Does Inter-Residue Hydrogen Bonding in ß-(1→4)-Linked Disaccharides Influence Linkage Conformation in Aqueous Solution?

Two disaccharides, methyl ß-d-galactopyranosyl-(1→4)-α-d-glucopyranoside (1) and methyl ß-d-galactopyranosyl-(1→4)-3-deoxy-α-d-ribo-hexopyranoside (3), were prepared with selective 13C-enrichment to allow measurement of six trans-O-glycosidic J-couplings (2JCOC, 3JCOCH, and 3JCOCC) in each compound. Density functional theory (DFT) was used to parameterize Karplus-like equations that relate these J-couplings to either ϕ or ψ. MA'AT analysis was applied to both linkages to determine mean values of ϕ and ψ in each disaccharide and their associated circular standard deviations (CSDs). Results show that deoxygenation at C3 of 1 has little effect on both the mean values and librational motions of the linkage torsion angles. This finding implies that, if inter-residue hydrogen bonding between O3H and O5' of 1 is present in aqueous solution and persistent, it plays little if any role in dictating preferred linkage conformation. Hydrogen bonding may lower the energy of the preferred linkage geometry but does not determine it to any appreciable extent. Aqueous 1-µs MD simulation supports this conclusion and also indicates greater conformational flexibility in deoxydisaccharide 3 in terms of sampling several, conformationally distinct, higher-energy conformers in solution. The populations of these latter conformers are low (3-14%) and could not be validated by MA'AT analysis. If the MD model is correct, however, C3 deoxygenation does enable conformational sampling over a wider range of ϕ/ψ values, but linkage conformation in the predominant conformer is essentially identical in both 1 and 3.

MA'AT Analysis: Unbiased Multi-State Conformational Modeling of Exocyclic Hydroxymethyl Group Conformation in Methyl Aldohexopyranosides.

MA'AT analysis (J. Chem. Inf. Model. 2022, 62, 3135-3141) has been applied to model exocyclic hydroxymethyl group conformation in methyl ß-D-glucopyranoside (ßGlcOMe), methyl ß-D-galactopyranoside (ßGalOMe), and methyl ß-D-mannopyranoside (ßManOMe) in an unbiased manner. Using up to eight NMR J-couplings sensitive to rotation about the C5-C6 bond (torsion angle ω), two-state models of ω were obtained that are qualitatively consistent with the relative populations of the gg, gt, and tg rotamers reported previously. MA'AT analysis gave consistent unbiased gt ⇌ tg models of ω in ßGalOMe, with gt more populated than tg and mean values of ω for each population similar to those obtained from aqueous 1-µs MD simulation. Using different combinations of J-couplings had little effect on the ßGalOMe model in terms of the mean values of ω and circular standard deviations (CSDs). In contrast, MA'AT analysis of ω in ßGlcOMe and ßManOMe produced more than one two-state model independent of the ensemble of J-values used in the analyses. These models were characterized by gg ⇌ gt conformer exchange as expected, but the mean values of ω in both conformers varied significantly in the different fits, especially for the gg rotamer. Constrained (biased) MA'AT analyses in which only staggered geometries about ω were allowed gave RMSDs slightly larger than those obtained from the unbiased fits, precluding an assignment of an unbiased model. It is unclear why MA'AT analysis gives consistent and predictable unbiased models of ω in ßGalOMe but not in ßGlcOMe and ßManOMe. One possibility is that the distribution of ω in one or both of the gg and gt conformers in the latter does not conform to a von Mises function (i.e., is not Gaussian-like), but rather to a broad and/or flat distribution that cannot be fit by the current version of MA'AT. Nevertheless, the results of this study provide new evidence of the ability of MA'AT analysis to treat multi-state conformational exchange using only experimental NMR data, extending recent MA'AT applications to furanosyl ring pseudorotation (Biochemistry 2022, 61, 239-251).

Methyl α-D-galactopyranosyl-(1→3)-ß-D-galactopyranoside and methyl ß-D-galactopyranosyl-(1→3)-ß-D-galactopyranoside: Glycosidic linkage conformation determined from MA'AT analysis.

MA'AT analysis has been applied to two biologically-important O-glycosidic linkages in two disaccharides, α-D-Galp-(1→3)-ß-D-GalpOMe (3) and ß-D-Galp-(1→3)-ß-D-GalpOMe (4). Using density functional theory (DFT) to obtain parameterized equations relating a group of trans-O-glycosidic NMR spin-couplings to either phi (ϕ') or psi (ψ'), and experimental 3JCOCH, 2JCOC, and 3JCOCC spin-couplings measured in aqueous solution in 13C-labeled isotopomers, probability distributions of ϕ' and ψ' in each linkage were determined and compared to those determined by aqueous 1-µs molecular dynamics (MD) simulation. Good agreement was found between the MA'AT and single-state MD conformational models of these linkages for the most part, with modest (approximately <15°) differences in the mean values of ϕ' and ψ', although the envelope of allowed angles (encoded in circular standard deviations or CSDs) is consistently larger for ϕ' determined from MA'AT analysis than from MD for both linkages. The MA'AT model of the α-Galp-(1→3)-ß-Galp linkage agrees well with those determined previously using conventional NMR methods (3JCOCH values and/or 1H-1H NOEs), but some discrepancy was observed for the ß-Galp-(1→3)-ß-Galp linkage, which may arise from errors in the conventions used to describe the linkage torsion angles. Statistical analyses of X-ray crystal structures show ranges of ϕ' and ψ' for both linkages that include the mean angles determined from MA'AT analyses, although both angles adopt a wide range of values in the crystalline state, with ϕ' in ß-Galp-(1→3)-ß-Galp linkages showing greater-than-expected conformational variability.

MA'AT Analysis: Probability Distributions of Molecular Torsion Angles in Solution from NMR Spectroscopy.

ConspectusMonosaccharides adopt multiple conformations in solution, and this structural complexity increases significantly when they are assembled into oligosaccharides and polysaccharides. Characterization of the conformational properties of saccharides in solution by NMR spectroscopy has been hampered by several complicating factors, including difficulty interpreting spectra because of significant signal overlap, population averaging of NMR parameters, and unique properties of the spectra that make accurate measurements of NMR parameters prone to error (e.g., non-first-order effects on J-couplings). Current conformational assignments rely heavily on theoretical calculations, especially molecular dynamics (MD) simulations, to interpret the experimental NMR parameters. While these studies assert that the available experimental data fit the calculated models well, a lack of independent experimental validation of the force fields from which MD models are derived and an inability to test all possible models that might be compatible with the experimental data in an unbiased manner make the approach less than ideal.NMR spin couplings or J-couplings have been used as structure constraints in organic and other types of molecules for more than six decades. The dihedral angle dependence of vicinal (three-bond) 1H-1H spin couplings (3JHH) first described by Karplus led to an explosion of applications for a wide range of conformational problems. Other vicinal J-couplings (e.g., 3JCCOP, 3JHCOP, and 3JCOCH) have been found to exhibit similar dihedral angle dependencies. 3J values have been used to assign the preferred conformation in molecules that are conformationally homogeneous. However, many molecules, particularly those in biological systems, are conformationally flexible, which complicates structural interpretations of J values in solution. Three-state staggered models are often assumed in order to deconvolute the conformationally averaged J values into conformer populations. While widely applied, this approach assumes highly idealized models of molecular torsion angles that are likely to be poor representations of those found in solution. In addition, this treatment often gives negative populations and neglects the presence of librational averaging of molecular torsion angles.Recent work in this research group has focused on the development of a hybrid experimental-computational method, MA'AT analysis, that provides probability distributions of molecular torsion angles in solution that can be superimposed on those obtained by MD. Ensembles of redundant NMR spin couplings, including 3J (vicinal), 2J (geminal), and sometimes 1J (direct) values, are used in conjunction with circular statistics to provide single- and multistate models of these angles. MA'AT analysis provides accurate mean torsion angles and circular standard deviations (CSDs) of each mean angle that describe the librational motion about the angle. Both conformational equilibria and dynamics are revealed by the method. In this Account, the salient features of MA'AT analysis are discussed, including some applications to conformational problems involving saccharides and peptides.

One-bond 13C-13C spin-coupling constants in saccharides: a comparison of experimental and calculated values by density functional theory using solid-state 13C NMR and X-ray crystallography.

Methyl aldohexopyranosides were 13C-labeled at contiguous carbons, crystallized, and studied by single-crystal X-ray crystallography and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy to examine the degree to which density functional theory (DFT) can calculate one-bond 13C-13C spin-coupling constants (1JCC) in saccharides with sufficient accuracy to permit their use in MA'AT analysis, a newly-reported hybrid DFT/NMR method that provides probability distributions of molecular torsion angles in solution (Zhang et al., J. Phys. Chem. B, 2017, 121, 3042-3058; Meredith et al., J. Chem. Inf. Model., 2022, 62, 3135-3141). Experimental 1JCC values in crystalline samples of the doubly 13C-labeled compounds were measured by solid-state 13C NMR and compared to those calculated from five different DFT models: (1) 1JCC values calculated from single structures identical to those observed in crystalline samples by X-ray crystallography (all atom refinement); (2) 1JCC values calculated from the single structures in (1) but after Hirshfeld atom refinement (HAR); (3) 1JCC values calculated from the single structures in (1) after DFT-optimization of hydrogen atoms only; and (4 and 5) 1JCC values calculated in rotamers of torsion angle θ2 (C1-C2-O2-O2H) or ω (C4-C5-C6-O6) from which either specific or generalized parameterized equations were obtained and used to calculate 1JCC values in the specific θ2 or ω rotamers observed in crystalline samples. Good qualitative agreement was observed between calculated 1JCC values and those measured by solid-state 13C NMR regardless of the DFT model, but in no cases were calculated 1JCC values quantitative, differing (over-estimated) on average by 4-5% from experimental values. These findings, and those reported recently from solution NMR studies (Tetrault et al., J. Phys. Chem. B 2022, 126, 9506-9515), indicate that improvements in DFT calculations are needed before calculated 1JCC values can be used directly as reliable constraints in MA'AT analyses of saccharides in solution.

One-Bond 13C-1H and 13C-13C Spin-Coupling Constants as Constraints in MA'AT Analysis of Saccharide Conformation.

MA'AT analysis uses ensembles of redundant experimental NMR spin-coupling constants, parametrized J-coupling equations obtained from density functional theory (DFT) calculations, and circular statistics to produce probability distributions of molecular torsion angles in solution and information on librational motions about these angles (Meredith et al., J. Chem. Info. Model. 2022, 62, 3135-3141). Current DFT methods give nearly quantitative two- and three-bond JHH, JCH, and 1JCC values for use in MA'AT analysis of saccharides. In contrast, the accuracy of DFT-calculated one-bond 1JCH and 1JCC values is more difficult to determine, preventing their use in MA'AT modeling. This report describes experimental and computational studies that address this problem using two approaches (Strategies 1 and 2). Differences [1JCHcalc - 1JCHexp] (Strategy 1) ranged from -1.2 to 2.5 Hz, giving an average difference of 0.8 ± 1.7 Hz. Percent differences ranged from -0.8% to 1.6%, giving an average % difference of 0.5 ± 1.1%. In comparison, [1JCHMA'AT - 1JCHexp] (Strategy 2) ranged from -1.8 to 0.2 Hz, giving an average difference of -1.2 ± 0.7 Hz. Percent differences ranged from -1.2% to 0.1%, giving an average % difference of -0.8 ± 0.5%. Strategy 1 gave an average difference of 2.1 Hz between calculated and experimental 1JCC values, with an average % difference of 5.1 ± 0.2%. Calculated 1JCC values were consistently larger than experimental values. Strategy 2 also gave calculated 1JCC values that were larger than the experimental values, with an average difference of 2.3 ± 0.6 Hz, and an average % difference of 5.6 ± 1.6%. The findings of both strategies are similar and indicate that 1JCH values in saccharides can be calculated nearly quantitatively, but 1JCC values appear to be consistently overestimated by ∼5% using current DFT methods.

Nonconventional NMR Spin-Coupling Constants in Oligosaccharide Conformational Modeling: Structural Dependencies Determined from Density Functional Theory Calculations.

Nonconventional NMR spin-coupling constants were investigated to determine their potential as conformational constraints in MA'AT modeling of the O-glycosidic linkages of oligosaccharides. Four (1 J C1',H1', 1 J C1',C2', 2 J C1',H2', and 2 J C2',H1') and eight (1 J C4,H4, 1 J C3,C4, 1 J C4,C5, 2 J C3,H4, 2 J C4,H3, 2 J C5,H4, 2 J C4,H5, and 2 J C3,C5) spin-couplings in methyl ß-d-galactopyranosyl-(1→4)-ß-d-glucopyranoside (methyl ß-lactoside) were calculated using density functional theory (DFT) to determine their dependencies on O-glycosidic linkage C-O torsion angles, ϕ and ψ, respectively. Long-range 4 J H1',H4 was also examined as a potential conformational constraint of either ϕ or ψ. Secondary effects of exocyclic (hydroxyl) C-O bond rotation within or proximal to these coupling pathways were investigated. Based on the findings of methyl ß-lactoside, analogous J-couplings were studied in five additional two-bond O-glycosidic linkages [ßGlcNAc-(1→4)-ßMan, 2-deoxy-ßGlc-(1→4)-ßGlc, αMan-(1→3)-ßMan, αMan-(1→2)-αMan, and ßGlcNAc(1→2)-αMan] to determine whether the coupling behaviors observed in methyl ß-lactoside were more broadly observed. Of the 13 nonconventional J-couplings studied, 7 exhibit properties that may be useful in future MA'AT modeling of O-glycosidic linkages, none of which involve coupling pathways that include the linkage C-O bonds. The findings also provide new insights into the general effects of exocyclic C-O bond conformation on the magnitude of experimental spin-couplings in saccharides and other hydroxyl-containing molecules.

N-Acetyl Side-Chain Conformation in Saccharides: Solution Models Obtained from MA'AT Analysis.

MA'AT analysis has been applied to model the conformational properties of N-acetyl side-chains in biologically important GlcNAc and ManNAc monosaccharides and in a ßGlcNAc-(1→4)-ßGlcNAc disaccharide. Density functional theory calculations were conducted to obtain parameterized equations that relate the magnitudes and signs of 10 spin-coupling constants to conformations of the C2-N2 bonds of GlcNAc and ManNAc. Six of these equations were used with experimental J-couplings, measured in H2O/2H2O and DMSO-d6 solvents in selectively 13C-labeled compounds, to model the C1-C2-N2-C1' torsion angle (θ1) in GlcNAc and ManNAc residues. MA'AT analysis gave mean values of θ1 of 106° for αGlcNAc and ∼116° for ßGlcNAc residues, with circular standard deviations (CSDs) of 21-22° in aqueous solution, in excellent agreement with those obtained by aqueous molecular dynamics (MD) simulation. Parameter space plots revealed unique MA'AT fits of the data, and root mean squared deviations (<0.2 Hz) were twofold smaller than those back-calculated from MD models, indicating that the MA'AT models better fit the experimental J-couplings. Context effects on both θ1 values were found to be small in a ßGlcNAc-(1→4)-ßGlcNAc disaccharide. MA'AT analysis gave a mean value of θ1 of 249° for αManNAc in H2O/2H2O, with a CSD of ∼19°, with both values in good agreement with MD. MA'AT models of N-acetyl side-chains are similar to those obtained previously for O-acetyl side-chains (J. Phys. Chem. B 2017, 121, 66-77). Both O- and N-acetylation conformationally constrain the C-O or C-N bonds relative to the same bonds in unsubstituted compounds. The present work confirms the ability of MA'AT analysis to reveal relatively small changes in mean molecular torsion angles in solution and provides additional evidence of the method as an experimental tool complementary to MD simulation.

MA'AT: A Web-Based Application to Determine Rotamer Population Distributions in Solution from Nuclear Magnetic Resonance Spin-Coupling Constants.

A hybrid experimental-computational method to determine conformational equilibria of molecules in solution has been developed based on the use of redundant nuclear magnetic resonance (NMR) spin-spin coupling constants (spin-couplings; J-couplings), density functional theory (DFT) calculations, and circular statistics. The mathematics that underpins the method, known as MA'AT analysis, is presented, and key components of a computer program that applies this algorithm are discussed. The method was tested using single-state and multi-state models to identify the factors required to obtain reliable results, to establish the limitations of the method, and to highlight techniques to evaluate the uniqueness of solution.

D-Mannosamine hydrochloride (2-amino-2-deoxy-D-mannose hydrochloride): ionic hydrogen bonding in saccharides involving chloride and aminium ions.

D-Mannosamine hydrochloride (2-amino-2-deoxy-D-mannose hydrochloride), C6H14NO5+·Cl-, (I), crystallized from a methanol/ethyl acetate/n-hexane solvent mixture at room temperature in a 4C1 chair conformation that is slightly distorted towards the C3,O5B form. A comparison of the structural parameters of (I) with the corresponding parameters in α-D-glucosamine hydrochloride, (II), and ß-D-galactosamine hydrochloride, (III)/(III'), was undertaken to evaluate the effects of ionic hydrogen bonding on structural properties. Three types of ionic hydrogen bonds are present in the crystals of (I)-(III)/(III'), i.e. N+-H...O, N+-H...Cl-, and O-H...Cl-. The exocyclic structural parameters in (I), (II), and (III)/(III') appear to be most influenced by this bonding, especially the exocyclic hydroxy groups, which adopt eclipsed conformations enabled by ionic hydrogen bonding to the chloride anion. Anomeric disorder was observed in crystals of (I), with an α:ß ratio of 37:63. However, anomeric configuration appears to exert minimal structural effects; that is, bond lengths, bond angles, and torsion angles are essentially identical in both anomers. The observed disorder at the anomeric C atom of (I) appears to be caused by the presence of the chloride anion and atom O3 or O4 in proximal voids, which provide opportunities for hydrogen bonding to atom O1 in both axial and equatorial orientations.

MA'AT Analysis of Aldofuranosyl Rings: Unbiased Modeling of Conformational Equilibria and Dynamics in Solution.

MA'AT analysis has been applied to methyl ß-d-ribofuranoside (3) and methyl 2-deoxy-ß-d-erythro-pentofuranoside (4) to demonstrate the ability of this new experimental method to determine multi-state conformational equilibria in solution. Density functional theory (DFT) was used to obtain parameterized equations for >20 NMR spin-coupling constants sensitive to furanose ring conformation in 3 and 4, and these equations were used in conjunction with experimental spin-couplings to produce unbiased MA'AT models of ring pseudorotation. These models describe two-state north-south conformational exchange consistent with results obtained from traditional treatments of more limited sets of NMR spin-couplings (e.g., PSEUROT). While PSEUROT, MA'AT, and aqueous molecular dynamics models yielded similar two-state models, MA'AT analysis gives more reliable results since significantly more experimental observables are employed compared to PSEUROT, and no assumptions are needed to render the fitting tractable. MA'AT models indicate a roughly equal distribution of north and south ring conformers of 4 in aqueous (2H2O) solution compared to ∼80% north forms for 3. Librational motion about the mean pseudorotation phase angles P of the preferred north and south conformers of 3 in solution is more constrained than that for 4. The greater rigidity of the ß-ribo ring may be caused by synergistic stereoelectronic effects and/or noncovalent (e.g., hydrogen-bonding) interactions in solution that preferentially stabilize north forms of 3. MA'AT analysis of oligonucleotides and other furanose ring-containing biomolecules promises to improve current experimental models of sugar ring behavior in solution and help reveal context effects on ring conformation in more complex biologically important systems.

Two-bond 13C-13C spin-coupling constants in saccharides: dependencies on exocyclic hydroxyl group conformation.

Seven doubly 13C-labeled isotopomers of methyl ß-D-glucopyranoside, methyl ß-D-xylopyranoside, methyl ß-D-galactopyranoside, methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside and methyl ß-D-galactopyranosyl-(1→4)-ß-D-xylopyranoside were prepared, crystallized, and studied by single-crystal X-ray crystallography and solid-state 13C NMR spectroscopy to determine experimentally the dependence of 2JC1,C3 values in aldopyranosyl rings on the C1-C2-O2-H torsion angle, θ2, involving the C2 carbon of the C1-C2-C3 coupling pathway. Using X-ray crystal structures to determine θ2 in crystalline samples and by selecting compounds that exhibit a relatively wide range of θ2 values in the crystalline state, 2JC1,C3 values measured in crystalline samples were plotted against θ2 and the resulting plot compared to that obtained from density functional theory (DFT) calculations. For θ2 values ranging from ∼90° to ∼240°, very good agreement was observed between the experimental and theoretical plots, providing strong validation of DFT-calculated spin-coupling dependencies on exocyclic C-O bond conformation involving the central carbon of geminal C-C-C coupling pathways. These findings provide new experimental evidence supporting the use of 2JCCC values as non-conventional spin-coupling constraints in MA'AT conformational modeling of saccharides in solution, and the use of NMR spin-couplings not involving coupled hydroxyl hydrogens as indirect probes of C-O bond conformation. Solvomorphism was observed in crystalline ßGal-(1→4)-ßGlcOCH3 wherein the previously-reported methanol solvate form was found to spontaneously convert to a monohydrate upon air-drying, leading to small but discernible conformational changes in, and a new crystalline form of, this disaccharide.

Methyl ß-lactoside [methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside] monohydrate: a solvomorphism study.

Methyl ß-lactoside [methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside] monohydrate, C13H24O11·H2O, (I), was obtained via spontaneous transformation of methyl ß-lactoside methanol solvate, (II), during air-drying. Cremer-Pople puckering parameters indicate that the ß-D-Galp (ß-D-galactopyranosyl) and ß-D-Glcp (ß-D-glucopyranosyl) rings in (I) adopt slightly distorted 4C1 chair conformations, with the former distorted towards a boat form (BC1,C4) and the latter towards a twist-boat form (O5SC2). Puckering parameters for (I) and (II) indicate that the conformation of the ßGalp ring is slightly more affected than the ßGlcp ring by the solvomorphism. Conformations of the terminal O-glycosidic linkages in (I) and (II) are virtually identical, whereas those of the internal O-glycosidic linkage show torsion-angle changes of 6° in both C-O bonds. The exocyclic hydroxymethyl group in the ßGalp residue adopts a gt conformation (C4' anti to O6') in both (I) and (II), whereas that in the ßGlcp residue adopts a gg (gauche-gauche) conformation (H5 anti to O6) in (II) and a gt (gauche-trans) conformation (C4 anti to O6) in (I). The latter conformational change is critical to the solvomorphism in that it allows water to participate in three hydrogen bonds in (I) as opposed to only two hydrogen bonds in (II), potentially producing a more energetically stable structure for (I) than for (II). Visual inspection of the crystalline lattice of (II) reveals channels in which methanol solvent resides and through which solvent might exchange during solvomorphism. These channels are less apparent in the crystalline lattice of (I).

Isopropyl 3-deoxy-α-D-ribo-hexopyranoside (isopropyl 3-deoxy-α-D-glucopyranoside): evaluating trends in structural parameters.

Isopropyl 3-deoxy-α-D-ribo-hexopyranoside (isopropyl 3-deoxy-α-D-glucopyranoside), C9H18O5, (I), crystallizes from a methanol-ethyl acetate solvent mixture at room temperature in a 4C1 chair conformation that is slightly distorted towards the C5SC1 twist-boat form. A comparison of the structural parameters in (I), methyl α-D-glucopyranoside, (II), α-D-glucopyranosyl-(1→4)-D-glucitol (maltitol), (III), and 3-deoxy-α-D-ribo-hexopyranose (3-deoxy-α-D-glucopyranose), (IV), shows that most endocyclic and exocyclic bond lengths, valence bond angles and torsion angles in the aldohexopyranosyl rings are more affected by anomeric configuration, aglycone structure and/or the conformation of exocyclic substituents, such as hydroxymethyl groups, than by monodeoxygenation at C3. The structural effects observed in the crystal structures of (I)-(IV) were confirmed though density functional theory (DFT) calculations in computed structures (I)c-(IV)c. Exocyclic hydroxymethyl groups adopt the gauche-gauche (gg) conformation (H5 anti to O6) in (I) and (III), and the gauche-trans (gt) conformation (C4 anti to O6) in (II) and (IV). The O-glycoside linkage conformations in (I) and (III) resemble those observed in disaccharides containing ß-(1→4) linkages.

Reconciling MA'AT and molecular dynamics models of linkage conformation in oligosaccharides.

MA'AT conformational models of the phi torsion angles of O-glycosidic linkages differ from those obtained from MD simulation. To determine the source of the discrepancy, MA'AT analyses were performed using DFT-derived equations obtained with and without psi constraints. The resulting phi models were essentially the same, indicating a force-field problem. Circular standard deviations (CSDs) were found to provide reliable estimates of torsional averaging.

Glycosidic linkage, N-acetyl side-chain, and other structural properties of methyl 2-acetamido-2-deoxy-ß-D-glucopyranosyl-(1→4)-ß-D-mannopyranoside monohydrate and related compounds.

The crystal structure of methyl 2-acetamido-2-deoxy-ß-D-glycopyranosyl-(1→4)-ß-D-mannopyranoside monohydrate, C15H27NO11·H2O, was determined and its structural properties compared to those in a set of mono- and disaccharides bearing N-acetyl side-chains in ßGlcNAc aldohexopyranosyl rings. Valence bond angles and torsion angles in these side chains are relatively uniform, but C-N (amide) and C-O (carbonyl) bond lengths depend on the state of hydrogen bonding to the carbonyl O atom and N-H hydrogen. Relative to N-acetyl side chains devoid of hydrogen bonding, those in which the carbonyl O atom serves as a hydrogen-bond acceptor display elongated C-O and shortened C-N bonds. This behavior is reproduced by density functional theory (DFT) calculations, indicating that the relative contributions of amide resonance forms to experimental C-N and C-O bond lengths depend on the solvation state, leading to expectations that activation barriers to amide cis-trans isomerization will depend on the polarity of the environment. DFT calculations also revealed useful predictive information on the dependencies of inter-residue hydrogen bonding and some bond angles in or proximal to ß-(1→4) O-glycosidic linkages on linkage torsion angles φ and ψ. Hypersurfaces correlating φ and ψ with the linkage C-O-C bond angle and total energy are sufficiently similar to render the former a proxy of the latter.

A convenient synthesis of short-chain α-(1 → 2) mannopyranosyl oligosaccharides.

Sugar 1,2-orthoesters are by-products of chemical glycosylation reactions that can be subsequently rearranged in situ to give trans glycosides. They have been used as donors in the synthesis of the latter glycosides with good regio- and stereo-selectivity. Alkyl α-(1 â†’ 2) linked mannopyranosyl disaccharides have been reported as the major products from the rearrangement of mannopyranosyl orthoesters. Recent studies in this laboratory have shown that α-(1 â†’ 2) linked mannopyranosyl di-, tri- and tetrasaccharides can be obtained in one step from mannopyranosyl allyl orthoester under optimized reaction conditions. In addition to the expected mono- and disaccharides (56%), allyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl-(1 â†’ 2)-3,4,6-tri-O-acetyl-α-d-mannopyranosyl-(1 â†’ 2)-tri-O-acetyl-α-d-mannopyranoside and allyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl-(1 â†’ 2)-3,4,6-tri-O-acetyl-α-d-mannopyranosyl-(1 â†’ 2)-3,4,6-tri-O-acetyl-α-d-mannopyranosyl-(1 â†’ 2)-3,4,6-tri-O-acetyl-α-d-mannopyranoside were obtained in 23% and 6% isolated yields, respectively, from the oligomerization of a ß-d-mannopyranosyl allyl 1,2-orthoester, along with small amounts of higher DP oligomers. Possible mechanisms for the oligomerization and side reactions are proposed based on NMR and mass spectrometric data.

13C-13C spin-coupling constants in crystalline 13C-labeled saccharides: conformational effects interrogated by solid-state 13C NMR spectroscopy.

Solid-state 13C NMR spectroscopy has been used in conjunction with selectively 13C-labeled mono- and disaccharides to measure 13C-13C spin-couplings (JCC) in crystalline samples. This experimental approach allows direct correlation of JCC values with specific molecular conformations since, in crystalline samples, molecular conformation is essentially static and can be determined by X-ray crystallography. JCC values measured in the solid-state in known molecular conformations can then be compared to corresponding JCC values calculated in the same conformations using density functional theory (DFT). The latter comparisons provide important validation of DFT-calculated J-couplings, which is not easily obtained by other approaches and is fundamental to obtaining reliable experiment-based conformational models from redundant J-couplings by MA'AT analysis. In this study, representative 1JCC, 2JCCC and 3JCOCC values were studied as either intra-residue couplings in the aldohexopyranosyl rings of monosaccharides or inter-residue (trans-glycoside) couplings in disaccharides. The results demonstrate that (a) accurate JCC values can be measured in crystalline saccharides that have been suitably labeled with 13C, and (b) DFT-calculated JCC values compare favorably with those determined by solid-state 13C NMR when molecular conformation is a constant in both determinations.

Structural properties of D-mannopyranosyl rings containing O-acetyl side-chains.

The crystal structures of 1,2,3,4,6-penta-O-acetyl-α-D-mannopyranose, C16H22O11, and 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl-(1→3)-1,2,4,6-tetra-O-acetyl-α-D-mannopyranose, C40H54O27, were determined and compared to those of methyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside, methyl α-D-mannopyranoside and methyl α-D-mannopyranosyl-(1→2)-α-D-mannopyranoside to evaluate the effects of O-acetylation on bond lengths, bond angles and torsion angles. In general, O-acetylation exerts little effect on the exo- and endocyclic C-C and endocyclic C-O bond lengths, but the exocyclic C-O bonds involved in O-acetylation are lengthened by ∼0.02 Å. The conformation of the O-acetyl side-chains is highly conserved, with the carbonyl O atom either eclipsing the H atom attached to a 2°-alcoholic C atom or bisecting the H-C-H bond angle of a 1°-alcoholic C atom. Of the two C-O bonds that determine O-acetyl side-chain conformation, that involving the alcoholic C atom exhibits greater rotational variability than that involving the carbonyl C atom. These findings are in good agreement with recent solution NMR studies of O-acetyl side-chain conformations in saccharides. Experimental evidence was also obtained to confirm density functional theory (DFT) predictions of C-O and O-H bond-length behavior in a C-O-H fragment involved in hydrogen bonding.

Conformational analysis of the disaccharide methyl α-D-mannopyranosyl-(1→3)-2-O-acetyl-ß-D-mannopyranoside monohydrate.

The crystal structure of methyl α-D-mannopyranosyl-(1→3)-2-O-acetyl-ß-D-mannopyranoside monohydrate, C15H26O12·H2O, (II), has been determined and the structural parameters for its constituent α-D-mannopyranosyl residue compared with those for methyl α-D-mannopyranoside. Mono-O-acetylation appears to promote the crystallization of (II), inferred from the difficulty in crystallizing methyl α-D-mannopyranosyl-(1→3)-ß-D-mannopyranoside despite repeated attempts. The conformational properties of the O-acetyl side chain in (II) are similar to those observed in recent studies of peracetylated mannose-containing oligosaccharides, having a preferred geometry in which the C2-H2 bond eclipses the C=O bond of the acetyl group. The C2-O2 bond in (II) elongates by ∼0.02 Šupon O-acetylation. The phi (φ) and psi (ψ) torsion angles that dictate the conformation of the internal O-glycosidic linkage in (II) are similar to those determined recently in aqueous solution by NMR spectroscopy for unacetylated (II) using the statistical program MA'AT, with a greater disparity found for ψ (Δ = ∼16°) than for φ (Δ = ∼6°).