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.

Rapid assembly of branched mannose oligosaccharides through consecutive regioselective glycosylation: A convergent and efficient strategy.

A convergent and efficient strategy for the synthesis of high-mannose oligosaccharides is described wherein regioselective glycosylations between trichloroacetimidate donors and partially protected acceptors are employed to reduce the number of protection-deprotection steps. Two representative branched mannose oligosaccharides, a mannose heptasaccharide (Man7) and a mannose nonasaccharide (Man9) were constructed via (4+3) and (5+4) glycosylations, respectively. These mannose-containing oligosaccharides were obtained in nine steps in ~25% overall yield and >98% purity on 60-70 mg scales to demonstrate the effectiveness of the strategy.

A chemical synthesis of a multiply 13 C-labeled hexasaccharide: a high-mannose N-glycan fragment.

As covalent modifiers of proteins, high-mannose N-glycans are important in maintaining protein structure and function in vivo. The conformations of these glycans can be studied by nuclear magnetic resonance spectroscopy using spin-spin couplings (J-couplings; scalar couplings) and other nuclear magnetic resonance parameters that are sensitive to the geometries of their constituent glycosidic linkages and other mobile elements in their structures. These analyses often require 13 C-labeling at specific carbon atoms, especially when measurements of 13 C-13 C J-couplings are of interest. The selection of particular 13 C isotopomers of a glycan depends on the type of question under scrutiny. A chemical synthesis of a mannose-containing hexasaccharide, α[1-13 C]Man(1→2)α[1,2-13 C2 ]Man(1→6)[α[1-13 C]Man(1→2)α[1,2-13 C2 ]Man(1→3)]α[1,2-13 C2 ]Man(1→6)ßManOCH3 , which is a nested fragment of the high-mannose N-glycans of human glycoproteins and contains eight 13 C-enriched carbon sites, is described in this report. The selected 13 C isotopomer was chosen to maximize the measurement of J-couplings sensitive to linkage conformations. This work demonstrates that chemical syntheses of multiply 13 C-labeled oligosaccharides are technically feasible and practical using present synthetic methods. The availability of this and other multiply 13 C-labeled mannose-containing oligosaccharides will promote future studies of their conformations in solution and in the bound state.

ET-MSF: a model stacking framework to identify electron transport proteins.

INTRODUCTION: The electron transport chain is closely related to cellular respiration and has been implicated in various human diseases. However, the traditional "wet" experimental method is time consuming. Therefore, it is key to identify electron transport proteins by computational methods. Many approaches have been proposed, but performance of them still has room for further improvement. Methodological issues: In our study, we propose a model stacking framework, which combines multiple base models. The protein features are extracted via PsePSSM from protein sequences. Features are fed into the base model including support vector machines (SVM), random forest (RF), XGBoost, etc. The results of base model are entered into logistic regression model for final process. RESULTS: On the independent dataset, the accuracy and Matthew's correlation coefficient (MCC) of proposed method are 95.70% and 0.8756, respectively. Furthermore, we show that the model stacking framework outperforms single machine learning classifiers statistically. CONCLUSION: Our models are better than most known strategies for identifying electron transport proteins. Our model can be used to more precisely identify electron transport proteins.

Identifying and Classifying Enhancers by Dinucleotide-Based Auto-Cross Covariance and Attention-Based Bi-LSTM.

Enhancers are a class of noncoding DNA elements located near structural genes. In recent years, their identification and classification have been the focus of research in the field of bioinformatics. However, due to their high free scattering and position variability, although the performance of the prediction model has been continuously improved, there is still a lot of room for progress. In this paper, density-based spatial clustering of applications with noise (DBSCAN) was used to screen the physicochemical properties of dinucleotides to extract dinucleotide-based auto-cross covariance (DACC) features; then, the features are reduced by feature selection Python toolkit MRMD 2.0. The reduced features are input into the random forest to identify enhancers. The enhancer classification model was built by word2vec and attention-based Bi-LSTM. Finally, the accuracies of our enhancer identification and classification models were 77.25% and 73.50%, respectively, and the Matthews' correlation coefficients (MCCs) were 0.5470 and 0.4881, respectively, which were better than the performance of most predictors.

Cryoprotectant biosynthesis and the selective accumulation of threitol in the freeze-tolerant Alaskan beetle, Upis ceramboides.

Adult Upis ceramboides do not survive freezing in the summer but tolerate freezing to -60 degrees C in midwinter. The accumulation of two cryoprotective polyols, sorbitol and threitol, is integral to the extraordinary cold-hardiness of this beetle. U. ceramboides are the only animals known to accumulate high concentrations of threitol; however, the biosynthetic pathway has not been studied. A series of (13)C-labeled compounds was employed to investigate this biosynthetic pathway using (13)C{(1)H} NMR spectroscopy. In vivo metabolism of (13)C-labeled glucose isotopomers demonstrates that C-3-C-6 of glucose become C-1-C-4 of threitol. This labeling pattern is expected for 4-carbon saccharides arising from the pentose phosphate pathway. In vitro experiments show that threitol is synthesized from erythrose 4-phosphate, a C(4) intermediate in the PPP. Erythrose 4-phosphate is epimerized and/or isomerized to threose 4-phosphate, which is subsequently reduced by a NADPH-dependent polyol dehydrogenase and dephosphorylated by a sugar phosphatase to form threitol. Threitol 4-phosphate appears to be the preferred substrate of the sugar phosphatase(s), promoting threitol synthesis over that of erythritol. In contrast, the NADPH-dependent polyol dehydrogenase exhibits broad substrate specificity. Efficient erythritol catabolism under conditions that promote threitol synthesis, coupled with preferential threitol biosynthesis, appear to be responsible for the accumulation of high concentrations of threitol (250 mm) without concomitant accumulation of erythritol.

Methyl 4-O-beta-D-galactopyranosyl alpha-D-mannopyranoside methanol 0.375-solvate.

Methyl beta-D-galactopyranosyl-(1-->4)-alpha-D-mannopyranoside methanol 0.375-solvate, C(13)H(24)O(11).0.375CH(3)OH, (I), was crystallized from a methanol-ethanol solvent system in a glycosidic linkage conformation, with varphi' (O5(Gal)-C1(Gal)-O1(Gal)-C4(Man)) = -68.2 (3) degrees and psi' (C1(Gal)-O1(Gal)-C4(Man)-C5(Man)) = -123.9 (2) degrees , where the ring is defined by atoms O5/C1-C5 (monosaccharide numbering); C1 denotes the anomeric C atom and C6 the exocyclic hydroxymethyl C atom in the betaGalp and alphaManp residues, respectively. The linkage conformation in (I) differs from that in crystalline methyl alpha-lactoside [methyl beta-D-galactopyranosyl-(1-->4)-alpha-D-glucopyranoside], (II) [Pan, Noll & Serianni (2005). Acta Cryst. C61, o674-o677], where varphi' is -93.6 degrees and psi' is -144.8 degrees . An intermolecular hydrogen bond exists between O3(Man) and O5(Gal) in (I), similar to that between O3(Glc) and O5(Gal) in (II). The structures of (I) and (II) are also compared with those of their constituent residues, viz. methyl alpha-D-mannopyranoside, methyl alpha-D-glucopyranoside and methyl beta-D-galactopyranoside, revealing significant differences in the Cremer-Pople puckering parameters, exocyclic hydroxymethyl group conformations and intermolecular hydrogen-bonding patterns.

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.

O-Benzoyl side-chain conformations in 2,3,4,6-tetra-O-benzoyl-ß-D-galactopyranosyl-(1→4)-1,2,6-tri-O-benzoyl-ß-D-glucopyranose (ethyl acetate solvate) and 1,2,4,6-tetra-O-benzoyl-ß-D-glucopyranose (acetone solvate).

The crystal structures of 2,3,4,6-tetra-O-benzoyl-ß-D-galactopyranosyl-(1→4)-1,2,6-tri-O-benzoyl-ß-D-glucopyranose ethyl acetate hemisolvate, C61H50O18·0.5C4H8O2, and 1,2,4,6-tetra-O-benzoyl-ß-D-glucopyranose acetone monosolvate, C34H28O10·C3H6O, were determined and compared to those of methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside (methyl ß-lactoside) and methyl ß-D-glucopyranoside hemihydrate, C7H14O6·0.5H2O, to evaluate the effects of O-benzoylation on bond lengths, bond angles and torsion angles. In general, O-benzoylation exerts little effect on exo- and endocyclic C-C and endocyclic C-O bond lengths, but exocyclic C-O bonds involved in O-benzoylation are lengthened by 0.02-0.04 Å depending on the site of substitution. The conformation of the O-benzoyl 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 an 1°-alcoholic C atom. Of the three bonds that determine the side-chain geometry, the C-O bond involving the alcoholic C atom exhibits greater rotational variability than the remaining C-O and C-C bonds involving the carbonyl C atom. These findings are in good agreement with recent solution NMR studies of the O-acetyl side-chain conformation in saccharides.

Synthesis of high-mannose oligosaccharides containing mannose-6-phosphate residues using regioselective glycosylation.

Molecular recognition of mannose-6-phosphate (M6P)-modified oligosaccharides by transmembrane M6P receptors is a key signaling event in lysosomal protein trafficking in vivo. Access to M6P-containing high-mannose N-glycans is essential to achieving a thorough understanding of the M6P ligand-receptor recognition process. Herein we report the application of a versatile and reliable chemical strategy to prepare asymmetric di-antennary M6P-tagged high-mannose oligosaccharides in >20% overall yield and in high purity (>98%). Regioselective chemical glycosylation coupled with effective phosphorylation and product purification protocols were applied to rapidly assemble these oligosaccharides. The development of this synthetic strategy simplifies the preparation of M6P-tagged high-mannose oligosaccharides, which will improve access to these compounds to study their structures and biological functions.

O-Acetyl Side-Chains in Monosaccharides: Redundant NMR Spin-Couplings and Statistical Models for Acetate Ester Conformational Analysis.

α- and ß-d-glucopyranose monoacetates 1-3 were prepared with selective 13C enrichment in the O-acetyl side-chain, and ensembles of 13C-1H and 13C-13C NMR spin-couplings (J-couplings) were measured involving the labeled carbons. Density functional theory (DFT) was applied to a set of model structures to determine which J-couplings are sensitive to rotation of the ester bond θ. Eight J-couplings (1JCC, 2JCH, 2JCC, 3JCH, and 3JCC) were found to be sensitive to θ, and four equations were parametrized to allow quantitative interpretations of experimental J-values. Inspection of J-coupling ensembles in 1-3 showed that O-acetyl side-chain conformation depends on molecular context, with flanking groups playing a dominant role in determining the properties of θ in solution. To quantify these effects, ensembles of J-couplings containing four values were used to determine the precision and accuracy of several 2-parameter statistical models of rotamer distributions across θ in 1-3. The statistical method used to generate these models has been encoded in a newly developed program, MA'AT, which is available for public use. These models were compared to O-acetyl side-chain behavior observed in a representative sample of crystal structures, and in molecular dynamics (MD) simulations of O-acetylated model structures. While the functional form of the model had little effect on the precision of the calculated mean of θ in 1-3, platykurtic models were found to give more precise estimates of the width of the distribution about the mean (expressed as circular standard deviations). Validation of these 2-parameter models to interpret ensembles of redundant J-couplings using the O-acetyl system as a test case enables future extension of the approach to other flexible elements in saccharides, such as glycosidic linkage conformation.

Conformational Populations of ß-(1→4) O-Glycosidic Linkages Using Redundant NMR J-Couplings and Circular Statistics.

Twelve disaccharides containing ß-(1→4) linkages and displaying systematic structural variations in the vicinity of these linkages were selectively labeled with 13C to facilitate measurements of multiple NMR spin-spin (scalar; J) coupling constants (JCH and JCC values) across their O-glycosidic linkages. Ensembles of spin-couplings (2JCOC, 3JCOCH, 3JCOCC) sensitive to the two linkage torsion angles, phi (ϕ) and psi (ψ), were analyzed by using parametrized equations obtained from density functional theory (DFT) calculations, Fredholm theory, and circular statistics to calculate experiment-based rotamer populations for ϕ and ψ in each disaccharide. With the statistical program MA'AT, torsion angles ϕ and ψ were modeled as a single von Mises distribution, which yielded two parameters, the mean position and the circular standard deviation (CSD) for each angle. The NMR-derived rotamer populations were compared to those obtained from 1 µs aqueous molecular dynamics (MD) simulations and crystallographic database statistical analyses. Conformer populations obtained exclusively from the MA'AT treatment of redundant J-couplings were in very good agreement with those obtained from the MD simulations, providing evidence that conformational populations can be determined by NMR for mobile molecular elements such as O-glycosidic linkages with minimal input from theory. The approach also provides an experimental means to validate the conformational preferences predicted from MD simulations. The conformational behaviors of ϕ in the 12 disaccharides were very similar, but those of ψ varied significantly, allowing a classification of the 12 disaccharides based on preferred linkage conformation in solution.

DFT and NMR studies of 2JCOH, 3JHCOH, and 3JCCOH spin-couplings in saccharides: C-O torsional bias and H-bonding in aqueous solution.

Density functional theory (DFT) has been used to investigate the structural dependencies of NMR spin-coupling constants (J-couplings) involving the exchangeable hydroxyl protons of saccharides. 3JHCOH, 3JCCOH, and 2JCOH values were calculated at different positions in model aldopyranosyl rings as a function of one or more torsion angles, and results support the use of a generalized Karplus equation to treat 3JHCOH involving the non-anomeric OH groups. The presence of O5 appended to the H1-C1-O1-H coupling pathway introduces asymmetry in 3JH1,O1H Karplus curves due to internal electronegative substituent effects on the gauche couplings, thus requiring separate equations to treat this coupling. 3JCCOH values depend not only on the C-C-O-H torsion angle but also on the orientation of terminal substituents on the coupled carbon, similar to 3JCOCC studied previously (Bose et al., J. Am. Chem. Soc. 1998, 120, 11158-11173). "In-plane" oxygen increased 3JCCOH by approximately 3-4 Hz, whereas "in-plane" carbon gave more modest enhancements ( approximately 1 Hz). Three Karplus equations were derived for non-anomeric 3JCCOH based on the nature and orientation of substituents on the coupled carbon. Like 3JH1,O1H, 3JC2,O1H is also subject to internal electronegative substituent effects on the gauche couplings, thus necessitating separate equations to treat this coupling. 2JCOH values were found not to be useful probes of C-O torsions as a result of their nonsystematic dependence on these torsions. Experimental measurements of 3JHCOH and 3JCCOH in doubly 13C-labeled methyl beta-lactoside 20 and its constituent 13C-labeled methyl aldopyranosides in H2O/acetone-d6 at -20 degrees C showed that some C-O torsion angles are influenced by molecular context and do not experience complete rotational averaging in solution. A strong bias in the H3-C3-O3-H torsion angle in the Glc residue of 20 favoring a gauche conformation suggests the presence of inter-residue H-bonding between O3HGlc and O5Gal. Quantitative analysis of 3JHCOH and 3JCCOH values in 20 indicates that approximately 85% of the forms in solution have geometries consistent with H-bonding. These results suggest that H-bonding between adjacent and/or remote residues may play a role in dictating preferred glycosidic bond conformation in simple and complex oligosaccharides in aqueous solution.

A disaccharide rearrangement catalyzed by molybdate anion in aqueous solution.

Reaction of a galactosylated 2-C-(hydroxymethyl)-tetrofuranose with paramolybdate ion-exchange resin in aqueous solution at 67 degrees C gave an equililibrium mixture containing the reactant aldofuranose (42%) and a 2-ketopentofuranose galactosylated at O1 (58%). Observation of this stereospecific rearrangement supports prior arguments that substituents at C2 of the branched-chain aldofuranose reactant are located in a sterically accessible pocket of the putative dimolybdate-saccharide reactive complex during epimerization. This rearrangement provides a new and convenient route to 2-ketosugars glycosylated at the exocyclic C1 position.

Methyl 4-O-beta-L-galactopyranosyl-beta-D-glucopyranoside (methyl beta-L-lactoside).

Methyl beta-L-lactoside, C13H24O11, (II), is described by glycosidic torsion angles phi (O5Gal-C1Gal-O4Glc-C4Glc) and psi (C1Gal-O1Gal-C4Glc-C5Glc) of 93.89 (13) and -127.43 (13) degrees , respectively, where the ring atom numbering conforms to the convention in which C1 is the anomeric C atom and C6 is the exocyclic hydroxymethyl (CH2OH) C atom in both residues (Gal is galactose and Glc is glucose). Substitution of L-Gal for D-Gal in the biologically relevant disaccharide, methyl beta-lactoside [Stenutz, Shang & Serianni (1999). Acta Cryst. C55, 1719-1721], (I), significantly alters the glycosidic linkage interface. In the crystal structure of (I), one inter-residue (intramolecular) hydrogen bond is observed between atoms H3OGlc and O5Gal. In contrast, in the crystal structure of (II), inter-residue hydrogen bonds are observed between atoms H6OGlc and O5Gal, H6OGlc and O6Gal, and H3OGlc and O2Gal, with H6OGlc serving as a donor with two intramolecular acceptors.

[13C,15N]2-Acetamido-2-deoxy-D-aldohexoses and their methyl glycosides: synthesis and NMR investigations of J-couplings involving 1H, 13C, and 15N.

[reaction: see text] A series of 2-amino-2-deoxy-D-[1-13C]aldohexoses and their methyl glycosides was prepared with use of a simplified cyanohydrin reduction route. Four d-aldopentosylamines (arabino, lyxo, ribo, xylo) were prepared from the corresponding D-aldopentoses by reaction with NH3(g) in MeOH solvent, isolated in solid form, and characterized by 13C and 1H NMR. Hydrolysis of beta-D-xylopyranosylamine was studied using 13C-labeled substrates to establish optimal solution conditions for cyanohydrin formation. Major hydrolytic intermediates were observed and identified by time-lapse 1D and 2D NMR analyses of reaction mixtures. The aldopentosylamines were subsequently employed in cyanohydrin reduction reactions with K13CN to yield C2-epimeric [1-13C]2-aminosugars, which were separated by chromatography on ion-exchange columns. N-Acetylation and methyl glycosidation followed by chromatography gave pure 2-acetamido-2-deoxy-D-[1-13C]aldohexopyranosides. J(CH) and J(CC) spin-spin coupling constants involving the labeled anomeric carbon were measured and compared to those observed previously in methyl D-[1-13C]aldohexopyranosides. In parallel studies, theoretical J-couplings were calculated in model N-acetylated aldopyranosides using density functional theory (DFT) to predict the effect of OH vs NHCOCH(3) substitution at C2 on J(CH) and J(CC) values in aldopyranosyl rings. The synthetic method was also modified to accommodate (15)N- and (13)C-labeling within the N-acetyl side-chain, and some J-couplings involving 1H, 13C, and 15N atoms in 2-[1,2-13C2;15N]acetamido-2-deoxy-D-[1-13C]glucose were measured and interpreted.

Methyl 4-O-beta-D-galactopyranosyl alpha-D-glucopyranoside (methyl alpha-lactoside).

Methyl alpha-lactoside, C13H24O11, (I), is described by glycosidic torsion angles varphi (O5gal-C1gal-O1gal-C4glc) and psi (C1gal-O1gal-C4glc-C5glc), which have values of -93.52 (13) and -144.83 (11) degrees, respectively, where the ring atom numbering conforms to the convention in which C1 is the anomeric C atom and C6 is the exocyclic hydroxymethyl (-CH2OH) C atom in both residues. The linkage geometry is similar to that observed in methyl beta-lactoside methanol solvate, (II), in which varphi is -88.4 (4) degrees and psi is -161.3 (4) degrees. As in (II), an intermolecular O3glc-H...O5gal hydrogen bond is observed in (I). The hydroxymethyl group conformation in both residues is gauche-trans, with torsion angles omegagal (O5gal-C5gal-C6gal-O6gal) and omega(glc) (O5glc-C5glc-C6glc-O6glc) of 69.15 (13) and 72.55 (14) degrees, respectively. The latter torsion angle differs substantially from that found for (II) [-54.6 (2) degrees; gauche-gauche]. Cocrystallization of methanol, which is hydrogen bonded to O6glc in the crystal structure of (II), presumably affects the hydroxymethyl conformation in the Glc residue in (II).

4J(COCCH) and 4J(CCCCH) as probes of exocyclic hydroxymethyl group conformation in saccharides.

[structure: see text] 1H NMR spectra of aldohexopyranosyl rings containing 13C-enrichment at either C1 or C3 reveal the presence of long-range 4J(C1,H6R/S) and 4J(C3,H6R/S) whose magnitudes depend mainly on the O5-C5-C6-O6 torsion angle. Using theoretical calculations (density functional theory, DFT; B3LYP/6-31G*) and conformationally constrained experimental model compounds, the magnitudes and signs of 4J(C1,H6R/S) and 4J(C3,H6R/S) have been established, and their dependencies on the geometry of the C1-O5-C5-C6-H6R/S and C3-C4-C5-C6-H6R/S coupling pathways, respectively, were determined. The latter dependencies mimic that observed previously for 4J(HH) in aliphatic compounds such as propane. DFT calculations also showed that inclusion of non-Fermi contact terms is important for accurate predictions of 4J(CH) values. Application to methyl alpha- and beta-D-glucopyranosides reveals different rotameric distributions about their hydroxymethyl groups, with the beta-anomer enriched in the gt rotamer, in agreement with recent multi-J redundant coupling analyses. 4J(C1,H6R/S) and 4J(C3,H6R/S) are expected to complement other recently developed J-couplings for the assignment of hydroxymethyl group conformation in oligosaccharides containing 1,6-glycosidic linkages.

Comparative overwintering physiology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols, dehydration and diapause.

The beetle Cucujus clavipes is found in North America over a broad latitudinal range from North Carolina (latitude approximately 35 degrees N) to near tree line in the Brooks Range in Alaska (latitude, approximately 67 degrees 30' N). The cold adaptations of populations from northern Indiana (approximately 41 degrees 45' N) and Alaska were compared and, as expected, the supercooling points (the temperatures at which they froze) of these freeze-avoiding insects were significantly lower in Alaska insects. Both populations produce glycerol, but the concentrations in Alaska larvae were much higher than in Indiana insects (approximately 2.2 and 0.5 mol l(-1), respectively). In addition, both populations produce antifreeze proteins. Interestingly, in the autumn both populations have the same approximate level of hemolymph thermal hysteresis, indicative of antifreeze protein activity, suggesting that they synthesize similar amounts of antifreeze protein. A major difference is that the Alaska larvae undergo extreme dehydration in winter wherein water content decreases from 63-65% body water (1.70-1.85 g H2O g(-1) dry mass) in summer to 28-40% body water (0.40-0.68 g H2O g(-1) dry mass) in winter. These 2.5-4.6-fold reductions in body water greatly increase the concentrations of antifreeze in the Alaska insects. Glycerol concentrations would increase to 7-10 mol l(-1) while thermal hysteresis increased to nearly 13 degrees C (the highest ever measured in any organism) in concentrated hemolymph. By contrast, Indiana larvae do not desiccate in winter. The Alaska population also undergoes a diapause while insects from Indiana do not. The result of these, and likely additional, adaptations is that while the mean winter supercooling points of Indiana larvae were approximately -23 degrees C, those of Alaska larvae were -35 to -42 degrees C, and at certain times Alaska C. clavipes did not freeze when cooled to -80 degrees C.