Drug resistant Mycobacterium tuberculosis strains have altered cell envelope hydrophobicity that influences infection outcomes in human macrophages.

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is considered one of the top infectious killers in the world. In recent decades, drug resistant (DR) strains of M.tb have emerged that make TB even more difficult to treat and pose a threat to public health. M.tb has a complex cell envelope that provides protection to the bacterium from chemotherapeutic agents. Although M.tb cell envelope lipids have been studied for decades, very little is known about how their levels change in relation to drug resistance. In this study, we examined changes in the cell envelope lipids [namely, phthiocerol dimycocerosates (PDIMs)], glycolipids [phosphatidyl-myo-inositol mannosides (PIMs)], and the PIM associated lipoglycans [lipomannan (LM); mannose-capped lipoarabinomannan (ManLAM)] of 11 M.tb strains that range from drug susceptible (DS) to multi-drug resistant (MDR) to pre-extensively drug resistant (pre-XDR). We show that there was an increase in the PDIMs:PIMs ratio as drug resistance increases, and provide evidence of PDIM species only present in the DR-M.tb strains studied. Overall, the LM and ManLAM cell envelope levels did not differ between DS- and DR-M.tb strains, but ManLAM surface exposure proportionally increased with drug resistance. Evaluation of host-pathogen interactions revealed that DR-M.tb strains have decreased association with human macrophages compared to DS strains. The pre-XDR M.tb strain with the largest PDIMs:PIMs ratio had decreased uptake, but increased intracellular growth rate at early time points post-infection when compared to the DS-M.tb strain H37Rv. These findings suggest that PDIMs may play an important role in drug resistance and that this observed increase in hydrophobic cell envelope lipids on the DR-M.tb strains studied may influence M.tb-host interactions.

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).

Age associated susceptibility to SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still an ongoing global health crisis. Clinical data indicate that the case fatality rate (CFR) is age dependent, with a higher CFR percentage in the elderly population. We compared the pathogenesis of SARS-CoV-2 in young and aged K18-hACE2 transgenic mice. We evaluated morbidity, mortality, viral titers, immune responses, and histopathology in SARS-CoV-2-infected young and old K18-hACE2 transgenic mice. Within the limitation of having a low number of mice per group, our results indicate that SARS-CoV-2 infection resulted in slightly higher morbidity, mortality, and viral replication in the lungs of old mice, which was associated with an impaired IgM response and altered cytokine and chemokine profiles. Results of this study increase our understanding of SARS-CoV-2 infectivity and immuno-pathogenesis in the elderly population.

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.

Serum biomarkers associated with aging and neurodegeneration in common marmosets (Callithrix jacchus).

The common marmoset (Callithrix jacchus), a small South American monkey, is an important nonhuman primate model in the study of aging and age-related neurodegenerative disease, including Alzheimer's disease, Parkinson's disease, and related dementias. Thorough characterization of the wild type marmoset brain agingmodel, including biomarkers of aging and neural degeneration, will further the marmoset's utility in translational research. We measured serum concentration of four key biomarkers of neural degeneration [total tau (T-tau), glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), and ubiquitin C-terminal hydrolase-L1 (UCH-L1)] via single molecule array from 24 marmosets (female n = 13, male n = 11) ranging in age from 1.3 to 18.7 years. Aged marmosets (>7 years) had significantly higher GFAP, NfL, UCH-L1, and T-tau than adult marmosets. Sex differences were not detected for any of these biomarker concentrations. These data provide an important initial range of reference values for GFAP, NfL, T-tau, and UCH-L1 to evaluate aging and neural health in marmosets, as well as evaluation of therapeutics in clinical models of disease.

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.

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.

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.

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.

Synthesis and O-Glycosidic Linkage Conformational Analysis of 13C-Labeled Oligosaccharide Fragments of an Antifreeze Glycolipid.

NMR studies of two 13C-labeled disaccharides and a tetrasaccharide were undertaken that comprise the backbone of a novel thermal hysteresis glycolipid containing a linear glycan sequence of alternating [ßXyl p-(1→4)-ßMan p-(1→4)] n dimers. Experimental trans-glycoside NMR J-couplings, parameterized equations obtained from density functional theory (DFT) calculations, and an in-house circular statistics package ( MA'AT) were used to derive conformational models of linkage torsion angles ϕ and ψ in solution, which were compared to those obtained from molecular dynamics simulations. Modeling using different probability distribution functions showed that MA'AT models of ϕ in ßMan(1→4)ßXyl and ßXyl(1→4)ßMan linkages are very similar in the disaccharide building blocks, whereas MA'AT models of ψ differ. This pattern is conserved in the tetrasaccharide, showing that linkage context does not influence linkage geometry in this linear system. Good agreement was observed between the MA'AT and MD models of ψ with respect to mean values and circular standard deviations. Significant differences were observed for ϕ, indicating that revision of the force-field employed by GLYCAM is probably needed. Incorporation of the experimental models of ϕ and ψ into the backbone of an octasaccharide fragment leads to a helical amphipathic topography that may affect the thermal hysteresis properties of the glycolipid.

Use of Circular Statistics To Model αMan-(1→2)-αMan and αMan-(1→3)-α/ßMan O-Glycosidic Linkage Conformation in 13C-Labeled Disaccharides and High-Mannose Oligosaccharides.

A new experimental method, MA' AT analysis, has been applied to investigate the conformational properties of O-glycosidic linkages in several biologically important mannose-containing di- and oligosaccharides. Methyl α-d-mannopyranosyl-(1→2)-α-d-mannopyranoside (2), methyl α-d-mannopyranosyl-(1→3)-α-d-mannopyranoside (3), and methyl α-d-mannopyranosyl-(1→3)-ß-d-mannopyranoside (4) were prepared with selective 13C-enrichment to enable the measurement of NMR scalar couplings across their internal O-glycosidic linkages. Density functional theory (DFT) was used to parameterize equations for JCH and JCC values in 2-4 that are sensitive to phi (ϕ) and psi (ψ). The experimental J-couplings and parameterized equations were treated using a circular statistics algorithm encoded in the MA' AT program. Conformations about ϕ and ψ treated using single-state von Mises models gave excellent fits to the ensembles of redundant J-couplings. Mean values and circular standard deviations (CSDs) for each linkage torsion angle ϕ (CSD) and ψ (CSD) in 2, -29° (25°) and 20° (22°); in 3, -36° (36°) and 8° (27°); in 4, -37° (34°) and 10° (26°); ϕ = H1'-C1'-O1'-CX and ψ = C1'-O1'-CX-HX (CX = aglycone carbon) were compared to histograms obtained from 1 µs aqueous molecular dynamics (MD) simulations and X-ray database statistical analysis. MA' AT-derived models of ψ were in very good agreement with the MD and X-ray data, but not those of ϕ, suggesting a need for force field revision. The effect of structural context on linkage conformation was also investigated in four selectively 13C-labeled homomannose tri- and tetrasaccharides using the MA' AT method. In the cases examined, context effects were found to be small.