Nuclear Overhauser Enhancement Experiments Research Articles

A concise methodology for the stereoselective synthesis of O-glycosylated amino acid building blocks: complete 1H NMR assignments and their application in solid-phase glycopeptide synthesis.

A facile strategy for the stereoselective synthesis of suitably protected O-glycosylated amino acid building blocks, namely, Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1-3)-Ac2-alpha or beta-D-GalN3]-OPfp and Nalpha-Fmoc-Thr-[Ac4-beta-D-Gal-(1-3)-Ac2-alpha or beta-D-GalN3]-OPfp is described. What is new and novel in this report is that Koenigs-Knorr type glycosylation of an aglycon serine/threonine derivative (i.e. Nalpha-Fmoc-Ser-OPfp or Nalpha-Fmoc-Thr-OPfp) with protected beta-D-Gal(1-3)-D-GalN3 synthon mediated by silver salts resulted in only alpha- and/or beta-isomers in excellent yields under two different reaction conditions. The subtle differences in stereoselectivity were demonstrated clearly when glycosylation was carried out using only AgClO4 at -40 degrees C which afforded a-isomer in a quantitative yield (alpha:beta = 5:1). On the other hand, the beta-isomer was formed exclusively when the reaction was performed in the presence of Ag2CO3/AgClO4 at room temperature. A complete assignment of 1H resonances to individual sugar ring protons and the characteristic anomeric alpha-1 H and beta-1 H in Ac4Galbeta(1-3)Ac2GalN3 alpha and/or beta linked to Ser/Thr building blocks was accomplished unequivocally by two-dimensional double-quantum filtered correlated spectroscopy and nuclear Overhauser enhancement and exchange spectroscopy NMR experiments. An unambiguous structural characterization and documentation of chemical shifts, including the coupling constants for all the protons of the aforementioned alpha- and beta-isomers of the O-glycosylated amino acid building blocks carrying protected beta-D-Gal(1-3)-D-GalN3, could serve as a template in elucidating the three-dimensional structure of glycoproteins. The synthetic utility of the building blocks and versatility of the strategy was exemplified in the construction of human salivary mucin (MUC7)-derived, O-linked glycopeptides with varied degrees of glycosylation by solid-phase Fmoc chemistry. Fmoc/tert-butyl-based protecting groups were used for the peptidic moieties in conjunction with acetyl sugar protection. The transformation of the 2-azido group into the acetamido derivative was carried out with thioacetic acid on the polymer-bound glycopeptides before the cleavage step. After cleaving the glycopeptide from the resin, the acetyl groups used for sugar OH-protection were removed with sodium methoxide in methanol. Finally, the glycopeptides were purified by reversed-phase high-performance liquid chromatography and their integrity was confirmed by proton NMR as well as by mass spectral analysis. Secondary structure analysis by circular dichroism of both the glycosylated and nonglycosylated peptides revealed that carbohydrates did not exert any profound structural effect on the peptide backbone conformation.

Conformer selection and differential restriction of ligand mobility by a plant lectin--conformational behaviour of Galbeta1-3GlcNAcbeta1-R, Galbeta1-3GalNAcbeta1-R and Galbeta1-2Galbeta1-R' in the free state and complexed with galactoside-specific mistletoe lectin as revealed by random-walk and conformational-clustering molecular-mechanics.

To study conformational parameters of ligands before and after complex formation with the galactoside-binding agglutinin of Viscum album L. (VAA) in solution, combined computer-assisted random walk molecular mechanics (RAMM) calculations extended by conformational clustering analysis (CCA), molecular dynamics (MD) simulations as well as two-dimensional rotating-frame nuclear Overhauser effect (ROE) and two-dimensional nuclear Overhauser effect (NOE) spectroscopy NMR experiments were employed. Derivatives of the naturally occurring disaccharides Galbeta1-3GlcNAcbeta1-R and Galbeta1-3GalNAcbeta1-R as well as of a synthetic high-affinity binding partner, i.e. the disaccharide Galbeta1-2Galbeta1-R', were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between phi/psi combinations of (40 degrees/60 degrees) and (40 degrees/-60 degrees). Calculations of the de-N-acetylated sugars revealed that presence of this group did not markedly influence the distribution of low-energy conformers in the phi, psi, epsilon plot. Occupation of side minima at phi/psi (180 degrees/0 degrees) or (0 degrees/180 degrees) is either unlikely or low according to the results of MD simulations and RAMM calculations extended by CCA. Notably, these side minima define conformations which are not stable during a MD simulation. Transitions to other minima occur already a few picoseconds after the start of the simulation. NMR experiments of the free-state ligand confirmed the validity of the data sets obtained by the calculations. Following the description of the conformational space in the free-state NMR experiments were performed for these disaccharides complexed with VAA. They yielded two interresidual contacts for Galbeta1-3GlcNAcbeta1-R and Galbeta1-2Galbeta1-R'. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the free state. One- and two-dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments at different mixing times excluded the influence of spin-diffusion effects. When the NOE build-up curves in the three studied cases were compared, the residual mobility of the penultimate carbohydrate unit of Galbeta1-3GalNAcbeta1-R was observed to be higher than that of the respective hexopyranose unit of the other two bound ligands. Due to the availability of the conformational parameters of Galbeta1-2Galbeta1-R' in association with a galectin, namely the beta-galactoside-binding protein from chicken liver, it is remarkable to note that this ligand displays different conformations in the binding sites of either the plant or the animal lectin. They correspond to local energy-minimum conformations in the phi,psi, epsilon plot and substantiate differential conformer selection by these two lectins with identical nominal monosaccharide specificity.

Olefin−Amino Acid Complexes of Platinum(II). 3. NOE Difference and Low-Temperature NMR Determinations of Rotamer Populations of Coordinated Olefins and the Stereoselectivity of Coordination of Prochiral Olefins

The exceptional stereoselectivity of coordination of 2-methyl-3-buten-2-ol in cis(N,olefin)-Pt(2-methyl-3-buten-2-ol)(sarcosine)X, 1 (X = Cl), has been attributed to stabilization of one rotamer of the H2-coordinated olefin by intramolecular hydrogen bonding between the OH oxygen on the olefinic alcohol and the N−H proton of coordinated sarcosine. Nuclear Overhauser enhancement (NOE) difference experiments on 1 have confirmed that the preferred conformation in solution corresponds to that found in the crystalline solid. Both NOE and low-temperature NMR experiments were used to establish approximate rotamer ratios for a series of complexes as a function of olefin structure, X substituent (Cl-, H2O, OH-), and solvent composition (D2O vs CD3OD). Olefins that show strong stereoselectivity in diastereomeric complexes containing sarcosine and proline also show a strong preference for one rotamer in complexes containing the achiral amino acid α-aminoisobutyric acid. The strong stereoselectivity and rotamer prefe...

Transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments reflect the size of the bound segment of the Forssman pentasaccharide in the binding site of Dolichos biflorus lectin.

A complex between the Forssman pentasaccharide alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D- Gal-(1-->4)-D-Glc and the seed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non-reducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNac compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the nonreducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non-reducing disaccharide fragment is buried in the lectin-binding pocket, whereas the reducing trisaccharide portion alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton-proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.

Configurational and Conformational Analysis of Macrocarpals H, I, and J from Eucalyptus globulus.

Configurational and conformational analysis of unique phloroglucinol-sesquiterpene coupled compounds, macrocarpals H, I, and J (1-3), showing potent antibacterial activity and inhibitory effect of glucosyltransferase, was made by nuclear Overhauser enhancement (NOE) experiments and computational chemical methods. The configuration at C-9' of the side chains of 1-3 was elucidated as 1: S, 2: R, and 3: S, by the structures obtained by the Monte Carlo (MC) calculation using MM2* force field implemented in MacroModel/Batchmin, followed by semiempirical molecular orbital calculations, whose stable conformation of macrocarpals H, I, and J was well satisfied by the NOEs observed around H-9'.

Stereoselective synthesis of (2 S,3 S)-γ-hydroxyvaline utilising an asymmetric radical hydrogen bromide addition

( S)-Valine has been utilised in the stereocontrolled synthesis (2 S,3 S)-γ-hydroxyvaline. The selectivity was achieved via 1,2-asymmetric induction in the anti-Markovnikov hydrobromination of a β,γ-dehydrovaline derivative. The relative and absolute stereochemistry of the γ-hydroxyvaline was determined using a variety of methods, including a nuclear Overhauser enhancement experiment with the diastereomeric lactones of γ-hydroxy- N-phthaloylvaline, and the synthetic material was shown to be identical to the natural product. 1,2 Asymmetric induction in anti-Markovnikov hydrobromination of the β-γ-dehydrovaline derivative shown has been utilised in the stereocontrolled synthesis of (2 S,3 S)-γ-hydroxyvaline from ( S)-valine.

The Conformation of Coenzyme A Bound to Chloramphenicol Acetyltransferase Determined by Transferred NOE Experiments

The conformation of coenzyme A bound to chloramphenicol acetyltrans ferase has been studied in solution by NMR methods. Transferred nuclear Overhauser enhancement (NOE) and rotating frame NOE (ROE) experiments were used to determine the conformation of the bound coenzyme. Experiments were carried out at five mixing times and two temperatures, and with normal and perdeuterated enzyme, to ensure (1) that the fast exchange condition was satisfied and (2) that the results were not complicated by spin diffusion involving enzyme protons. The data were analysed using a general approach involving combined exchange and relaxation matrices. For the binary complex of coenzyme A (CoA) and enzyme, the conformation of CoA was calculated by using distance constraints derived from the intensities of 71 NOE and 33 ROE cross-peaks between coenzyme protons. The conformation of the adenosine moiety of CoA in the structure deduced by NMR is very close to that seen in the crystal structure of this complex, while the pantetheine moiety is clearly less extended. Essentially the same conformation was obtained whether or not the calculations included the protein (with appropriate intermolecular energy terms). The difference between the NMR and X-ray structures is interpreted in terms of the existence of two conformations of the CoA-enzyme complex. Support for this model comes from measurements of the coenzyme dissociation rate constant; NMR (lineshape analysis and transferred NOE experiments) gives estimates of k off∼3700 s −1at 298 K and ∼500 s −1at 280 K, both significantly greater than estimates by fluorescence stopped-flow measurements. For the ternary complex of CoA, chloram phenicol and enzyme, 71 NOE cross-peaks between protons of coenzyme A and a further ten cross-peaks between protons of coenzyme A and chloramphenicol were measured. Starting with a model derived from the crystal structures of the two binary complexes (in the absence of crystallographic data for the ternary complex) the conformations and relative positions of the two ligands were refined using the distance constraints derived from these NOEs. The conformation of the adenosine part of CoA is the same as in the binary complex, while the pantetheine arm is more extended and approaches close to the bound chloramphenicol molecule. The model of the ternary complex is discussed in terms of the information available on the mechanism of the enzyme.

Synthesis and stereochemical assignment of 7‐arylidene and 7‐heteroarylidene morphinan‐6‐ones

AbstractA number of (E)‐7‐arylidenenaltrexones were synthesized by azeotropic distillation of water from a benzene solution of naltrexone and an aromatic aldehyde (benzaldehyde, 4‐chloro‐ and 4‐fluorobenzaldehyde, 3‐and 4‐pyridinecarboxaldehyde and 1‐methyl‐2‐imidazolecarboxaldehyde) using piperidine as a catalyst. In addition, (E)‐7‐benzylidenenaloxone was prepared by the previously published Claisen‐Schmidt condensation using sodium hydroxide in methanol. The stereochemistry of these arylidene derivatives 3–9 was determined to be (E) by means of nuclear Overhauser enhancement experiments. The 13C nmr spectra of (E)‐3–9 are recorded in deuteriochloroform and those of the hydrochlorides in deuteriodimethyl sulfoxide.

Syntheses and unusual spectroscopic properties of novel ketobacteriopurpurins

Nuclear Overhauser enhancement experiments show that migratory aptitudes of alkyl groups in various vis-dihydroxybacteriopurpurins are different for individual stereoisomers. Long wavelength absorption maxima of ketobacteriopurpurins obtained from pinacol-pinacolone rearrangements of the corresponding diols are influenced by the position of the keto group in the macrocycle; differences in these maxima are diminished by introduction of electron-withdrawing substituents.

Synthesis of 4-phosphazenyl-2,3-dihydropyrrol-2-ones and 5-phosphazenyl-2-pyridones from β-[(N-acyl)phosphazenyl]enamines and dimethyl acetylenedicarboxylate

4-Phosphazenyl-2,3-dihydropyrrol-2-ones and 5-phosphazenyl-2-pyridones are obtained by addition of dimethyl acetylenedicarboxylate to [(N-acyl)phosphazenyl]enamines. In refluxing dichloromethane the reaction affords exclusively 2,3-dihydropyrrol-2-ones, while in toluene at 110 °C a mixture of 2-pyridones and 2,3-dihydropyrrol-2-ones is formed. They are easily separated by fractional recrystallization. The structures of all new compounds have been unequivocally assigned based on two-dimensional 1H,13C correlation and steady-state nuclear Overhauser enhancement experiments. For 2-pyridones the absolute sign of 4JPH has been determined.

Structural elucidation of the backbone oligosaccharide from the lipopolysaccharide of Moraxella catarrhalis serotype A

The cell-surface lipopolysaccharide produced by Moraxella catarrhalis serotype A is composed of a hydrophobic lipid A moiety and an oligosaccharide, but lacks high-molecular-weight O-polysaccharide chains. The oligosaccharide component is composed of D-glucose, D-galactose, D-glucosamine, and 3-deoxy-D-manno-octulosonic acid. The carbohydrate backbone was obtained from the lipopolysaccharide by employing a reaction sequence involving deacylation, dephosphorylation, and reduction of the reducing D-glucosamine terminus of the lipid A moiety. Structural analysis of the backbone oligosaccharide employed a combination of microanalytical methods and nuclear magnetic resonance spectroscopy. Homo- and hetero-nuclear chemical shift correlation techniques and nuclear Overhauser enhancement (NOE) experiments led to the unambiguous assignment of the 1H and l3C resonances associated with each of the component glycosyl residues and established their sequence within the backbone oligosaccharide as shown,[Formula: see text]This lipopolysaccharide was found to consist of a highly branched, D-glucose-containing inner-core region which possesses a unique and unusual solution conformation. This was established by measurement of transglycosidic NOEs and three-bond 1H−13C coupling constants, in conjunction with molecular modeling. A D-galactose-containing disaccharide identified as a terminal group of the lipopolysaccharide was structurally identical to antigenic epitopes expressed by certain mammalian epithelial cells and may be related to the virulence potential of this human pathogen.

Synthesis and X-ray structures of mono- and bis-acetonitrile derivatives of the cationic rhenium allyl complex [η 5-C 5Me 5) Re(CO) 2(η 3-C 3H 5)][BF 4

Replacement of CO ligands in the η 3-allyl complex [Cp*Re(CO) 2(η −C 3H 5)][BF 4] 1 has been accomplished by reacting 1 in acetonitrile with PhIO or Me 3NO to yield endo isomers of [Cp*Re(CO)(NCMe)(η 3-C 3H 5)][BF 4] 2 and [Cp*Re(NCMe) 2(η 3-C 3H 5)][BF 4] 3 respectively. The assignment of the allyl proton resonances and the isomeric forms of 2 and 3 have been deduced with the aid of 1H nuclear magnetic resonance nuclear Overhauser enhancement experiments. The X-ray crystal structures of 2 and 3 confirm that these are endo isomers. Poor quality crystals of 2 (with 0.5 toluene solvate) are monoclinic, of space group P2 1/ c, with Z = 4, a = 8.381(2) Å, b = 15.389(2) Å c = 17.161(3) Å, β = 91.71(2)° and V= 2212.4 Å 3. The structure refined to R F = 0.060 for 1950 data ( I o ⩾ 2.5σ(I o)). Crystals of 3 are orthorhombic, of space group P2 12 12 1, with Z = 4, a = 11.296(2) Å, b = 12.012(2) Å, c = 14.760(2) Å and V= 2002.7 Å 3. The structure refined to R F = 0.027 for 1815 data ( I o >/ 2.5σ( I o)). The rhenium atom is bonded to the allyl group in 3 with an ReC(7) distance of 2.124(11) Å to the central carbon atom and an ReC(6) distance of 2.177(12) Å and an Re-C(8) distance of 2.201(12) Å to the terminal carbon atoms. The acetonitrile groups are bound with an ReN(1) distance of 2.089(7) Å and an ReN(2) distance of 2.086(10) Å, and NC with an N(1)C(9) distance of 1.125(11) Å and an N(2)C(11) distance of 1.134(14) Å.

Nuclear magnetic resonance studies of the structure of B50/neuromodulin and its interaction with calmodulin.

B50/neuromodulin is a neuronal phosphoprotein that is found in association with the inner membrane of nerve cells. In this work, we have studied the structure of bovine B50 in aqueous solution (pH 7.5) by 1H nuclear magnetic resonance (NMR) spectroscopy and our results indicate that B50 is an unstructured protein under these conditions. One-dimensional 1H-NMR titration studies of the interaction between B50 and calmodulin (CaM) have shown that B50 does not interact with (or) interacts very weakly with apo-CaM in solution; neither does B50 interact with Ca(2+)-CaM. These NMR data are consistent with an earlier observation that B50 is not capable of binding apo-CaM in vitro unless some nonionic detergent is present. We have also detected aromatic NMR peaks for a new posttranslational modification that might involve the His residues of the protein. The interaction of a 14-residue peptide (I38-L51) encompassing the CaM-binding domain of B50 with CaM was also studied by NMR. We have found from two-dimensional transferred nuclear Overhauser enhancement experiments that the B50 peptide binds weakly to apo-CaM in an alpha-helical conformation; the alpha-helix appears to be induced by the binding of the peptide to apo-CaM.

NMR Studies of Cyclodextrin Inclusion Complex with Ethyl Hexanoate in Ethanol Solution

The formation of a βCD inclusion complex for aliphatic ethyl ester, with ethyl hexanoate as a guest molecule was investigated. Proton T1 measurements by the IR method and 1D difference nuclear Overhauser enhancement experiments showed that ethyl hexanoate exists with the groups (H-2 and H-3) around the ester bond nearer to the narrower βCD cavity and the other protons are proximal to the H(CD)-3 and H(CD)-5 protons within the cavity with a folded structure. The complexation of βCD was greatly affected by the addition of ethanol. With increasing ethanol concentration, a marked increase in volatility of ethyl hexanoate from the βCD solution was observed; the volatility in 30% ethanol was about 3 times larger than in 0% one. On the basis of the observed NOE and T1 values, the lowering of the abilitys of βCD complexation in ethanol solution is assumed to be caused by the situation shift of guest molecule toward the narrower side or outside of the cavity.

Solution structure of a conserved DNA sequence from the HIV-1 genome: restrained molecular dynamics simulation with distance and torsion angle restraints derived from two-dimensional NMR spectra.

The three-dimensional solution structure of a trisdecamer DNA duplex sequence, d(AGCTTGCCTTGAG).d(CTCAAGGCAAGCT), from a conserved region of HIV-1 genome's long terminal repeat, has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances derived from two-dimensional nuclear Overhauser enhancement (2D NOE) experiments, using the iterative complete relaxation matrix algorithm MARDIGRAS, and torsion angles for sugar rings, estimated from stimulated fitting of double-quantum-filtered correlation (2QF-COSY) spectra, were obtained [Mujeeb, A., Kerwin, S. M., Egan, W. M., Kenyon, G. L., & James, T. L. (1992) Biochemistry 31, 9325-38]. These structural restraints have now been employed as the basis for structure refinement using restrained molecular dynamics (rMD) to search conformational space for structures consistent with the experimental restraints. Specifically, upper and lower bounds on the restraints were incorporated into the AMBER (version 4.0) total potential energy function of the system, the bounds being used to define the width of a flat-well penalty term in the AMBER force field. Confidence in the time-averaged structure obtained is engendered by convergence to essentially the same structure (root-mean-square deviation approximately 0.9 A) when two quite different DNA models, A-DNA and B-DNA (RMSD approximately 6.5 A), were employed as starting structures and when various initial trajectories were used for the rMD runs. The derived structure is further supported by the total energy calculated, the restraint violation energy, the restraint deviations, and the fit with experimental data. For the latter, the sixth-root residual index indicated a good fit of the determined structure with experimental 2D NOE spectral intensities (R1x < 0.07), and the RMS difference between vicinal proton coupling constants calculated for the derived structure and experimental coupling constants were also in reasonable agreement (JRMS = 0.9 Hz). While the structure of the trisdecamer is basically in the B-DNA family, some structural parameters manifest interesting local variations. The helix parameters indicate that, compared with classical B-DNA, the structure is longitudinally more compressed. Local structural variations at the two TG steps in particular together create bending into the major groove of the duplex. Comparison of the two-CTTG-tetrads in the duplex reveals that they have similar structures, with the TT moieties being almost identical; however, the -CTTG-pur sequence has a larger roll and slide for the -TG- step than for the -CTTG-pyr sequence, in accord with published X-ray crystallographic conclusions.

Characterization of the cis-syn and cis-anti diastereoisomers of 5-methoxypsoralen pyrone-side monocycloadducts to thymidine.

The photoreaction of 5-methoxypsoralen (5-MOP) with thymidine as a DNA model compound was investigated under dry-state conditions. In this respect, a thin film of thymidine and 5-MOP in a ratio 10:1 was exposed to 350-nm UV light. Four [2 + 2] photocycloadducts were isolated in a 0.5-2.2% yield with respect to 5-MOP by HPLC and characterized as two pairs of cis-syn and cis-anti diastereoisomers, respectively, on the basis of extensive spectroscopic measurements, including UV, fast atom bombardment mass spectrometry, 1H and 13C NMR, and CD. Information concerning the absolute configuration of the four photocycloadducts was inferred from detailed nuclear Overhauser enhancement experiments. This is indicative of a 3R,4S,5R,6S and a 3S,4R,5S,6R configuration for the cis-anti cycloadducts and a 3S,4R,5R,6S) and a 3R,4S,5S,6R configuration for the cis-syn cycloadducts. In addition, conformational features of the four photocycloadducts were obtained from consideration of various 1H NMR measurements including NOE data.

Phage display and selection of a site-directed randomized single-chain antibody Fv fragment for its affinity improvement.

The affinity of an antibody Fv fragment was improved by semirational design involving site-directed randomization and phage display. On the basis of the predicted model of an anti-2-phenyloxazol-5-one (phOx) antibody Fv fragment, into which the ligand was inserted with the help of nuclear Overhauser enhancement (NOE) data, residues close to the hapten were identified. Seven of these residues in the third hypervariable regions of light and heavy chains were randomized in polymerase chain reactions (PCR) using degenerate oligonucleotides. Resulting clones were expressed as single-chain Fv (scFV) fragments on the surface of filamentous phage and selected for binding to phOx-conjugated bovine serum albumin. Selected Fv fragments were analyzed for hapten affinity by fluorescence quenching, and several mutants with improved affinities were identified. Phage selection on the basis of binding was very successful when phage scFv mutants differed in affinity by at least a factor of 6. Smaller differences did not result in predominant selection of the best binder. Combination of the two point mutations most crucial for improved hapten binding decreased the dissociation constant of the Fv for phOx 11-14-fold. Hapten binding of the improved Fv was analyzed in NOE experiments.

Stereochemistry of the solid state photodimerization of thymoquinone

Thymoquinone (2-methyl-5- iso-propyl-1,4-benzoquinone) undergoes a stereospecific solid state photochemical [2+2]cyclodimerization. The product is the anti H-T (head-tail) cyclobutane derivative. The relative orientation of the quinone rings was elucidated by 1H NMR and nuclear Overhauser enhancement experiments done on the monoepoxide derivative of the dimer.

Reactions of aromatic ketones with 3‐mercapto‐1,2‐propanediol. Synthesis of cis‐ and trans‐2‐alkyl‐2‐aryl‐(1,3‐oxathiolane‐5‐methanols and 1,3‐dioxolane‐4‐methanethiols)

AbstractAromatic ketones react with 3‐mercapto‐1,2‐propanediol (1) in refluxing benzene under the catalytic influence of a sulfonic acid and with azeotropic removal of water to yield a mixture comprised predominantly of cis‐ and trans‐2‐alkyl‐2‐aryl‐1,3‐oxathiolane‐5‐methanols 7, accompanied by lesser amounts of cis‐ and trans‐2‐alkyl‐2‐aryl‐1,3‐dioxolane‐4‐methanethiols 8 (up to 30%). It was discovered that 8 is the kinetic product and is isomerized by 4‐toluenesulfonic acid in hot benzene to the thermodynamically more stable 7. Under these conditions, ortho‐ and α‐substituted aromatic ketones tend to produce more of 8, which can be attributed to steric hindrance encountered by the thiol as it attacks the ketone. Ketalizations of 1‐aryl‐2‐(1H‐imidazol‐1‐yl)‐1‐ as well as 1‐aryl‐2‐(1,H‐1,2,4‐triazol‐1‐yl)‐1‐ethanones by 1 fail under these conditions, even after 24 hours of reflux in toluene. However, 1‐(4‐chlorophenyl)‐3‐(1H‐imidazol‐1‐yl)‐1‐propanone and 1‐(4‐bromophenyl)‐4‐(1H‐imidazol‐1‐yl)‐1‐butanone are ketalized by 1 as expected. Interestingly, the reaction of 2‐bromo‐4′‐chloroace‐tophenone with 1 produces 1‐(4‐chlorophenyl)‐2,8‐dioxa‐6‐thiabicyclo[3.2.1]octane. Characterization of all isomers and separation of some diastereomers is described. Nuclear Overhauser enhancement experiments are utilized to establish the stereochemistry of 1,3‐oxathiolanes.

Simulating the effect of the two-spin approximation on the generation of protein structures from NOE data

The use of structural information derived from NMR data, when combined with computer modeling techniques, has enabled the structures of many proteins to be determined ( 1-6). Among the most useful structural information is interproton distances which can be derived from nuclear Overhauser enhancement experiments. For small rigid molecules, the intensity of the NOE between protons i andj is proportional to the inverse sixth power of the distance between the two protons. In macromolecules, indirect magnetization transfer (spin diffusion) from nearby protons and intramolecular dynamics affect the magnitude of the NOE between protons i andj and contribute to errors in estimating interproton distances from NOE cross peaks ( 7-9). Several methods have been proposed for dealing with the spin-diffusion problem. These methods make use of the relaxation matrix to incorporate multispin effects (10-14). The simplest but least systematic approach is to calculate a theoretical spectrum from the relaxation matrix using a trial structure, and to adjust distances by trial and error between pairs of protons whose calculated cross peaks have the largest deviations from the experimental values ( 15-18). In another approach theoretical cross peaks from a trial structure are merged with experimental NOES ( I2,19,20), and after the solution of the Bloch equations is inverted to obtain the relaxation matrix, the resulting distances can be added as constraints in a molecular-dynamics or distance geometry refinement. The most rigorous and computationally intensive procedures involve the direct refinement of calculated against experimental NOE spectra, either by analytically evaluating the gradients of the calculated NOES with respect to coordinates (21-23) or by using a Monte-Carlo procedure (24). Despite the recent focus on incorporating spin-diffusion effects in the refinement of macromolecular structures, it is unclear how much error is introduced by spin diffusion. This is of interest because, although the direct refinement of calculated against experimental NOE intensities is in principle the more “correct” procedure, it is much more computationally intensive than re-