides 3 and 6), and along the strands at positions i and i + 2 (Boc-L-L-V-(D)P-G-Y-L-W-OMe: peptide 4). Two peptides served as controls (Boc-L-L-V-(D)P-G-Y-W-V-OMe: peptide 5; Boc-L-Y-V-(D)P-G-L-L-V-OMe: peptide 7) for aromatic interactions. All studies have been carried out using solution NMR methods in CDCl(3) + 10% DMSO-d(6) and have been additionally examined in CD(3)OH for peptides 1 and 2. Inter-ring proton-proton nuclear Overhauser effects (NOEs) and upfield shifted aromatic proton resonances have provided firm evidence for specific aromatic interactions. Calculated NMR structures for peptides 1 and 2, containing aromatic pairs at facing non-hydrogen bonded positions, revealed that T-shaped arrangements of the interacting pairs of rings are favored, with ring current effects leading to extremely upfield chemical shifts and temperature dependences for specific aromatic protons. Anomalous far-UV CD spectra appeared to be a characteristic feature in peptides where the two aromatic residues are spatially proximal. The observation of the close approach of aromatic rings in organic solvents suggests that interactions of an electrostatic nature may be favored. This situation may be compared to the case of aqueous solutions, where clustering of aromatic residues is driven by solvophobic (hydrophobic) forces.     

Evaluation of NMR diffusion measurements for the conformational analysis of flexible peptides

The use of diffusion coefficients measured with pulsed-field gradient NMR spectroscopy for the determination of the relative population of conformers in solutions of the human Growth Hormone peptide fragment, hGH(9–19), has been studied in aqueous and in trifluoroethanol (TFE)/ water solutions. The peptide is a good model compound for this study because it adopts a predominantly random coil conformation in aqueous solution and is helical in TFE. The results of the diffusion measurements suggest that the peptide exhibits predominantly random coil structures in aqueous solution and adopts a more helical conformation in solutions containing increasing mole fractions of TFE, consistent with the qualitative findings of the standard CD and NMR experiments to probe peptide conformation. These results indicate that diffusion coefficients measured with NMR can provide additional information about temperature- and solvent-induced changes in the extent of the helical conformer for hGH(9–19) in aqueous solution and in solutions containing various mole fraction of TFE, respectively.     

Evaluation of NMR diffusion measurements for the conformational analysis of flexible peptides

The use of diffusion coefficients measured with pulsed-field gradient NMR spectroscopy for the determination of the relative population of conformers in solutions of the human Growth Hormone peptide fragment, hGH(9-19), has been studied in aqueous and in trifluoroethanol (TFE)/ water solutions. The peptide is a good model compound for this study because it adopts a predominantly random coil conformation in aqueous solution and is helical in TFE. The results of the diffusion measurements suggest that the peptide exhibits predominantly random coil structures in aqueous solution and adopts a more helical conformation in solutions containing increasing mole fractions of TFE, consistent with the qualitative findings of the standard CD and NMR experiments to probe peptide conformation. These results indicate that diffusion coefficients measured with NMR can provide additional information about temperature- and solvent-induced changes in the extent of the helical conformer for hGH(9-19) in aqueous solution and in solutions containing various mole fraction of TFE, respectively.     

Calibration by NMR for quantitative analysis: p-toluenesulfonic acid as a reference substance

We have demonstrated the usefulness of p-toluenesulfonic acid (TsOH) as a reference substance to calibrate a water-soluble standard by NMR for quantitative analysis. In order to make TsOH convenient for this purpose, we first of all established its molar extinction coefficient under these conditions (epsilon = 351 at 262 nm). This was done by comparing it by quantitative NMR (qNMR) with a compound of known molar extinction coefficient, guanosine monophosphate. TsOH was then used as the standard in a second qNMR experiment to measure an aqueous solution of phosphoglycolate, a metabolite of interest in our laboratory. Perhaps due to its content of water, the purity of this compound was found to be 82%, lower than the value of 94% from the manufacturer.     

DFT analysis of NMR scalar interactions across the glycosidic bond in DNA

The relationship between the glycosidic torsion angle chi, the three-bond couplings (3)J(C2/4-H1') and (3)J(C6/8-H1'), and the one-bond coupling (1)J(C1'-H1') in deoxyribonucleosides and a number of uracil cyclo-nucleosides has been analyzed using density functional theory. The influence of the sugar pucker and the hydroxymethyl conformation has also been considered. The parameters of the Karplus relationships between the three-bond couplings and chi depend strongly on the aromatic base. (3)J(C2/4-H1') reveals different behavior for deoxyadenosine, deoxyguanosine, and deoxycytidine as compared to deoxythymidine and deoxyuridine. In the case of (3)J(C6/8-H1'), an opposite trans to cis ratio of couplings is obtained for pyrimidine nucleosides in contrast to purine nucleosides. The extremes of the Karplus curves are shifted by ca. 10 degrees with respect to syn and anti-periplanar orientations of the coupled nuclei. The change in the sugar pucker from S to N decreases (3)J(C2/4-H1') and (3)J(C6/8-H1'), while increasing (1)J(C1'-H1') for the syn rotamers, whereas all of the trends are reversed for the anti rotamers. The influence of the sugar pucker on (1)J(C1'-H1') is interpreted in terms of interactions between the n(O4'), sigma*(C1'-H1') orbitals. The (1)J(C1'-H1') are related to chi through a generalized Karplus relationship, which combines cos(chi) and cos(2)(chi) functions with mutually different phase shifts that implicitly accounts for a significant portion of the related sugar pucker effects. Most of theoretical (3)J(C2/4-H1') and (3)J(C6/8-H1') for uracil cyclo-nucleosides compare well with available experimental data. (3)J(C6/8-H1') couplings for all C2-bridged nucleosides are up to 3 Hz smaller than in the genuine nucleosides with the corresponding chi, revealing a nonlocal aspect of the spin-spin interactions across the glycosidic bond. Theoretical (1)J(C1'-H1') are underestimated with respect to the experiment by ca. 10% but reproduce the trends in (1)J(C1'-H1') vs chi.     

A NMR reverse diffusion filter for the simplification of spectra of complex mixtures and the study of drug receptor interactions

A reverse diffusion filter NMR experiment (Drev) is proposed for the study of small molecules in binding with macromolecules. The filtering efficiency of Drev to eliminate the signals of the macromolecule is shown to be superior to conventional transverse relaxation filters at least for macromolecules containing a significant fraction of flexible residues. The Drev filter was also a useful complement for ligand‐based NMR screening in combination with saturation transfer difference experiments. Copyright © 2011 John Wiley & Sons, Ltd.     

Slow diffusion by singlet state NMR spectroscopy

Small diffusion coefficients can be measured by using populations of singlet states that have a relaxation time constant, T(s), which can be much longer than the longitudinal relaxation time, T1. Spatial information can be encoded with pulsed field gradients in the manner of stimulated echo sequences. Singlet states can be excited via double-quantum coherences to enhance the efficiency of phase encoding and decoding.     

Measurements of anisotropic interactions in solid-state NMR

A two-dimensional NMR method is reported which allows us to separately obtain individual NMR powder patterns of chemically distinct nuclei by switching the sample-spinning axis on and off the magic angle. As an example, the measurement of C-H dipolar interactions in urea-trans-4-octene clathrate is reported.     

NMR studies of interionic interactions in N-methylformamide

The NMR chemical shifts of alkali and thallium(I) salts with various monovalent anions have been measured in N-methylformamide solution. Lithium-7 chemical shifts are virtually concentration and counter-ion independent, presumably due to an absence of direct cation-anion interactions. The sodium-23, potassium-39 and cesium-133 chemical shifts of the salts studied depend on the anion and vary linearly with the concentration. The observed behavior can be accounted for by the formation of collisional ion pairs. On the other hand, the thallium-205 chemical shifts of thallium(I) nitrate and perchlorate were anion-dependent and varied non-linearly with the salt concentration. These results are indicative of contact ion pair formation; formation constants were calculated to be 2.6±0.4 M ^–1 for TlNO ₃ and 1.7±0.5 M ^–1 for TlClO ₄ . The cesium-133 NMR spectra of several mixed electrolyte systems also have been measured in N-methylformamide solution. The¹³³Cs chemical shifts also change linearly with the concentrations of the salts added to 0.10 M CsI/NMF solutions. The influence of the anions on the chemical shifts is the same as that observed for cesium salts alone.     

Alkylidene derivatives of syn-sesquinorbornatriene. 13C NMR and theoretical analysis of homoconjugative orbital interactions

Despite downward folding of the central π linkage in 7 and 13, polarization of the exocyclic olefinic bonds does not surpass that seen in 1 and is not at all affected by cumulative carbomethoxy substitution of the peripheral unsaturated centers as in 9 and 10.     

Properties of small molecular drug loading and diffusion in a fluorinated PEG hydrogel studied by 1H molecular diffusion NMR and 19F spin diffusion NMR

R(f)-PEG (fluoroalkyl double-ended poly(ethylene glycol)) hydrogel is potentially useful as a drug delivery depot due to its advanced properties of sol-gel two-phase coexistence and low surface erosion. In this study, (1)H molecular diffusion nuclear magnetic resonance (NMR) and (19)F spin diffusion NMR were used to probe the drug loading and diffusion properties of the R(f)-PEG hydrogel for small anticancer drugs, 5-fluorouracil (FU) and its hydrophobic analog, 1,3-dimethyl-5-fluorouracil (DMFU). It was found that FU has a larger apparent diffusion coefficient than that of DMFU, and the diffusion of the latter was more hindered. The result of (19)F spin diffusion NMR for the corresponding freeze-dried samples indicates that a larger portion of DMFU resided in the R(f) core/IPDU intermediate-layer region (where IPDU refers to isophorone diurethane, as a linker to interconnect the R(f) group and the PEG chain) than that of FU while the opposite is true in the PEG-water phase. To understand the experimental data, a diffusion model was proposed to include: (1) hindered diffusion of the drug molecules in the R(f) core/IPDU-intermediate-layer region; (2) relatively free diffusion of the drug molecules in the PEG-water phase (or region); and (3) diffusive exchange of the probe molecules between the above two regions. This study also shows that molecular diffusion NMR combined with spin diffusion NMR is useful in studying the drug loading and diffusion properties in hydrogels for the purpose of drug delivery applications.     

Simultaneous enhancement of chemical shift dispersion and diffusion resolution in mixture analysis by diffusion-ordered NMR spectroscopy

Mixture analysis by high resolution diffusion-ordered NMR spectroscopy (HR-DOSY) requires differences in both chemical shift and diffusion coefficient; resolution can be greatly enhanced by exploiting the chemical specificity of lanthanide shift reagent binding to increase chemical shift and diffusion dispersion simultaneously.     

NMR study of diffusion of butane in linear polyethylene

The diffusion coefficient of butane in linear polyethylene at room temperature as a function of the vapor pressure of butane was measured by the spin-echo method with a pulsed magnetic field gradient. For the Special morphology of randomly oriented stacks of parallel lamellas the detour factor is 1/3. As long as the blocking factor and migration through the lamellas can be neglected, the local diffusion coefficient D_a of the small molecules through the amorphous layers in the direction parallel to the lamellas is three times the apparent diffusion coefficient D derived from the decay of the amplitude of the spin echo under the assumption of an infinitely extended homogeneous medium. The diffusion coefficient and the spin–spin relaxation time both increase exponentially with increasing pressure, i.e., butane concentration in the polymer, while the spin-lattice relaxation time is pressure independent and seems to be determined by interaction with the amorphous polyethylene matrix.     

NMR diffusion and relaxation study of drug-protein interaction.

In this work, NMR diffusion and relaxation measurements are applied to the study of the interaction between the anti-inflammatory drug salicylate and the human serum albumin (HSA) in solutions. The self-diffusion coefficients and the spin-lattice relaxation rates of salicylate are measured as a function of the concentration. The dissociation constant, Kd, for drug/HSA complexes and the number of binding sites, n, are evaluated.     

An NMR determination of CO diffusion on platinum electrocatalysts

We report the first direct measurement of CO diffusion on nanoparticle Pt electrocatalysts at the solid/liquid interface, carried out using 13C nuclear magnetic resonance (NMR) with a spin-labeling pulse sequence. Diffusion parameters were measured in the temperature range of 253-293 K for CO adsorbed on commercial Pt-black under saturation coverage. 2H NMR of the same system indicates that the electrolyte remains in the liquid state at temperatures where the CO diffusion experiments were performed. The CO diffusion parameters follow typical Arrhenius behavior with an activation energy of 6.0 +/- 0.4 kcal/mol and a pre-exponential factor of (1.1 +/- 0.6) x 10-8 cm2/s. Exchange between different CO populations, driven by a chemical potential gradient, is suggested to be the main mechanism for CO diffusion. The presence of the electrolyte medium considerably slows down the diffusion of CO as compared to that seen on surfaces of bulk metals under UHV conditions. This work opens up a new approach to the study of surface diffusion of adsorbed molecules on nanoparticle electrode catalysts, including the possibility of correlating diffusion parameters to catalytic activity in real world applications of broad general interest.     

NMR relaxation and pulsed-gradient diffusion study of polyethylene nanocomposites

We performed pulsed-gradient spin-echo nuclear-magnetic-resonance (NMR) experiments on zinc oxide filled polyethylene. The molecular weights of the polyethylene samples ranged between 808 and 33,000 gmol, and four different zinc oxide samples were used: 27-, 33-, 51-, and 2500-nm-diameter particles. The results of these experiments showed that the diffusion coefficients of the polyethylene chains did not change with nanofiller content, but a drastic change is observed in the NMR relaxation spectrum in spin-spin-relaxation experiments. At fixed zinc oxide content and polyethylene molecular weight (close to entanglement), the system with the smallest zinc oxide showed the most rigid environment. At high polyethylene molecular weights, this effect was still observable but the difference between the three investigated systems was very small, suggesting that the system was dominated by entanglements.     

Quantitative measurement of molecular diffusion coefficients by NMR spectroscopy

An offset-independent adiabatic inversion pulse is used in the diffusion experiment to uniformly excite a sample region that is sufficiently long to ignore the ending effects, yet is short enough to have a homogeneous RF field and to represent the pulsed field gradient with a linear approximation. Under these conditions, the diffusion decay of the peak intensity appears to be Gaussian as a function of the effective gradient field ge as if all the molecules inside the selected region experienced the same ge. Quantitative measurement of molecular diffusion coefficients is therefore made possible.