A solid state 13C NMR, crystallographic, and quantum chemical investigation of chemical shifts and hydrogen bonding in histidine dipeptides

We report the first solid-state NMR, crystallographic, and quantum chemical investigation of the origins of the 13C NMR chemical shifts of the imidazole group in histidine-containing dipeptides. The chemical shift ranges for Cgamma and Cdelta2 seen in eight crystalline dipeptides were very large (12.7-13.8 ppm); the shifts were highly correlated (R2= 0.90) and were dominated by ring tautomer effects and intermolecular interactions. A similar correlation was found in proteins, but only for buried residues. The imidazole 13C NMR chemical shifts were predicted with an overall rms error of 1.6-1.9 ppm over a 26 ppm range, by using quantum chemical methods. Incorporation of hydrogen bond partner molecules was found to be essential in order to reproduce the chemical shifts seen experimentally. Using AIM (atoms in molecules) theory we found that essentially all interactions were of a closed shell nature and the hydrogen bond critical point properties were highly correlated with the N...H...O (average R2= 0.93) and Nepsilon2...H...N (average R2= 0.98) hydrogen bond lengths. For Cepsilon1, the 13C chemical shifts were also highly correlated with each of these properties (at the Nepsilon2 site), indicating the dominance of intermolecular interactions for Cepsilon1. These results open up the way to analyzing 13C NMR chemical shifts, tautomer states (from Cdelta2, Cepsilon1 shifts), and hydrogen bond properties (from Cepsilon1 shifts) of histidine residue in proteins and should be applicable to imidazole-containing drug molecules bound to proteins, as well.     

Hydrogen-bonding partner of the proton-conducting histidine in the influenza m2 proton channel revealed from (1)h chemical shifts

The influenza M2 protein conducts protons through a critical histidine (His) residue, His37. Whether His37 only interacts with water to relay protons into the virion or whether a low-barrier hydrogen bond (LBHB) also exists between the histidines to stabilize charges before proton conduction is actively debated. To address this question, we have measured the imidazole (1)H(N) chemical shifts of His37 at different temperatures and pH using 2D (15)N-(1)H correlation solid-state NMR. At low temperature, the H(N) chemical shifts are 8-15 ppm at all pH values, indicating that the His37 side chain forms conventional hydrogen bonds (H-bonds) instead of LBHBs. At ambient temperature, the dynamically averaged H(N) chemical shifts are 4.8 ppm, indicating that the H-bonding partner of the imidazole is water instead of another histidine in the tetrameric channel. These data show that His37 forms H-bonds only to water, with regular strength, thus supporting the His-water proton exchange model and ruling out the low-barrier H-bonded dimer model.     

Structure distribution in an elastin-mimetic peptide (VPGVG)3 investigated by solid-state NMR

Elastin is an extracellular-matrix protein that imparts elasticity to tissues. We have used solid-state NMR to determine a number of distances and torsion angles in an elastin-mimetic peptide, (VPGVG)3, to understand the structural basis of elasticity. C-H and C-N distances between the V6 carbonyl and the V9 amide segment were measured using 13C-15N and 13C-1H rotational-echo double-resonance experiments. The results indicate the coexistence of two types of intramolecular distances: a third of the molecules have short C-H and C-N distances of 3.3 +/- 0.2 and 4.3 +/- 0.2 A, respectively, while the rest have longer distances of about 7 A. Complementing the distance constraints, we measured the (phi, psi ) torsion angles of the central pentameric unit using dipolar correlation NMR. The -angles of P7 and G8 are predominantly ~150, thus restricting the majority of the peptide to be extended. Combining all torsion angles measured for the five residues, the G8 C chemical shift, and the V6-V9 distances, we obtained a bimodal structure distribution for the PG residues in VPGVG. The minor form is a compact structure with a V6-V9 C=O-HN hydrogen bond and can be either a type II -turn or a previously unidentified turn with Pro (phi = -70, psi= 20 +/- 20) and Gly ( phi= -100 +/- 20, psi = -20 +/- 20). The major form is an extended and distorted beta-strand without a V6-V9 hydrogen bond and differs from the ideal parallel and antiparallel beta-strands. The other three residues in the VPGVG unit mainly adopt antiparallel beta-sheet torsion angles. Since (VPGVG)3 has the same 13C and 15N isotropic and anisotropic chemical shifts as the elastin-mimetic protein (VPGXG)n (X = V and K, n = 195), the observed conformational distribution around Pro and Gly sheds light on the molecular mechanism of elastin elasticity.     

Magic angle spinning solid-state NMR spectroscopy for structural studies of protein interfaces. resonance assignments of differentially enriched Escherichia coli thioredoxin reassembled by fragment complementation

De novo site-specific backbone and side-chain resonance assignments are presented for U-15N(1-73)/U-13C,15N(74-108) reassembly of Escherichia coli thioredoxin by fragment complementation, determined using solid-state magic angle spinning NMR spectroscopy at 17.6 T. Backbone dihedral angles and secondary structure predicted from the statistical analysis of 13C and 15N chemical shifts are in general agreement with solution values for the intact full-length thioredoxin, confirming that the secondary structure is retained in the reassembled complex prepared as a poly(ethylene glycol) precipitate. The differential labeling of complementary thioredoxin fragments introduced in this work is expected to be beneficial for high-resolution structural studies of protein interfaces formed by protein assemblies by solid-state NMR spectroscopy.     

Synthesis and structure of 1-[(3-hydroxybenzo[<Emphasis Type="Italic">b</Emphasis>]thiophen-2-yl)methylidene]-3-oxo-5-phenyl-1-pyrazolidinium-2-ide

Potentially tautomeric azomethine imine, 1-[(3-hydroxybenzo[b]thiophen-2-yl)methylidene]-3-oxo-5-phenyl-1-pyrazolidinium-2-ide has been prepared. According to X-ray diffraction, ¹H, ¹³C, and ¹⁵N NMR and electronic spectroscopy, the compound exists as 3-hydroxybenzothiophene structure containing intermolecular hydrogen bond between the hydroxy group and the carbonyl oxygen of the pyrazolidone ring in crystal state. Quantum chemical calculations predict the possibility of OH and NH tautomeric forms.     

Quantifying weak hydrogen bonding in uracil and 4-cyano-4'-ethynylbiphenyl: a combined computational and experimental investigation of NMR chemical shifts in the solid state

Weak hydrogen bonding in uracil and 4-cyano-4'-ethynylbiphenyl, for which single-crystal diffraction structures reveal close CH...O=C and C[triple bond]CH...N[triple bond]C distances, is investigated in a study that combines the experimental determination of 1H, 13C, and 15N chemical shifts by magic-angle spinning (MAS) solid-state NMR with first-principles calculations using plane-wave basis sets. An optimized synthetic route, including the isolation and characterization of intermediates, to 4-cyano-4'-ethynylbiphenyl at natural abundance and with 13C[triple bond]13CH and 15N[triple bond]C labeling is described. The difference in chemical shifts calculated, on the one hand, for the full crystal structure and, on the other hand, for an isolated molecule depends on both intermolecular hydrogen bonding interactions and aromatic ring current effects. In this study, the two effects are separated computationally by, first, determining the difference in chemical shift between that calculated for a plane (uracil) or an isolated chain (4-cyano-4'-ethynylbiphenyl) and that calculated for an isolated molecule and by, second, calculating intraplane or intrachain nucleus-independent chemical shifts that quantify the ring current effects caused by neighboring molecules. For uracil, isolated molecule to plane changes in the 1H chemical shift of 2.0 and 2.2 ppm are determined for the CH protons involved in CH...O weak hydrogen bonding; this compares to changes of 5.1 and 5.4 ppm for the NH protons involved in conventional NH...O hydrogen bonding. A comparison of CH bond lengths for geometrically relaxed uracil molecules in the crystal structure and for geometrically relaxed isolated molecules reveals differences of no more than 0.002 A, which corresponds to changes in the calculated 1H chemical shifts of at most 0.1 ppm. For the C[triple bond]CH...N[triple bond]C weak hydrogen bonds in 4-cyano-4'-ethynylbiphenyl, the calculated molecule to chain changes are of similar magnitude but opposite sign for the donor 13C and acceptor 15N nuclei. In uracil and 4-cyano-4'-ethynylbiphenyl, the CH hydrogen-bonding donors are sp2 and sp hybridized, respectively; a comparison of the calculated changes in 1H chemical shift with those for the sp3 hybridized CH donors in maltose (Yates et al. J. Am. Chem. Soc. 2005, 127, 10216) reveals no marked dependence on hybridization for weak hydrogen-bonding strength.     

Chemical shifts in nucleic acids studied by density functional theory calculations and comparison with experiment

NMR chemical shifts are highly sensitive probes of local molecular conformation and environment and form an important source of structural information. In this study, the relationship between the NMR chemical shifts of nucleic acids and the glycosidic torsion angle, χ, has been investigated for the two commonly occurring sugar conformations. We have calculated by means of DFT the chemical shifts of all atoms in the eight DNA and RNA mono-nucleosides as a function of these two variables. From the DFT calculations, structures and potential energy surfaces were determined by using constrained geometry optimizations at the BP86/TZ2P level of theory. The NMR parameters were subsequently calculated by single-point calculations at the SAOP/TZ2P level of theory. Comparison of the (1) H and (13) C?NMR shifts calculated for the mono-nucleosides with the shifts determined by NMR spectroscopy for nucleic acids demonstrates that the theoretical shifts are valuable for the characterization of nucleic acid conformation. For example, a clear distinction can be made between χ angles in the anti and syn domains. Furthermore, a quantitative determination of the χ angle in the syn domain is possible, in particular when (13) C and (1) H chemical shift data are combined. The approximate linear dependence of the C1' shift on the χ angle in the anti domain provides a good estimate of the angle in this region. It is also possible to derive the sugar conformation from the chemical shift information. The DFT calculations reported herein were performed on mono-nucleosides, but examples are also provided to estimate intramolecularly induced shifts as a result of hydrogen bonding, polarization effects, or ring-current effects.     

Platinum-modified adenines: unprecedented protonation behavior revealed by NMR spectroscopy and relativistic density-functional theory calculations

Two novel Pt(IV) complexes of aromatic cytokinins with possible antitumor properties were prepared by reaction of selected aminopurines with K(2)PtCl(6). The structures of both complexes, 9-[6-(benzylamino)purine] pentachloroplatinate (IV) and 9-[6-(furfurylamino)purine] pentachloroplatinate (IV), were characterized in detail by using two-dimensional NMR spectroscopy ((1)H, (13)C, (15)N, and (195)Pt) in solution and CP/MAS NMR techniques in the solid state. We report for the first time the X-ray structure of a nucleobase adenine derivative coordinated to Pt(IV) via the N9 atom. The protonation equilibria for the complexes in solution were characterized by using NMR spectroscopy (isotropic chemical shifts and indirect nuclear spin-spin coupling constants) and the structural conclusions drawn from the NMR analysis are supported by relativistic density-functional theory (DFT) calculations. Because of the presence of the Pt atom, hybrid GGA functionals and scalar-relativistic and spin-orbit corrections were employed for both the DFT calculations of the molecular structure and particularly for the NMR chemical shifts. In particular, the populations of the N7-protonated and neutral forms of the complexes in solution were characterized by correlating the experimental and the DFT-calculated NMR chemical shifts. In contrast to the chemical exchange process involving the N7-H group, the hydrogen atom at N3 was determined to be unexpectedly rigid, probably because of the presence of the stabilizing intramolecular interaction N3-H···Cl. The described methodology combining the NMR spectroscopy and relativistic DFT calculations can be employed for characterizing the tautomeric and protonation equilibria in a large family of transition-metal-modified purine bases.     

NMR “Crystallography” for Uniformly (13C, 15N)-Labeled Oriented Membrane Proteins

In oriented-sample (OS) solid-state NMR of membrane proteins, the angular-dependent dipolar couplings and chemical shifts provide a direct input for structure calculations. However, so far only ¹H–¹⁵N dipolar couplings and ¹⁵N chemical shifts have been routinely assessed in oriented ¹⁵N-labeled samples. The main obstacle for extending this technique to membrane proteins of arbitrary topology has remained in the lack of additional experimental restraints. We have developed a new experimental triple-resonance NMR technique, which was applied to uniformly doubly (¹⁵N, ¹³C)-labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible ¹H_α–¹³C_α dipolar couplings have been measured, which make it possible to determine the torsion angles between the peptide planes without assuming α-helical structure a priori. The fitting of three angular restraints per peptide plane and filtering by Rosetta scoring functions has yielded a consensus α-helical transmembrane structure for Pf1 protein.     

Interaction of Cd and Zn with biologically important ligands characterized using solid-state NMR and ab initio calculations

For the first time, coordination geometry and structure of metal binding sites in biologically relevant systems are studied using chemical shift parameters obtained from solid-state NMR experiments and quantum chemical calculations. It is also the first extensive report looking at metal-imidazole interaction in the solid state. The principal values of the (113)Cd chemical shift anisotropy (CSA) tensor in crystalline cadmium histidinate and two different cadmium formates (hydrate and anhydrate) were experimentally measured to understand the effect of coordination number and geometry on (113)Cd CSA. Further, (13)C and (15)N chemical shifts have also been experimentally determined to examine the influence of cadmium on the chemical shifts of (15)N and (13)C nuclei present near the metal site in the cadmium-histidine complex. These values were then compared with the chemical shift values obtained from the isostructural bis(histidinato)zinc(II) complex as well as from the unbound histidine. The results show that the isotropic chemical shift values of the carboxyl carbons shift downfield and those of amino and imidazolic nitrogens shift upfield in the metal (Zn,Cd)-histidine complexes relative to the values of the unbound histidine sample. These shifts are in correspondence with the anticipated values based on the crystal structure. Ab initio calculations on the cadmium histidinate molecule show good agreement with the (113)Cd CSA tensors determined from solid-state NMR experiments on powder samples. (15)N chemical shifts for other model complexes, namely, zinc glycinate and zinc hexaimidazole chloride, are also considered to comprehend the effect of zinc binding on (15)N chemical shifts.     

Factor analysis of deuterium isotope effects on 13C NMR chemical shifts in Schiff bases

We have analyzed deuterium isotope effects on (13)C chemical shifts in a series of o-hydroxy Schiff bases by applying factor analysis. Two orthogonal factors were obtained that explain about 80 and 10 % of the variance of the data. The numerical values of these factors can be related to 1H NMR chemical shifts of the proton involved in the intramolecular bonds delta(XH) (X = O or N). Such a relation allows one to identify clusters of compounds with different tautomeric forms of hydrogen bonding. Application of a similar approach to solution 13C NMR chemical shifts produces three important factors, which have a different structure to factors describing isotope effects. This illustrates well the different nature of chemical shifts and isotope effects. The three factors explain about 54, 15, and 13 % of variance. They can be rationalized and are strongly related to the electronic properties and location of substituents.     

NMR studies of solvent-assisted proton transfer in a biologically relevant Schiff base: toward a distinction of geometric and equilibrium H-bond isotope effects

The tautomeric equilibrium in a Schiff base, N-(3,5-dibromosalicylidene)-methylamine 1, a model for the hydrogen bonded structure of the cofactor pyridoxal-5'-phosphate PLP which is located in the active site of the enzyme, was measured by means of 1H and 15N NMR and deuterium isotope effects on 15N chemical shifts at variable temperature and in different organic solvents. The position of the equilibrium was estimated using the one-bond 1J(OHN) and vicinal 3J(H(alpha)CNH) scalar coupling constants. Additionally, DFT calculations of a series of Schiff bases, N-(R1-salicylidene)-alkyl(R2)amines, were performed to obtain the hydrogen bond geometries. The latter made it possible to investigate a broad range of equilibrium positions. The increase of the polarity of the aprotic solvent shifts the proton in the intramolecular OHN hydrogen bond closer to the nitrogen. The addition of methanol and of hexafluoro-2-propanol to 1 in aprotic solvents models the PLP-water interaction in the enzymatic active site. The alcohols, which vary in acidity and change the polarity around the hydrogen bond, also stabilize the equilibrium, so that the proton is shifted to the nitrogen.     

Solid-state NMR and quantum chemical investigations of 13Calpha shielding tensor magnitudes and orientations in peptides: determining phi and psi torsion angles

We report the experimental determination of the (13)C(alpha) chemical shift tensors of Ala, Leu, Val, Phe, and Met in a number of polycrystalline peptides with known X-ray or de novo solid-state NMR structures. The 700 Hz dipolar coupling between (13)C(alpha) and its directly bonded (14)N permits extraction of both the magnitude and the orientation of the shielding tensor with respect to the C(alpha)-N bond vector. The chemical shift anisotropy (CSA) is recoupled under magic-angle spinning using the SUPER technique (Liu et al., J. Magn. Reson. 2002, 155, 15-28) to yield quasi-static chemical shift powder patterns. The tensor orientation is extracted from the (13)C-(14)N dipolar modulation of the powder line shapes. The magnitudes and orientations of the experimental (13)C(alpha) chemical shift tensors are found to be in good accord with those predicted from quantum chemical calculations. Using these principal values and orientations, supplemented with previously measured tensor orientations from (13)C-(15)N and (13)C-(1)H dipolar experiments, we are able to predict the (phi, psi, chi(1)) angles of Ala and Val within 5.8 degrees of the crystallographic values. This opens up a route to accurate determination of torsion angles in proteins based on shielding tensor magnitude and orientation information using labeled compounds, as well as the structure elucidation of noncrystalline organic compounds using natural abundance (13)C NMR techniques.     

醇与极性有机溶剂间氢键的多核NMR研究

醇是一类重要的有机溶剂,对其结构和性质的研究已有很长历史。由于OH的存在,醇分子间存在着较强的氢键缔合作用,使其结构变得复杂,因而较难对它得到一个很清楚的认识。用NMR方法研究氢键也有几十年历史。早在五十年代,Arnold,Becker等就用~1HNMR研究了EtOH在CCl_4中的行为,测量了化学位移随浓度的变化。Becker认为当醇浓度很稀时,体系中只存在单体-二聚体平衡,井结合IR数据求得了平衡常数及缔合物位移。有     

Solid-state NMR and computational studies of 4-methyl-2-nitroacetanilide

Studies on the solid-state structure of two polymorphs of 4-methyl-2-nitroacetanilide (MNA) were conducted using magic-angle spinning (13)C, (15)N and (1)H NMR spectroscopy, together with first-principles computations of NMR shielding (including use of a program that takes explicit account of the translational symmetry inherent in crystalline structures). The effects on (13)C chemical shifts of side-chain rotations have been explored. Information derived from these studies was then incorporated within a systematic space-search methodology for elucidation of trial crystallographic structures from powder XRD.     

Reaction of o-phenylenediamine with diacetyl monoxime: characterisation of the product by solid-state 13C and 15N MAS NMR

2,3-dimethylquinoxaline (DMQ) and dimethylglyoxime (DMGH2) form a 1:1 hydrogen-bonded complex in the solid state, which is completely dissociated in methanol solution. There are small differences in solid-state 13C shifts between the separated components DMQ and DMGH2 and the complex. The changes in 15N solid-state chemical shifts are more significant: the hydrogen bond imparting a low frequency shift of ca 19 ppm. The effect of direct protonation on the DMQ solid-state 15N shifts was measured, and the experimental 15N data correlated with those from GIAO molecular orbital (MO) calculations.     

Toward direct determination of conformations of protein building units from multidimensional NMR experiments. V. NMR chemical shielding analysis of N-formyl-serinamide,a model for polar side-chain containing peptides

Knowledge of chemical shift-structure relationships could greatly facilitate the NMR chemical shift assignment and structure refinement processes that occur during peptide/protein structure determination via NMR spectroscopy. To determine whether such correlations exist for polar side chain containing amino acid residues the serine dipeptide model, For-L-Ser-NH(2), was studied. Using the GIAO-RHF/6-31+G(d) and GIAO-RHF/TZ2P levels of theory the NMR chemical shifts of all hydrogen ((1)H(N), (1)H(alpha), (1)H(beta1), (1)H(beta2)), carbon ((13)C(alpha), (13)C(beta), (13)C') and nitrogen ((15)N) atoms have been computed for all 44 stable conformers of For-L-Ser-NH(2). An attempt was made to establish correlation between chemical shift of each nucleus and the major conformational variables (omega(0), phi, psi, omega(1), chi,(1) and chi(2)). At both levels of theory a linear correlation can be observed between (1)H(alpha)/phi, (13)C(alpha)/phi, and (13)C(alpha)/psi. These results indicate that the backbone and side-chain structures of For-L-Ser-NH(2) have a strong influence on its chemical shifts.     

Benzoxazine oligomers: evidence for a helical structure from solid-state NMR spectroscopy and DFT-based dynamics and chemical shift calculations

A combination of molecular modeling, DFT calculations, and advanced solid-state NMR experiments is used to elucidate the supramolecular structure of a series of benzoxazine oligomers. Intramolecular hydrogen bonds are characterized and identified as the driving forces for ring-shape and helical conformations of trimeric and tetrameric units. In fast MAS (1)H NMR spectra, the resonances of the protons forming the hydrogen bonds can be assigned and used for validating and refining the structure by means of DFT-based geometry optimizations and (1)H chemical-shift calculations. Also supporting these proposed structures are homonuclear (1)H[bond](1)H double-quantum NMR spectra, which identify the local proton-proton proximities in each material. Additionally, quantitative (15)N[bond](1)H distance measurements obtained by analysis of dipolar spinning sideband patterns confirm the optimized geometry of the tetramer. These results clearly support the predicted helical geometry of the benzoxazine polymer. This geometry, in which the N...H...O and O...H...O hydrogen bonds are protected on the inside of the helix, can account for many of the exemplary chemical properties of the polybenzoxazine materials. The combination of advanced experimental solid-state NMR spectroscopy with computational geometry optimizations, total energy, and NMR spectra calculations is a powerful tool for structural analysis. Its results provide significantly more confidence than the individual measurements or calculations alone, in particular, because the microscopic structure of many disordered systems cannot be elucidated by means of conventional methods due to lack of long-range order.     

Intramolecular hydrogen-bonding interactions in 2-nitrosophenol and nitrosonaphthols: ab initio, density functional, and nuclear magnetic resonance theoretical study

Intramolecular hydrogen bonding (IHB) interactions and molecular structures of 2-nitrosophenol, nitrosonaphthols, and their quinone-monooxime tautomers were investigated at ab initio and density functional theory (DFT) levels. The geometry optimization of the structures studied was performed without any geometrical restrictions. Possible conformations with different types of the IHB of the tautomers were considered to understand the nature of the HB among these conformers. The effect of solvent on hydrogen bond energies, conformational equilibria, and tautomerism in aqueous solution were studied. Natural bond orbital analysis was performed to study the IHB in the gaseous phase and in aqueous medium. The NMR 1H, 13C, 15N, and 17O chemical shifts in the gaseous phase and in solution for the studied compounds were calculated using the gauge-including atomic orbitals approach implemented in the Gaussian 03 program package. The optimized geometrical parameters and 1H NMR chemical shifts are in good agreement with previous theoretical and experimental data.     

1,1′,1′′-(2,4,6-Trihydroxybenzene-1,3,5-triyl)triethanone tautomerism revisited

It has recently been suggested that 1,1′,1′′-(2,4,6-trihydroxybenzene-1,3,5-triyl)triethanone may be tautomeric. Using ¹³C NMR chemical shifts and deuterium isotope effects on ¹³C chemical shifts, it is demonstrated that this is not the case. This compound occurs as a strongly hydrogen bonded benzene structure with hydrogen bonds between OH groups and the acetyl groups in both non-polar and hydrogen donating solvents. Quantum-chemical calculations using MP2 and M06-2X methods show substantial preference for the phenol structure in both the gas phase, and in cyclohexane and methanol. In addition, conventional UV–vis spectroscopy data suggest not tautomeric, but aggregation behaviour of the molecule in methanol and acetonitrile.