Determination of multiple torsion-angle constraints in U-(13)C,(15)N-labeled peptides: 3D (1)H-(15)N-(13)C-(1)H dipolar chemical shift NMR spectroscopy in rotating solids

We demonstrate constraint of peptide backbone and side-chain conformation with 3D (1)H-(15)N-(13)C-(1)H dipolar chemical shift, magic-angle spinning NMR experiments. In these experiments, polarization is transferred from (15)N[i] by ramped SPECIFIC cross polarization to the (13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1] resonances and evolves coherently under the correlated (1)H-(15)N and (1)H-(13)C dipolar couplings. The resulting set of frequency-labeled (15)N(1)H-(13)C(1)H dipolar spectra depend strongly upon the molecular torsion angles phi[i], chi1[i], and psi[i - 1]. To interpret the data with high precision, we considered the effects of weakly coupled protons and differential relaxation of proton coherences via an average Liouvillian theory formalism for multispin clusters and employed average Hamiltonian theory to describe the transfer of (15)N polarization to three coupled (13)C spins ((13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1]). Degeneracies in the conformational solution space were minimized by combining data from multiple (15)N(1)H-(13)C(1)H line shapes and analogous data from other 3D (1)H-(13)C(alpha)-(13)C(beta)-(1)H (chi1), (15)N-(13)C(alpha)-(13)C'-(15)N (psi), and (1)H-(15)N[i]-(15)N[i + 1]-(1)H (phi, psi) experiments. The method is demonstrated here with studies of the uniformly (13)C,(15)N-labeled solid tripeptide N-formyl-Met-Leu-Phe-OH, where the combined data constrains a total of eight torsion angles (three phi, three chi1, and two psi): phi(Met) = -146 degrees, psi(Met) = 159 degrees, chi1(Met) = -85 degrees, phi(Leu) = -90 degrees, psi(Leu) = -40 degrees, chi1(Leu) = -59 degrees, phi(Phe) = -166 degrees, and chi1(Phe) = 56 degrees. The high sensitivity and dynamic range of the 3D experiments and the data analysis methods provided here will permit immediate application to larger peptides and proteins when sufficient resolution is available in the (15)N-(13)C chemical shift correlation spectra.     

Geminal 2JCCH spin-spin coupling constants as probes of the phi glycosidic torsion angle in oligosaccharides

Two-bond (13)C-(1)H NMR spin-spin coupling constants ((2)J(CCH)) between C2 and H1 of aldopyranosyl rings depend not only on the relative orientation of electronegative substituents on the C1-C2 fragment but also on the C-O torsions involving the same carbons. The latter dependencies were elucidated theoretically using density functional theory and appropriate model pyranosyl rings representing the four relative configurations at C1 and C2, and a 2-deoxy derivative, to probe the relationship between (2)J(C2,H1) magnitude and sign and the C1-O1 (phi, phi) and C2-O2 (alpha) torsion angles. Related calculations were also conducted for the reverse coupling pathway, (2)J(C1,H2). Computed J-couplings were validated by comparison to experimentally measured couplings. (2)J(CCH) displays a primary dependence on the C-O torsion involving the carbon bearing the coupled proton and a secondary dependence on the C-O torsion involving the coupled carbon. These dependencies appear to be caused mainly by the effects of oxygen lone pairs on the C-H and C-C bond lengths along the C-C-H coupling pathway. New parameterized equations are proposed to interpret (2)J(C1,H2) and (2)J(C2,H1) in aldopyranosyl rings. The equation for (2)J(C2,H1) has particular value as a potential NMR structure constraint for the C1-O1 torsion angle (phi) comprising the glycosidic linkages of oligosaccharides.     

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.     

A conformational dynamics study of alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution by NMR experiments and molecular simulations

The conformational preference of alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution has been studied by NMR spectroscopy using one-dimensional (1)H,(1)H T-ROESY experiments and measurement of trans-glycosidic (3)J(C,H) coupling constants. Molecular dynamics (MD) simulations with a CHARMM22 type of force field modified for carbohydrates were performed with water as the explicit solvent. The homonuclear cross-relaxation rates, interpreted as effective proton-proton distances, were compared to those obtained from simulation. Via a Karplus torsional relationship, (3)J(C,H) values were calculated from simulation and compared to experimental data. Good agreement was observed between experimental data and the MD simulation, except for one inter-residue T-ROE between protons in the terminal sugar residues. The results show that the trisaccharide exhibits substantial conformational flexibility, in particular along the psi glycosidic torsion angles. Notably, for these torsions, a high degree of correlation (77%) was observed in the MD simulation revealing either psi(2)(+) psi(3)(+) or psi(2)(-)psi(3)(-) states. The simulations also showed that non-exoanomeric conformations were present at the phi torsion angles, but to a limited extent, with the phi(3) state populated to a larger extent than the phi(2) state. Further NMR analysis of the trisaccharide by translational diffusion measurements and (13)C T(1) relaxation experiments quantified global reorientation using an anisotropic model together with interpretation of the internal dynamics via the "model-free" approach. Fitting of the dynamically averaged states to experimental data showed that the psi(2)(+)psi(3)(+) state is present to approximately 49%, psi(2)(-) psi(3)(-) to approximately 39%, and phi(3) (non-exo) to approximately 12%. Finally, using a dynamic and population-averaged model, (1)H,(1)H T-ROE buildup curves were calculated using a full relaxation matrix approach and were found to be in excellent agreement with experimental data, in particular for the above inter-residue proton-proton interaction between the terminal residues.     

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.     

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.     

Alpha-gamma hybrid peptides that contain the conformationally constrained gabapentin residue: characterization of mimetics of chain reversals

The crystal structures of four dipeptides that contain the stereochemically constrained gamma-amino acid residue gabapentin (1-(aminomethyl)cyclohexaneacetic acid Gpn) are described. The molecular conformation of Piv-Pro-Gpn-OH (1), reveals a beta-turn mimetic conformation, stabilized by a ten atom C[bond]H...O hydrogen bond between the Piv CO group and the pro S hydrogen of the Gpn CH(2)[bond]CO group. The peptides Boc-Gly-Gpn-OH (2), Boc-Aib-Gpn-OH (3), and Boc-Aib-Gpn-OMe (4) form compact, folded structures, in which a distinct reversal of polypeptide chain direction is observed. In all cases, the Gpn residue adopts a gauche,gauche (g,g) conformation about the C(gamma)[bond]C(beta) (theta(1)) and C(beta)[bond]C(alpha) (theta(2)) bonds. Two distinct Gpn conformational families are observed. In peptides 1 and 3, the average backbone torsion angle values for the Gpn residue are phi=98 degrees, theta(1)=-62 degrees, theta(2)=-73 degrees, and psi=79 degrees, while in peptide 2 and 4 the average values are phi=-103 degrees, theta(1)=-46 degrees, theta(2)=-49 degrees, and psi=-92 degrees. In the case of 1 and 3, an intramolecular nine-membered O[bond]H...O hydrogen bond is formed between the C[double bond]O of the preceding residue and the terminal carboxylic acid OH group. All four alpha-gamma dipeptide sequences yield compact folded backbone conformations; this suggests that the Gpn residue may be employed successfully in the design of novel folded structures.     

Torsional angular dependence of 1J(Se,Se) and Fermi contact control of 4J(Se,Se): analysis of nJ(Se,Se) (n=1-4) based on molecular orbital theory

(n)J(Se,Se) (n=1-4) nuclear couplings between Se atoms were analyzed by using molecular orbital (MO) theory as the first step to investigating the nature of bonded and nonbonded (n)J(Se,Se) interactions between Se atoms. The values were calculated by employing Slater-type triple xi basis sets at the DFT level, which were applied to structures optimized with the Gaussian 03 program. The contribution from each occupied MO (psi(i)) and psi(i)-->psi(a) (psi(a)=unoccupied MO) transition was evaluated separately. 1J(Se,Se) was calculated for the MeSeSeMe model compound, which showed a typical dependence on the torsion angle (phi(C(Me)SeSeC(Me))). This dependence explains the small values (< or =64 Hz) of 1Jobsd(Se,Se) observed for RSeSeR' and large values (330-380 Hz) of 1Jobsd(Se,Se) observed for 4-substituted naphtho[1,8-c,d]-1,2-diselenoles, which correspond to synperiplanar diselenides. The HOMO-->LUMO and HOMO-1-->LUMO transitions contribute the most to 1J(Se,Se) at phi=0 and 180 degrees to give large values of 1J(Se,Se), whereas various transitions contribute and cancel each other out at phi=90 degrees to give small values of 1J(Se,Se). Large 4Jobsd(Se,Se) values were also observed in the nonbonded Se...Se, Se...Se=O, and O=Se...Se=O interactions at naphthalene 1,8-positions. The Fermi contact (FC) term contributes significantly to 4J(Se,Se), whereas the paramagnetic spin-orbit (PSO) term contributes significantly to 1J(Se,Se). 2J(Se,Se) and 3J(Se,Se) were analyzed in a similar manner and a torsional angular dependence was confirmed for 3J(Se,Se). Depending on the structure, the main contribution to (n)J(Se,Se) (n=2, 3) is from the FC term, with a lesser contribution from the PSO term. Analysis of each transition enabled us to identify and clearly visualize the origin and mechanism of the couplings.     

DFT and NMR parameterized conformation of valeranone

A Monte Carlo random search using molecular mechanics, followed by geometry optimization of each minimum energy structure employing density functional theory (DFT) calculations at the B3LYP/6-31G* level and a Boltzmann analysis of the total energies, generated accurate molecular models which describe the conformational behavior of the antispasmodic bicyclic sesquiterpene valeranone (1). The theoretical H-C-C-H dihedral angles gave the corresponding 1H, 1H vicinal coupling constants using a generalized Karplus-type equation. In turn, the 3J(H,H) values were used as initial input data for the spectral simulation of 1, which after iteration provided an excellent correlation with the experimental 1H NMR spectrum. The calculated 3J(H,H) values closely predicted the experimental values, excepting the coupling constant between the axial hydrogen alpha to the carbonyl group and the equatorial hydrogen beta to the carbonyl group (J(2beta, 3beta)). The difference is explained in terms of the electron density distribution found in the highest occupied molecular orbital (HOMO) of 1. The simulated spectrum, together with 2D NMR experiments, allowed the total assignment of the 1H and 13C NMR spectra of 1.     

Structure determination of a peptide model of the repeated helical domain in Samia cynthia ricini silk fibroin before spinning by a combination of advanced solid-state NMR methods

Fibrous proteins unlike globular proteins, contain repetitive amino acid sequences, giving rise to very regular secondary protein structures. Silk fibroin from a wild silkworm, Samia cynthia ricini, consists of about 100 repeats of alternating polyalanine (poly-Ala) regions of 12-13 residues in length and Gly-rich regions. In this paper, the precise structure of the model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, was determined using a combination of three kinds of solid-state NMR studies; a quantitative use of (13)C CP/MAS NMR chemical shift with conformation-dependent (13)C chemical shift contour plots, 2D spin diffusion (13)C solid-state NMR under off magic angle spinning and rotational echo double resonance. The structure of the model peptide corresponding to the silk fibroin structure before spinning was determined. The torsion angles of the central Ala residue, Ala(19), in the poly-Ala region were determined to be (phi, psi) = (-59 degrees, -48 degrees ) which are values typically associated with alpha-helical structures. However, the torsion angles of the Gly(25) residue adjacent to the C-terminal side of the poly-Ala chain were determined to be (phi, psi) = (-66 degrees, -22 degrees ) and those of Gly(12) and Ala(13) residues at the N-terminal of the poly-Ala chain to be (phi, psi) = (-70 degrees, -30 degrees ). In addition, REDOR experiments indicate that the torsion angles of the two C-terminal Ala residues, Ala(23) and Ala(24), are (phi, psi) = (-66 degrees, -22 degrees ) and those of N-terminal two Ala residues, Ala(13) and Ala(14) are (phi, psi) = (-70 degrees, -30 degrees ). Thus, the local structure of N-terminal and C-terminal residues, and also the neighboring residues of alpha-helical poly-Ala chain in the model peptide is a more strongly wound structure than found in typical alpha-helix structures.     

Toward direct determination of conformations of protein building units from multidimensional NMR experiments VI: chemical shift analysis of his to gain 3D structure and protonation state information

NMR--chemical shift structure correlations were investigated by using GIAO-RB3LYP/6-311++G(2d,2p) formalism. Geometries and chemical shifts (CSI values) of 103 different conformers of N'-formyl-L-histidinamide were determined including both neutral and charged protonation forms. Correlations between amino acid torsional angle values and chemical shifts were investigated for the first time for an aromatic and polar amino acid residue whose side chain may carry different charges. Linear correlation coefficients of a significant level were determined between chemical shifts and dihedral angles for CSI[(1)H(alpha)]/phi, CSI[(13)C(alpha)]/phi, and CSI[(13)C(alpha)]/psi. Protonation of the imidazole ring induces the upfield shift of CSI[(13)C(alpha)] for positively charged histidines and an opposite effect for the negative residue. We investigated the correspondence of theoretical and experimental (13)C(alpha), (13)C(beta), and (1)H(alpha) chemical shifts and the nine basic conformational building units characteristic for proteins. These three chemical shift values allow the identification of conformational building units at 80% accuracy. These results enable the prediction of additional regular secondary structural elements (e.g., polyProlineII, inverse gamma-turns) and loops beyond the assignment of chemical shifts to alpha-helices and beta-pleated sheets. Moreover, the location of the His residue can be further specified in a beta-sheet. It is possible to determine whether the appropriate residue is located at the middle or in a first/last beta-strand within a beta-sheet based on calculated CSI values. Thus, the attractive idea of establishing local residue specific backbone folding parameters in peptides and proteins by employing chemical shift information (e.g., (1)H(alpha) and (13)C(alpha)) obtained from selected heteronuclear correlation NMR experiments (e.g., 2D-HSQC) is reinforced.     

Mapping the backbone dihedral free-energy surfaces in small peptides in solution using adiabatic free-energy dynamics

Ramachandran surfaces for the alanine di- and tripeptides in gas phase and solution are mapped out using the recently introduced adiabatic free-energy dynamics (AFED) approach introduced by Rosso et al. (J. Chem. Phys. 2002, 116, 4389) as applied to the CHARMM22 force field. It is shown that complete surfaces can be mapped out with an order of magnitude of greater efficiency with the AFED approach than they can using the popular umbrella sampling method. In the alanine dipeptide, it is found, in agreement with numerous other studies using the CHARMM22 force field, that the lowest free-energy structure is the extended beta conformation, (phi, psi) = (-81, 81), while in solution, the extended beta, (phi, psi) = (-81, 153) and right-handed alpha-helical, (phi, psi) = (-81, 63) conformations are nearly isoenergetic. In solution, a secondary minimum at (phi, psi) = (63, -81), corresponding to a C(7)ax conformation, occurs approximately 2.3 kcal/mol above the global free-energy minimum. The alanine tripeptide, a system that has received considerably less attention in the literature, is found to exhibit a similar structure to the alanine dipeptide with the extended beta conformation being the free-energy minimum in the gas phase and the beta and right-handed alpha-helical conformations being isoenergetic in solution. These studies indicate that the AFED method can be a powerful tool for studying multidimensional free-energy surfaces in complex systems.     

Electronic influences on 3J(C,H) coupling constants via -S-, -S(O)- and -SO2-: their determination, calculation and comparison of detection methods

3J(C,H) coupling constants via a sulfur atom in two series of compounds, both including a sulfide, a sulfoxide and a sulfone, were detected experimentally and calculated by quantum mechanical methods. In the first series (1-3) the coupling between a hydrogen, bonded to an sp3 carbon, and an sp2 carbon is treated; the second series (4-6) deals with the coupling between a hydrogen, bonded to an sp3 carbon, and an sp3 carbon. Different pulse sequences (broadband HMBC, SelJres, 1D HSQMBC, J-HMBC-2, selective J-resolved long-range experiment and IMPEACH-MBC) proved to be useful in determining the long-range 3J(C,H) coupling constants. However, the dynamic behaviour of two of the compounds (4 and 6) led to weighted averages of the two coupling constants expected (concerning equatorial and axial positions of the corresponding hydrogens). DFT calculations proved to be useful to calculate not only the 3J(C,H) coupling constants but also the different contributions of FC, PSO, DSO and SD terms; the calculation of the Fermi contact term (FC) was found to be sufficient for the correct estimation of 3J(C,H) coupling constants.     

Relative signs of 29Si-13C couplings

Two pulse sequences applicable to the determination of relative signs of coupling constants, gHSQC-RELAY(P) and gHSQC-RELAY(D), were developed and tested. These sequences are suitable for determination of relative signs of long-range coupling constants (<2 Hz) between two heteronuclei of low abundance (such as (29)Si and (13)C), and are applicable even to cases in which one of the heteronuclei ((29)Si) does not exhibit coupling with some of the detected protons ((1)H). The two sequences differ in the manner in which they suppress undesirable homonuclear coherence transfers. Each of the sequences can be combined with an isotope filter for better suppression of the centerlines arising from more abundant NMR-inactive isotopes. The sequences were tested on ethoxytrimethylsilane and (E)-(buta-1,3-dienyloxy)trimethylsilane, and we conclude that (2)J((29)Si-O-(13)C) is positive while (3)J((29)Si-O-C-(13)C) is negative in both compounds.     

Ramachandran-type plots for glycosidic linkages: Examples from molecular dynamic simulations using the Glycam06 force field

The goals of this article are to (1) provide further validation of the Glycam06 force field, specifically for its use in implicit solvent molecular dynamic (MD) simulations, and (2) to present the extension of G.N. Ramachandran's idea of plotting amino acid phi and psi angles to the glycosidic phi, psi, and omega angles formed between carbohydrates. As in traditional Ramachandran plots, these carbohydrate Ramachandran-type (carb-Rama) plots reveal the coupling between the glycosidic angles by displaying the allowed and disallowed conformational space. Considering two-bond glycosidic linkages, there are 18 possible conformational regions that can be defined by (alpha, phi, psi) and (beta, phi, psi), whereas for three-bond linkages, there are 54 possible regions that can be defined by (alpha, phi, psi, omega) and (beta, phi, psi, omega). Illustrating these ideas are molecular dynamic simulations on an implicitly hydrated oligosaccharide (700 ns) and its eight constituent disaccharides (50 ns/disaccharide). For each linkage, we compare and contrast the oligosaccharide and respective disaccharide carb-Rama plots, validate the simulations and the Glycam06 force field through comparison to experimental data, and discuss the general trends observed in the plots.     

Dynamic stereochemistry of erigeroside by measurement of 1H-1H and 13C-1H coupling constants

Erigeroside was extracted from Satureja khuzistanica Jamzad (Marzeh Khuzistani in Persian, family of lamiaceae), and (1)H, (13)C, (13)C{(1)H}, (1)H-(1)H COSY, HMQC and J-HMBC were obtained to identify this compound and determine a complete set of J-coupling constants ((1)J(C-H), (2)J(C-H), (3)J(C-H) and (3)J(H-H)) values within the exocyclic hydroxymethyl group (CH(2)OH) and anomeric center. In parallel, density functional theory (DFT) using B3LYP functional and split-valance 6-311++G** basis set has been used to optimized the structures and conformers of erigeroside. In all calculations solvent effects were considered using a polarized continuum (overlapping spheres) model (PCM). The dependencies of (1)J, (2)J and (3)J involving (1)H and (13)C on the C(5')-C(6') (omega), C(6')-O(6') (theta) and C(1')-O(1') (phi) torsion angles in erigeroside were computed using DFT method. Complete hyper surfaces for (1)J(C1',H1'), (2)J(C5',H6'R), (2)J(C5',H6'S), (2)J(C6',H5'), (3)J(C4',H6'R), (3)J(C4',H6'S) and (2)J(H6'R-H5'S) as well as (3)J(H5',H6'R) were obtained and used to derive Karplus equations to correlate these couplings to omega, theta and phi. These calculated J-couplings are in agreement with experimental values. These results confirm the reliability of DFT calculated coupling constants in aqueous solution.     

Dihedral psi angle dependence of the amide III vibration: a uniquely sensitive UV resonance Raman secondary structural probe.

UV resonance Raman studies of peptide and protein secondary structure demonstrate an extraordinary sensitivity of the amide III (Am III) vibration and the C(alpha)H bending vibration to the amide backbone conformation. We demonstrate that this sensitivity results from a Ramachandran dihedral psi angle dependent coupling of the amide N-H motion to (C)C(alpha)H motion, which results in a psi dependent mixing of the Am III and the (C)C(alpha)H bending motions. The vibrations are intimately mixed at psi approximately 120 degrees, which is associated with both the beta-sheet conformation and random coil conformations. In contrast, these motions are essentially unmixed for the alpha-helix conformation where psi approximately -60 degrees. Theoretical calculations demonstrate a sinusoidal dependence of this mixing on the psi angle and a linear dependence on the distance separating the N-H and (C)C(alpha)H hydrogens. Our results explain the Am III frequency dependence on conformation as well as the resonance Raman enhancement mechanism for the (C)C(alpha)H bending UV Raman band. These results may in the future help us extract amide psi angles from measured UV resonance Raman spectra.     

Eremophilane esters of Robinsonecio gerberifolius and their rearranged products. Study of the coupling constants (2)J(H, H), (3)J(H, H) and (4)J(H, H)

In the course of the basic hydrolysis of four eremophilane esters isolated from Robinsonecio gerberifolius, some rearrangements, eliminations, and additions occurred. Five compounds were obtained, three of them not previously described. Additionally, a new sesquiterpene was produced by autooxidation of compound 1. The (1)H and (13)C NMR spectra of these compounds were completely assigned by utilization of HMQC, HMBC, COSY, DEPT, and NOESY techniques. The long-range coupling constants of the peroxide 10 are reported, and all its coupling constants (2)J(H, H), (3)J(H, H), and (4)J(H, H) are calculated at the B3LYP/6-31G(d,p) level of theory. Their magnitude is explained in terms of electronic delocalization and the additivity of stereoelectronic effects.     

Correlated C-C and C-O bond conformations in saccharide hydroxymethyl groups: parametrization and application of redundant 1H-1H, 13C-1H, and 13C-13C NMR J-couplings

Methyl alpha- and beta-pyranosides of d-glucose and d-galactose 1-4 were prepared containing single sites of (13)C-enrichment at C4, C5, and C6 (12 compounds), and (1)H and (13)C[(1)H] NMR spectra were obtained to determine a complete set of J-couplings ((1)J, (2)J, and (3)J) involving the labeled carbon and nearby protons and carbons within the exocyclic hydroxymethyl group (CH(2)OH) of each compound. In parallel theoretical studies, the dependencies of (1)J, (2)J, and (3)J involving (1)H and (13)C on the C5-C6 (omega) and C6-O6 (theta;) torsion angles in aldohexopyranoside model compounds were computed using density functional theory (DFT) and a special basis set designed to reliably recover the Fermi contact contribution to the coupling. Complete hypersurfaces for (1)J(C5,C6), (2)J(C5,H6)(R), (2)J(C5,H6)(S), (2)J(C6,H5), (2)J(C4,C6), (3)J(C4,H6)(R), (3)J(C4,H6)(S), and (3)J(C6,H4), as well as (2)J(H6)(R)(,H6)(S), (3)J(H5,H6)(R), and (3)J(H5,H6)(S), were obtained and used to parametrize new equations correlating these couplings to omega and/or theta;. DFT-computed couplings were also tested for accuracy by measuring J-couplings in (13)C-labeled 4,6-O-ethylidene derivatives of d-glucose and d-galactose in which values of omega and theta; were constrained. Using a new computer program, Chymesa, designed to utilize multiple J-couplings sensitive to exocyclic CH(2)OH conformation, the ensemble of experimental couplings observed in 1-4 were analyzed to yield preferred rotamer populations about omega and theta;. Importantly, due to the sensitivity of some couplings, most notably (2)J(H6)(R)(,H6)(S), (2)J(C5,H6)(R), and (2)J(C5,H6)(S), to both omega and theta;, unique information on correlated conformation about both torsion angles was obtained. The latter treatment represents a means of evaluating correlated conformation in 1,6-linked oligosaccharides, since psi and theta; are redundant in these linkages. In the latter regard, multiple, redundant scalar couplings originating from both sides of the glycosidic linkage can be used collectively to evaluate conformational correlations between psi/theta; and C5-C6 bond rotamers.