1H NMR detection of immobilized water molecules within a strong distal hydrogen-bonding network of substrate-bound human heme oxygenase-1

Solution 1H NMR is used to probe the environments of the donor protons of eight strong hydrogen bonds on the distal side of the heme substrate in the cyanide-inhibited, substrate-bound complex of human heme oxygenase, hHO. It is demonstrated that significant magnetization transfer from the bulk water signal to the eight labile protons does not result from chemical exchange, but from direct nuclear Overhauser effect due to the dipolar interaction of these labile protons with "ordered" water molecules. The enzyme labile proton to water proton distances are estimated at approximately 3 A. It is proposed that the role of the strong hydrogen-bonding network is to immobilize numerous water molecules which both stabilize the activated hydroperoxy species and funnel protons to the active site.     

Observation of intermolecular interactions in large protein complexes by 2D-double difference nuclear Overhauser enhancement spectroscopy: application to the 44 kDa interferon-receptor complex

NMR detection of intermolecular interactions between protons in large protein complexes is very challenging because it is difficult to distinguish between weak NOEs from intermolecular interactions and the much larger number of strong intramolecular NOEs. This challenging task is exacerbated by the decrease in signal-to-noise ratio in the often used isotope-edited and isotope-filtered experiments as a result of enhanced T(2) relaxation. Here, we calculate a double difference spectrum that shows exclusively intermolecular NOEs and manifests the good signal-to-noise ratio in 2D homonuclear NOESY spectra even for large proteins. The method is straightforward and results in a complete picture of all intermolecular interactions involving non exchangeable protons. Ninety-seven such (1)H-(1)H NOEs were assigned for the 44 KDa interferon-α2/IFNAR2 complex and used for docking these two proteins. The symmetry of the difference spectrum, its superb resolution, and unprecedented signal-to-noise ratio in this large protein/receptor complex suggest that this method is generally applicable to study large biopolymeric complexes.     

High-sensitivity observation of dipolar exchange and NOEs between exchangeable protons in proteins by 3D solid-state NMR spectroscopy

A highly sensitive new 1H-detected 3D solid-state NMR method is described for characterizing 1H-1H spin exchange in nanocrystalline samples of 15N- and 2H-enriched protein. Long-range contacts are observed in human ubiquitin. The method is also used to show that numerous NOEs between backbone amides and crystal water protons can be observed.     

Infrared spectra of protiated and deuterated cobalt acetate dihydrate

The infrared spectra of cobalt acetate dihydrate provide a direct evidence for the existence of quite strong hydrogen bonds formed by the water protons. An intense band is, namely, found around 2750 cm^?1, several additional bands are present at lower frequencies and bands originating from water librations appear above 1000 cm^?1. The existence of a band around 3310 cm^?1 on the other hand, indicates that much weaker hydrogen bonds are also present. On deuteration the protons involved in stronger hydrogen bonds are apparently replaced by deuterons to a higher degree than those forming weaker H-bonds.     

Theoretical IR spectra for water clusters (H2O)n (n = 6-22, 28, 30) and identification of spectral contributions from different H-bond conformations in gaseous and liquid water

The vibrational IR spectra in the O-H stretching region are computed for water clusters containing 6-22, 28, and 30 molecules using quantum-chemical calculations (B3LYP and an augmented basis set). For the cluster with 20 molecules, several different structures were studied. The vibrational spectrum was partitioned into contributions from different molecules according to their coordination properties. The frequency shifts depend on the number of donated/accepted H-bonds primarily of the two molecules participating in the H-bond, but also of the surrounding molecules H-bonding to these molecules. The frequencies of H-bonds between two molecules of the same coordination type are spread over a broad interval. The most downshifted hydrogen-bond vibrations are those donated by a single-donor 3-coordinated molecule where the H-bond is accepted by a single-acceptor molecule. The H-bonded neighbors influence the downshift, and their contribution can be rationalized in the same way as for the central dimer. Single donors/acceptors cause larger downshifts than 4-coordinated molecules, and the least downshift is obtained for double donors/acceptors. This result is at variance with the conception that experimental liquid water spectra may be divided into components for which larger downshifts imply higher H-bond coordination. A mean spectral contribution for each coordination type for the donor molecule was derived and fitted to the experimental liquid water IR spectrum, which enabled an estimation of the distribution of H-bond types and average number of H-bonds (3.0 +/- 0.2) in the liquid.     

1H and 13C NMR spectral study of some 3,5-bis[(E)-thienylmethylene]piperidin-4-ones

(1)H and (13)C NMR spectra have been recorded for 3,5-bis[(E)-thienylmethylene]piperidin-4-one (1a), 3',3″-dimethyl-3,5-bis[(E)-thienylmethylene]piperidin-4-one (1b), 5',5″-dibromo-3,5-bis[(E)-thienylmethylene]piperidin-4-one (1c), their 1-methyl derivatives 2a-c and 3,5-bis[(E)-thienylmethylene]-2r,6c-diphenylpiperidin-4-one (3a). For selected compounds 2D spectra have been recorded. The spectral data are used to study the configuration and conformation of these molecules. The chemical shifts are discussed in light of steric, electronic and magnetic anisotropic effects. The magnetic anisotropic effects of thiophene ring and phenyl group are noteworthy. (1)H-(1)H COSY spectrum of 2b suggests that long-range (1)H-(1)H coupling, up to seven bonds, is possible in it. HMBC spectrum of 2b displays the magnetic nonequivalence of C-2 and C-6 and protons at these carbons.     

Oxazolidin-2-one-containing pseudopeptides that fold into beta-bend ribbon spirals

Three sets of oligomers containing the 4-carboxy-5-methyloxazolidin-2-one (Oxd) moiety have been synthesized with the aim of checking whether these molecules are able to fold in ordered structures: A set [Boc-(L-Ala-L-Oxd)(n)-OR], B set [Boc-(L-Ala-D-Oxd)(n)-OR], and C set [Boc-(Aib-L-Oxd)(n)-OR] preferential conformations have been analyzed with IR absorption, NMR, and CD. We have noticed that in these oligomers three stabilizing effects are active: (i) the rigid Oxd -CO-N(CH<)-CO- moiety, which always tend to assume a trans conformation; (ii) the formation of Oxd C=O...H-(alpha)C intramolecolar H-bonds; (iii) the alternate formation of 1 <-- 4 intramolecular C=O...H-N H-bonds. Through the analysis of the experimental data, we could demonstrate that only the oligomers of the B set are able to meet all three requirements listed above. By a deeper insight into the CD spectra, we gathered that the secondary structure adopted by the B set oligomers is a beta-bend ribbon spiral, which is a subtype of the 3(10)-helix.     

Molecular structure of the solvated proton in isolated salts. Short,strong, low barrier (SSLB) H-bonds

Large, inert, weakly basic carborane anions of the icosahedral type CHB(11)R(5)X(6)(-) (R = H, Me; X = Cl, Br) allow ready isolation and structural characterization of discrete salts of the solvated proton, [H(solvent)(x)][CHB(11)R(5)X(6)], (solvent = common O-atom donor). These oxonium ion Br?nsted acids are convenient reagents for the tuned delivery of protons to organic solvents with a specified number of donor solvent molecules and with acidities leveled to those of the chosen donor solvent. They have greater thermal stability than the popular [H(OEt(2))(2)][BAr(F)] acids based on fluorinated tetraphenylborate counterions because carborane anions can sustain much higher levels of acidity. When organic O-atom donors such as diethyl ether, tetrahydrofuran, benzophenone, and nitrobenzene are involved, the coordination number of the proton (x) in [H(solvent)(x)()](+) is two. A mixed species involving the [H(H(2)O)(diethyl ether)](+) ion has also been isolated. These solid-state structures provide expectations for the predominant molecular structures of solvated protons in solution and take into account that water is an inevitable impurity in organic solvents. The O.O distances are all short, lying within the range from 2.35 to 2.48 A. They are consistent with strong, linear O.H.O hydrogen bonding. Density functional theory calculations indicate that all H(solvent)(2)(+) cations have low barriers to movement of the proton within an interval along the O.H.O trajectory, i.e., they are examples of so-called SSLB H-bonds (short, strong, low-barrier). Unusually broadened IR bands, diagnostic of SSLB H-bonds, are observed in these H(solvent)(2)(+) cations.     

Hydrogen Bonding Assembled 3D Supramolecular Structures Formed by 5-Amino-2,4,6-triiodoisophthalic Acid and N-Heterocyclic Aromatic Ligands

Three salts constructed by 5-amino-2,4,6-triiodoisophthalic acid(ATIPA) with N-heterocycles aromatic coformers such as pyridine tetrazolium, tetramethylpyraziiie and cyanuric acid were synthesized by slowing evaporation of solvent. X-Ray single crystal analysis shows that hydrogen protons of the carboxyl groups transfer to nitrogen atoms of the N-heterocyclic coformers to form N-H…0 hydrogen bonds in all the three compounds. A huge amount of H-bonds play significant role in tlie construction of these compounds and all of them generate 3D structures through strong O-H…N, O-H…O, N-H…O and weak C-H…O hydrogen bonds. Moreover, solvent water molecules are indispensable in the formation of compounds 1 and 3, which constitutes different supramolecular synthons to bridge individual molecules and chains to form stable structures. In addition, these crystal structures were further characterized by themiogravimetric analysis and infrared spectroscopy.     

Helical aromatic oligoamides: reliable, readily predictable folding from the combination of rigidified structural motifs

Factors responsible for the folding of aromatic oligoamides with backbones rigidified by local three-center H-bonds were investigated. The stability of the three-center H-bonds was quantified by the half-lives of amide proton-deuterium exchange reactions, which show that the three-center H-bonds were largely intact at room temperature in the oligomer examined. This result is consistent with our current and previous 2D NMR studies. The overall helical conformation of nonamer 1 was found by variable-temperature NOESY studies to be dynamic. As temperature rose, the end-to-end NOEs rapidly disappeared, while the amide side chain NOEs were still readily detectable, corresponding to the "breath" and stretching of the helix by slightly twisting the local H-bonded rings. Based on the simple repetition of the same structural motif and local conformational preference, undecamer 2 was found to fold into well-defined helical conformation. The predictability of the folding of these backbone-rigidified aromatic oligoamides was demonstrated by a simple modeling method using structural parameters from oligomers with known crystal structures. The reliability and generality of the modeling methods were shown by the excellent agreement between the modeled structures corresponding to 1 and 2 and data from NOESY studies.     

Molecular modelling and 1H-NMR: ultimate tools for the investigation of tolbutamide: beta-cyclodextrin and tolbutamide: hydroxypropyl-beta-cyclodextrin complexes

A structural study of the inclusion compound of tolbutamide (TBM) with beta-cyclodextrin (beta-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was attempted by means of 1H-nuclear magnetic resonance (1H-NMR) experiments and computer molecular modelling. To establish the stoichiometry and stability constant of the beta-CD:TBM complex, the continuous variation method was used. The presence of true inclusion complexes between TBM and beta-CD or HP-beta-CD in solution was clearly evidenced by the 1H-NMR technique. Changes in chemical shifts of H-3 and H-5 protons, located inside the CD cavity, associated with variations in the chemical shifts of TBM aromatic protons provided clear evidence of inclusion complexation, suggesting that the phenyl moiety of the drug molecule was included in the hydrophobic cavity of CDs. This view was further supported by the observation of intermolecular NOEs between TBM and beta-CD and by the aid of a molecular modelling program, which established the most probable structure of the complex. The molecular graphic computation confirmed that the minimum energy, positioning TBM relative to beta-CD, occurs when the aromatic ring of TBM is included within the beta-CD cavity by its wider side, leaving the aliphatic chain externally, which is in good agreement with the results of 1H-NMR studies.     

Ab initio calculations on the vibrational behavior of ladder type oligo(p-phenylenes)

Ab initio calculations of the Raman spectra of ladder type oligo (p-phenylenes) (LOPP) were done in order to study the influence of the side chains attached on the methine bridge and the aromatic rings. We found that the aromatic CC stretching modes and the interring CC stretching modes are significantly influenced. Due to the attachment of an aromatic side chain on the methin bridge, an additional strong mode appears in the interring CC stretching region. Side chains on the aromatic rings cause a splitting of the aromatic CC stretching mode.     

Protein-ligand NOE matching: a high-throughput method for binding pose evaluation that does not require protein NMR resonance assignments

Given the three-dimensional (3D) structure of a protein, the binding pose of a ligand can be determined using distance restraints derived from assigned intra-ligand and protein-ligand nuclear Overhauser effects (NOEs). A primary limitation of this approach is the need for resonance assignments of the ligand-bound protein. We have developed an approach that utilizes data from 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectra for evaluating ligand binding poses without requiring protein NMR resonance assignments. Only the 1H NMR assignments of the bound ligand are essential. Trial ligand binding poses are generated by any suitable method (e.g., computational docking). For each trial binding pose, the 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectrum is predicted, and the predicted and observed patterns of protein-ligand NOEs are matched and scored using a fast, deterministic bipartite graph matching algorithm. The best scoring (lowest "cost") poses are identified. Our method can incorporate any explicit restraints or protein assignment data that are available, and many extensions of the basic procedure are feasible. Only a single sample is required, and the method can be applied to both slowly and rapidly exchanging ligands. The method was applied to three test cases: one complex involving muscle fatty acid-binding protein (mFABP) and two complexes involving the leukocyte function-associated antigen 1 (LFA-1) I-domain. Without using experimental protein NMR assignments, the method identified the known binding poses with good accuracy. The addition of experimental protein NMR assignments improves the results. Our "NOE matching" approach is expected to be widely applicable; i.e., it does not appear to depend on a fortuitous distribution of binding pocket residues.     

NMR Investigations of Inclusion Complexes between β-Cyclodextrin and Naphthalene/Anthraquinone Derivatives

A ¹H and ¹³C NMR study on the inclusion complex between β-cyclodextrin and naphthalene, 1,5-dichloronaphthalene and 9,10-anthraquinone was carried out in order to define the stoichiometry of the association and the possible conformations. The upfield variation of the chemical shifts from the protons locate inside the cavity (H-3, H-5) coupled with the downfield variation of the other protons which locate outer sphere of the β-CD (H-1, H-2, H-4 and H-6,6′) provided clear evidence of the inclusion phenomena. The NMR spectra revealed the formation of 1:1 inclusion complex in which aromatic ring of the guest is tightly held by the host cavity. The signal degeneration of ¹H & ¹³C NMR spectra still exist for naphthalene and 1,5-dichloronaphthalene upon complexation revealed a symmetrical conformation of the guest molecules in the cavity of β-cyclodextrin, respectively. However, in 9,10-anthraquinone, the signal degeneration of ¹H & ¹³C NMR spectra was removed upon complexation which revealed an unsymmetrical conformation of the guest molecule inside the cavity. According to theoretical calculations and NMR studies, the possible conformations of the inclusion complexes are given here.     

Gradient-purged isotope filter experiments for the detection of bound water in proteins

A combination of gradient-purged isotope-filtered NMR experiments is presented, which allows for the detection of long-lived bound water molecules in proteins. The discrimination of direct water–protein exchange from NOE effects between bound water and protein protons is achieved by NOE/ROE cancellation during the mixing time in one of the otherwise identical experiments. The method was applied successfully to ¹³C/¹⁵N-labelled serine protease PB92, and allowed for the identification of 22 protein–water NOEs in this 269-residue enzyme.     

Effects of protonation and co-anion on the 1H and 13C chemical shifts of 2r,6c- diphenylpiperidin-4-ones: evidence for the formation of ion pair

(1)H and (13)C NMR spectra have been recorded for 2r,6c-diphenylpiperidin-4-one (1a), 3t-alkyl-2r,6c-diphenylpiperidin-4-ones 1b-d, 3t-alkyl-2r,6c-diphenyl-4-oxopiperidinium nitrates 2b and 2d, 3t-alkyl-2r,6c-diphenylpiperidin-4-one hydrochlorides 3a-c and 3t-methyl-2r,6c-diphenyl-4-oxopiperidinium picrate 4b in DMSO-d(6). For 1b, 2b and 3b, (1)H and (13)C NMR spectra have been recorded in CH(3)OD also. For 1b, 1d, 2b, 2d, 3b and 4b, 2D spectra have also been recorded. In DMSO-d(6) the protons of the piperidine ring and the ortho protons of the phenyl groups are markedly deshielded due to protonation. Protonation shields all the carbons of the piperidine ring and the ipso carbons of the phenyl groups markedly but deshields the other aromatic carbons slightly. The deshieldings on H-3a, H-5a and the ortho protons of the phenyl groups are less in the nitrate and picrate than in the corresponding hydrochloride. The effects on (13)C chemical shifts are not influenced by the co-anion. These observations suggest that the nitrates and pictrate exist as ion pairs in DMSO-d(6) and the nitrate and picrate ions shield, H-3a, H-5a and the ortho protons by magnetic anistropic effect. In CH(3)OD for 2b and 3b in addition to the ion-pair containing free ions two ion-pairs containing solvated ions are also present. The effects of protonation in the ion pairs containing the solvated ions are significantly different from those in the ion-pair containing free ions.     

Creation and investigation of protein-core mimetics with parallel and antiparallel aligned amino acids

Mimetic protein cores were created that align a set of l-Phe, d-Phe, or l-Leu residues in a parallel or an antiparallel arrangement in chloroform. Not all cores show a single conformation at room temperature. Stable structures require a synergistic relationship between the H-bonding groups and the residues within the core. The spatial arrangement of the side chains dictates whether a zippered or a crossed pattern of H-bonds is observed for these cores. Variable-temperature (1)H NMR experiments were used to determine the strengths of the H-bonds. The existence of H-bonds was verified through FTIR spectroscopic analysis. Large temperature coefficients exist for some protons of aromatic rings that are held in a T-shaped arrangement. A comparison of these temperature coefficients shows that a more stable core is obtained by combining benzenoid and nitrobenzenoid rings as compared to benzenoid rings. Structures were determined using a combination of 2D NMR analysis and molecular modeling.     

Synthesis and Structures of Two New Molybdophosphates

A one-dimension solid and a two-dimension solid consisting of polyoxoanions bridged by {CoO3N2} and {CoO4} groups respectively have been synthesized by the hydrothermal method and structurally characterized by X-Ray crystallography. Each structure has the [(PO4)4Mo6O15]^12- cluster that is commonly observed in many molybdophosphates. In compound 1, [(PO4)4Mo6O15]^12- clusters are linked by {CoO3N2} groups to form a chain-like structure, and the chains are hydrogen-bonded into a network. Compound 2 is a layer structure with channnels constructed from [(PO4)4Mo6O15]^12- clusters and {CoO4} groups, the organic molecules and the lattice water molecules are distributed disorderly in the interlamellar region and form strong hydrogen bonds with inorganic framework. The IR and the fluorescent spectra were investigated and discussed.     

Multinuclear magnetic resonance and theoretical calculations in the study of structure and tautomerism of some 2-amino-N'-(aryl)-benzamidines

1H, 13C and 15N NMR spectra of eight 2-amino-N'-(aryl)-benzamidines and of the parent compound were recorded, and unequivocal chemical shift assignments through the use of COSY, 1H-J resolved, HETCOR and COLOC sequences were performed. 1H and 13C chemical shifts for the nuclei of the benzamidine aromatic ring were not affected by the substituents present at N'-phenyl group, while the substituent effects in the chemical shifts of the same nuclei of N'-phenyl ring were very similar to the ones reported for the corresponding monosubstituted benzenes, indicating that there is no interaction between the two aromatic rings. 15N NMR spectra (DEPT sequence) show just two hydrogenated nitrogen atoms, which confirm that the amino form is the most stable tautomer, but the observation of a sharp signal and two broad signals (15N decoupled spectra), and the corresponding broad signal for the =C-NH(2) protons (in the 1H spectra), indicates the occurrence of tautomerism between the amino and imino forms, observable for some of the studied benzamidines. Theoretical calculations lead to the conclusion that these compounds occur mostly as the amino tautomer with Z configuration, which is stabilized by hydrogen bonding.     

Dicondensed indolinobenzospiropyrans as precursors of thermo- and photochromic spiropyrans. Part II: Assignment of (1)H and (13)C NMR spectra

The (1)H and (13)C NMR spectra of dicondensed indolinobenzospiropyrans as precursors of thermo- and photochromic spiropyrans, DC1-DC5, were completely assigned. Especially, the (1)H assignment and coupling characteristics of the diastereotopic protons at the carbon-3 position of the benzopyran rings were achieved by conducting (1)H-(1)H COSY and nOe experiments. The dihedral angles (theta(1), theta(2) and theta(3)) calculated from the experimental values of the vicinal coupling constants ((3)J) of DC5 are in good agreement with the observed values in the solid state. All of the carbons in the DC dye molecules were investigated through a combination of heteronuclear 2D-shift correlation spectroscopy (HETCOR) and DEPT135.