Carbohydrate Conformation Research Articles

Martini 3 Coarse-Grained Force Field for Carbohydrates.

The Martini 3 force field is a full reparametrization of the Martini coarse-grained model for biomolecular simulations. Due to the improved interaction balance, it allows for a more accurate description of condensed phase systems. In the present work, we develop a consistent strategy to parametrize carbohydrate molecules accurately within the framework of Martini 3. In particular, we develop a canonical mapping scheme which decomposes arbitrarily large carbohydrates into a limited number of fragments. Bead types for these fragments have been assigned by matching physicochemical properties of mono- and disaccharides. In addition, guidelines for assigning bonds, angles, and dihedrals were developed. These guidelines enable a more accurate description of carbohydrate conformations than in the Martini 2 force field. We show that models obtained with this approach are able to accurately reproduce osmotic pressures of carbohydrate water solutions. Furthermore, we provide evidence that the model differentiates correctly the solubility of the polyglucoses dextran (water-soluble) and cellulose (water insoluble but soluble in ionic liquids). Finally, we demonstrate that the new building blocks can be applied to glycolipids. We show they are able to reproduce membrane properties and induce binding of peripheral membrane proteins. These test cases demonstrate the validity and transferability of our approach.

Stereoelectronic Effects Impact Glycan Recognition.

Recognition of distinct glycans is central to biology, and lectins mediate this function. Lectin glycan preferences are usually centered on specific monosaccharides. In contrast, human intelectin-1 (hItln-1, also known as Omentin-1) is a soluble lectin that binds a range of microbial sugars, including β-d-galactofuranose (β-Galf), d-glycerol 1-phosphate, d-glycero-d-talo-oct-2-ulosonic acid (KO), and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). Though these saccharides differ dramatically in structure, they share a common feature-an exocyclic vicinal diol. How and whether such a small fragment is sufficient for recognition was unclear. We tested several glycans with this epitope and found that l-glycero-α-d-manno-heptose and d-glycero-α-d-manno-heptose possess the critical diol motif yet bind weakly. To better understand hItln-1 recognition, we determined the structure of the hItln-1·KO complex using X-ray crystallography, and our 1.59 Å resolution structure enabled unambiguous assignment of the bound KO conformation. This carbohydrate conformation was present in >97% of the KDO/KO structures in the Protein Data Bank. Bioinformatic analysis revealed that KO and KDO adopt a common conformation, while heptoses prefer different conformers. The preferred conformers of KO and KDO favor hItln-1 engagement, but those of the heptoses do not. Natural bond orbital (NBO) calculations suggest these observed conformations, including the side chain orientations, are stabilized by not only steric but also stereoelectronic effects. Thus, our data highlight a role for stereoelectronic effects in dictating the specificity of glycan recognition by proteins. Finally, our finding that hItln-1 avoids binding prevalent glycans with a terminal 1,2-diol (e.g., N-acetyl-neuraminic acid and l-glycero-α-d-manno-heptose) suggests the lectin has evolved to recognize distinct bacterial species.

Adsorption of difructose dianhydrides on hydrophobic Y-zeolites

The chemical conversion of fructose to a mixture of isomeric difructose dianhydrides (DFAs) is limited by thermodynamical reason, and the enzymatic production of DFAIII from inulin is restricted by the yield of the final crystallization. Both processes require a separation process for the isolation of pure samples. Hydrophobic Y-zeolites are shown here to adsorb DFAs from aqueous solution. The interaction of the zeolite with DFAs is significantly greater than the interaction with fructose and higher fructose containing oligosaccharides and varies with the structure of the DFA isomer, the β,β-(1-2':2-1′)-connected difructopyranose dianhydride having an exceptional high affinity. Compared to sucrose the DFA adsorption is significantly slower. Molecular modeling of the DFAs by ab initio (GTO 3-31G*) and MM+ force field revealed a stiff but stretched geometry which enables the molecules to enter the 0.74 nm pore openings without great conformational change. An application of the DFA adsorption was realized for isolating biotechnologically produced DFA III. DFA III can be selectively adsorbed from the residue after its crystallization and desorbed with ethanol. The application of the method to the isolation of DFAs from a mixture of 13 isomers, obtained by chemical condensation of fructose, yields a preferential sorption of 2 DFA isomers. • DFA adsorption by hydrophobic interaction. • Much greater adsorption compared to fructose. • Cylindrical carbohydrate conformation. • Molecular diameter matches pore opening.

Enhanced Ordering in Monolayers Containing Glycosphingolipids: Impact of Carbohydrate Structure

The influence of carbohydrate structure on the ordering of glycosphingolipids (GSLs) and surrounding phospholipids was investigated in monolayers at the air-water interface. Binary mixtures composed of GSLs, chosen to span a range of carbohydrate complexity, and zwitterionic dipalmitoylphosphatidylcholine phospholipid, were studied. X-ray reflectivity was used to measure the out-of-plane structure of the monolayers and characterize the extension and conformation of the GSL carbohydrates. Using synchrotron grazing incidence x-ray diffraction, the in-plane packing of the lipid acyl chains and the area per molecule within ordered domains were characterized at different mole ratios of the two components. Our findings indicate that GSL-containing mixtures, regardless of the carbohydrate size, enhance the ordering of the surrounding lipids, resulting in a larger fraction of ordered phase of the monolayer and greater dimensions of the ordered domains. Reduction of the averaged area per molecule within the ordered domains was also observed but only in the cases where there was a size mismatch between the phospholipid headgroups and GSL components, suggesting that the condensation mechanism involves the relief of steric interactions between headgroups in mixtures.

Conformational Dynamics and Exchange Kinetics of N-Formyl and N-Acetyl Groups Substituting 3-Amino-3,6-dideoxy-α-d-galactopyranose, a Sugar Found in Bacterial O-Antigen Polysaccharides.

Three dimensional shape and conformation of carbohydrates are important factors in molecular recognition events and the N-acetyl group of a monosaccharide residue can function as a conformational gatekeeper whereby it influences the overall shape of the oligosaccharide. NMR spectroscopy and quantum mechanics (QM) calculations are used herein to investigate both the conformational preferences and the dynamic behavior of N-acetyl and N-formyl substituents of 3-amino-3,6-dideoxy-α-d-galactopyranose, a sugar and substitution pattern found in bacterial O-antigen polysaccharides. QM calculations suggest that the amide oxygen can be involved in hydrogen bonding with the axial OH4 group primarily but also with the equatorial OH2 group. However, an NMR J coupling analysis indicates that the θ1 torsion angle, adjacent to the sugar ring, prefers an ap conformation where conformations <180° also are accessible, but does not allow for intramolecular hydrogen bonding. In the formyl-substituted compound 4JHH coupling constants to the exo-cyclic group were detected and analyzed. A van't Hoff analysis revealed that the trans conformation at the amide bond is favored by ΔG° ≈ - 0.8 kcal·mol-1 in the formyl-containing compound and with ΔG° ≈ - 2.5 kcal·mol-1 when the N-acetyl group is the substituent. In both cases the enthalpic term dominates to the free energy, irrespective of water or DMSO as solvent, with only a small contribution from the entropic term. The cis-trans isomerization of the θ2 torsion angle, centered at the amide bond, was also investigated by employing 1H NMR line shape analysis and 13C NMR saturation transfer experiments. The extracted transition rate constants were utilized to calculate transition energy barriers that were found to be about 20 kcal·mol-1 in both DMSO-d6 and D2O. Enthalpy had a higher contribution to the energy barriers in DMSO-d6 compared to in D2O, where entropy compensated for the loss of enthalpy.

Approximate quantum chemical methods for modelling carbohydrate conformation and aromatic interactions: β-cyclodextrin and its adsorption on a single-layer graphene sheet.

Noncovalent functionalization of graphene by carbohydrates such as β-cyclodextrin (βCD) has the potential to improve graphene dispersibility and its use in biomedical applications. Here we explore the ability of approximate quantum chemical methods to accurately model βCD conformation and its interaction with graphene. We find that DFTB3, SCC-DFTB and PM3CARB-1 methods provide the best agreement with density functional theory (DFT) in calculation of relative energetics of gas-phase βCD conformers; however, the remaining NDDO-based approaches we considered underestimate the stability of the trans,gauche vicinal diol conformation. This diol orientation, corresponding to a clockwise hydrogen bonding arrangement in the glucosyl residue of βCD, is present in the lowest energy βCD conformer. Consequently, for adsorption on graphene of clockwise or counterclockwise hydrogen bonded forms of βCD, calculated with respect to this unbound conformer, the DFTB3 method provides closer agreement with DFT values than PM7 and PM6-DH2 approaches. These findings suggest approximate quantum chemical methods as potentially useful tools to guide the design of carbohydrate-graphene interactions, but also highlights the specific challenge to NDDO-based methods in capturing the relative energetics of carbohydrate hydrogen bond networks.

Abstract 3909: Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study

Abstract Chlorotoxin is a scorpion-derived peptide that preferentially binds to tumor cells of neuroectodermal origin, like glioma, but not to normal non-transformed cells. We have previously reported the development of a targeted nanodelivery system using chlorotoxin. Chlorotoxin has been studied as an imaging and targeting agent for drug and radioisotope delivery, but the mechanism of interaction of chlorotoxin with cancer cells has not been well understood and it needs to be further evaluated in order to optimize its use as a targeting compound for cancer cells. In this study, we used U87 human glioma cell line to evaluate the binding kinetics of chlorotoxin by Attenuated Total Reflection Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. As a sensitive and label free technique, ATR-FTIR is a powerful diagnostic tool for the comparison of cancer cells with normal cells. First, we characterized the signature spectra of chlorotoxin and U87 cells by assigning and evaluating the infrared absorption bands and their second derivatives. Next, we studied the spectral differences between U87 cells incubated with and without chlorotoxin for incremental time periods ranging from 15 minutes to 24 hours. We also examined the metabolic changes in U87 cells induced with chlorotoxin at different incubation time points by measuring the ratios of integrated areas corresponding to different chemical conformations of proteins, lipids, carbohydrates, and nucleic acids. Our results revealed spectral changes in U87 cells at different stages of incubation with chlorotoxin. The most notable change occurred after 30 minutes of incubation, where the band assigned to CH3 bending of lipid membranes shifted from 1456 cm-1 to 1450 cm-1. Consequently, a new shoulder appeared at wavenumber of 1466 cm-1 assigned to CH2 bending of lipid membranes for U87 cells treated with chlorotoxin. These changes indicate a direct interaction due to the presence of a molecular binding site between chlorotoxin and the lipids of the cell membrane. Another significant alternation occurred after 1 hour of incubation of U87 cells with chlorotoxin in the spectral region assigned for nucleic acids. This interaction included a large upshift of the band at 1036 cm-1 to 1051 cm-1 assigned to C-O stretching vibrations of ribose ring in RNA due to the hydrogen bonding with chlorotoxin. These findings suggest at least two different interactions of chlorotoxin with U87 human glioma cells that can be further explored for improving chlorotoxin containing drug delivery systems. We are currently using the ATR-FTIR method to conduct control experiments by measuring possible interactions of chlorotoxin with normal human astrocytes. Citation Format: Rana Falahat, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3909.

Gene-Silencing-Induced Changes in Carbohydrate Conformation in Relation to Bioenergy Value and Carbohydrate Subfractions in Modeled Plant (Medicago sativa) with Down-Regulation of HB12 and TT8 Transcription Factors.

Gene silencing with RNA interference (RNAi) technology may be capable of modifying internal structure at a molecular level. This structural modification could affect biofunctions in terms of biodegradation, biochemical metabolism, and bioactive compound availability. The objectives of this study were to (1) Detect gene silencing-induced changes in carbohydrate molecular structure in an alfalfa forage (Medicago sativa spp. sativa: alfalfa) with down-regulation of genes that encode transcription factors TT8 and HB12; (2) Determine gene silencing-induced changes in nutrient bioutilization and bioavailability in the alfalfa forage (Medicago sativa); and (3) Quantify the correlation between gene silencing-induced molecular structure changes and the nutrient bioutilization and bioavailability in animals of ruminants. The experimental treatments included: T1 = Non-transgenic and no-gene silenced alfalfa forage (code “NT”); T2 = HB12-RNAi forage with HB12 gene down regulation (code “HB12”); T3 = TT8-RNAi forage with TT8 gene down regulation (code “TT8”). The HB12 and TT8 gene silencing-induced molecular structure changes were determined by non-invasive and non-destructive advanced molecular spectroscopy in a middle infrared radiation region that focused on structural, non-structural and total carbohydrate compounds. The nutrient bioutilization and bioavailability of the modified forage were determined using NRC-2001 system in terms of total digestive nutrient (TDN), truly digestible fiber (tdNDF), non-fiber carbohydrate (tdNDF), fatty acid (tdFA), crude protein (tdCP) and bioenergy profiles (digestible energy, metabolizable energy, net energy) for ruminants. The carbohydrate subfractions were evaluated using the updated CNCPS 6.0 system. The results showed that gene silencing significantly affected tdNFC (42.3 (NT) vs. 38.7 (HB12) vs. 37.4% Dry Matter (TT8); p = 0.016) and tdCP (20.8 (NT) vs. 19.4 (HB12) vs. 22.3% DM (TT8); p = 0.009). The gene-silencing also affected carbohydrate CA4 (7.4 (NT) vs. 4.2 (HB12) and 4.4% carbohydrate (CHO) (TT8), p = 0.063) and CB1 fractions (5.3 (NT) vs. 2.0 (HB12) and 2.6% CHO (TT8), p = 0.006). The correlation study showed that the structural CHO functional group peak area intensity at ca. 1315 cm−1 was significantly correlated to the TDN1x (r = −0.83, p = 0.042) and the tdNFC (r = −0.83, p = 0.042), the structural CHO functional group height intensity at ca. 1370 cm−1 was significantly correlated to the tdNDF (r = −0.87, p = 0.025). The A_Non-stCHO to A_StCHO ratio and A_Non-stCHO to A_CHO ratio were significantly correlated to the tdFA (r = 0.83–0.91, p < 0.05). As to carbohydrate fractions, both CA4 and CB1 correlated with carbohydrate spectral intensity of the H_1415 and the H_1315 (p = 0.039; p = 0.059, respectively), CB3 tended to correlate with the H_1150, H_1100 and H_1025 (p < 0.10). In conclusion, RNAi-mediated silencing of HB12 and TT8 modified not only inherent CHO molecular structure but also the biofunctions. The CHO molecular structure changes induced by RNAi gene silencing were associated with biofunctions in terms of the carbohydrate subfractions and nutrient digestion.

Solution conformation of carbohydrates: a view by using NMR assisted by modeling.

Structural elucidation of complex carbohydrates in solution is not a trivial task. From the NMR view point, the limited chemical shift dispersion of sugar NMR spectra demands the combination of a variety of NMR techniques as well as the employment of molecular modeling methods. Herein, a general protocol for assignment of resonances and determination of inter-proton distances within the saccharides by homonuclear and heteronuclear experiments (i.e., (1)H and (13)C) is described. In addition, several computational tools and procedures for getting a final ensemble of geometries that represent the structure in solution are presented.

Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.

Recent Research and Progress in Food, Feed and Nutrition with Advanced Synchrotron-based SR-IMS and DRIFT Molecular Spectroscopy

Ultraspatially resolved synchrotron radiation based infrared microspectroscopy is able to detect the structure features of a food or feed tissue at cellular and molecular levels. However, to date, this advanced synchrotron-based technique is almost unknown to food and feed scientists. The objective of this article was to introduce this novel analytical technology, ultra-spatially resolved synchrotron radiation based infrared microspectroscopy (SR-IMS) to food, feed, conventional nutrition, and molecular nutrition scientists. The emphasis of this review focused on the following areas: (1) Principles of molecular spectroscopy for food and feed structure research, such as protein molecular structure, carbohydrate conformation, heating induced protein structure changes, and effect of gene-transformation on food and feed structure; (2) Molecular spectral analysis methodology; (3) Biological applications of synchrotron SR-IMS and DRIFT spectroscopy; and (4) Recent progress in food, feed and nutrition research program. The information described in this article gives better insight in food structure research progress and update.

Lanthanide-chelating carbohydrate conjugates are useful tools to characterize carbohydrate conformation in solution and sensitive sensors to detect carbohydrate-protein interactions.

The increasing interest in the functional versatility of glycan epitopes in cellular glycoconjugates calls for developing sensitive methods to define carbohydrate conformation in solution and to characterize protein-carbohydrate interactions. Measurements of pseudocontact shifts in the presence of a paramagnetic cation can provide such information. In this work, the energetically privileged conformation of a disaccharide (lactose as test case) was experimentally inferred by using a synthetic carbohydrate conjugate bearing a lanthanide binding tag. In addition, the binding of lactose to a biomedically relevant receptor (the human adhesion/growth-regulatory lectin galectin-3) and its consequences in structural terms were defined, using Dy(3+), Tb(3+), and Tm(3+). The described approach, complementing the previously tested protein tagging as a way to exploit paramagnetism, enables to detect binding, even weak interactions, and to characterize in detail topological aspects useful for physiological ligands and mimetics in drug design.

Specific rotation of monosaccharides: a global property bringing local information.

Carbohydrates generally occur in several conformations that may differ among themselves by energy values that are smaller than the accuracy of the most sophisticated theoretical methods used to determine them. In addition, the preferential orientations of the hydroxyl groups of these molecules cannot be identified by any experimental technique. Therefore, a method that is able to validate the absolute conformations (i.e., consisting of the orientations of the hydroxyl groups) of carbohydrates would be helpful to improve our knowledge about monosaccharides. SR has been used for this purpose, and here, we present a test to measure the specific rotation (SR) ability of a molecule that possesses not only many conformations, but also four adjacent chiral centers. The results show that the final SR value is a weighted average of a global property (obtained for each conformation), and the latter by its turn is influenced by each chiral center in a multi chiral system. By comparing the SR values calculated for the most abundant anomers of xylopyranose with those of the corresponding monochiral analogs obtained by saturation of three different chiral centers each time, the influence of each center on the global property is confirmed.

Using ATR–FT/IR to detect carbohydrate-related molecular structure features of carinata meal and their in situ residues of ruminal fermentation in comparison with canola meal

There is no information on the co-products from carinata bio-fuel and bio-oil processing (carinata meal) in molecular structural profiles mainly related to carbohydrate biopolymers in relation to ruminant nutrition. Molecular analyses with Fourier transform infrared spectroscopy (FT/IR) technique with attenuated total reflectance (ATR) and chemometrics enable to detect structural features on a molecular basis. The objectives of this study were to: (1) determine carbohydrate conformation spectral features in original carinata meal, co-products from bio-fuel/bio-oil processing; and (2) investigate differences in carbohydrate molecular composition and functional group spectral intensities after in situ ruminal fermentation at 0, 12, 24 and 48h compared to canola meal as a reference. The molecular spectroscopic parameters of carbohydrate profiles detected were structural carbohydrates (STCHO, mainly associated with hemi-cellulosic and cellulosic compounds; region and baseline ca. 1483–1184cm−1), cellulosic compounds (CELC, region and baseline ca. 1304–1184cm−1), total carbohydrates (CHO, region and baseline ca. 1193–889cm−1) as well as the spectral ratios calculated based on respective spectral intensity data. The results showed that the spectral profiles of carinata meal were significantly different from that of canola meal in CHO 2nd peak area (center at ca. 1091cm−1, region: 1102–1083cm−1) and functional group peak intensity ratios such as STCHO 1st peak (ca. 1415cm−1) to 2nd peak (ca. 1374cm−1) height ratio, CHO 1st peak (ca. 1149cm−1) to 3rd peak (ca. 1032cm−1) height ratio, CELC to total CHO area ratio and STCHO to CELC area ratio, indicating that carinata meal may not in full accord with canola meal in carbohydrate utilization and availability in ruminants. Carbohydrate conformation and spectral features were changed by significant interaction of meal type and incubation time and almost all the spectral parameters were significantly decreased (P<0.05) during 48h ruminal degradation in both carinata meal and canola meal. Although carinata meal differed from canola meal in some carbohydrate spectral parameters, multivariate results from agglomerative hierarchical cluster analysis and principal component analysis showed that both original and in situ residues of two meals were not fully distinguished from each other within carbohydrate spectral regions. It was concluded that carbohydrate structural conformation could be detected in carinata meal by using ATR–FT/IR techniques and further study is needed to explore more information on molecular spectral features of other functional group such as protein structure profile and their association with potential nutrient supply and availability of carinata meal in animals.

Effect of altered carbohydrate traits in hulless barley (Hordeum vulgare L.) on nutrient profiles and availability and nitrogen to energy synchronization

Four hulless barley cultivars (zero-amylose waxy, CDC Fibar; 5%-amylose waxy, CDC Rattan; normal-amylose, CDC McGwire and high-amylose, HB08302) were developed at the Crop Development Centre, University of Saskatchewan, with differences in carbohydrates traits on the basis of amylose (1–20% DM) and β-glucan (5–10% DM) content. The objectives of this research were to determine: 1) the effect of the alteration of these carbohydrate traits in hulless barley on nutrient availability in ruminants, and 2) carbohydrate structure conformation on hourly effective rumen degradation ratio, in comparison with hulled barley-CDC Copeland. Among the hulless barley lines, HB08302 was greater (P < 0.05) in rumen bypass crude protein (BCP: 52.1% CP), effective degradability (ED) of neutral detergent fibre (EDNDF: 74 g/kg DM) than these in CDC Fibar, whereas CDC Fibar showed greater (P < 0.05) effective degradable crude protein (EDCP: 90 g/kg DM) than the other hulless barley lines. Compared with hulless barley, the hulled CDC Copeland showed relatively greater (P < 0.05) BNDF (62.5% NDF or 98 g/kg DM), starch degradation rate (Kd: 17.4%/h) and EDST (75.9% ST) but reduced (P < 0.05) rumen undegradable protein (RUP: 49 g/kg DM). With the respect to hourly effective degradation (ED) ratios, hulless barley cultivars exhibited relatively optimal hourly ED ratios, ranging from 23 to 34 g N/kg CHO, higher than the hulled barley. Among hulless barley cultivars, CDC Fibar and Rattan had the highest (P < 0.05) ratios (34 g N/kg CHO), while the ratio for CDC McGwire (23 g N/kg CHO) was the lowest (P > 0.05). In conclusion, hulless barley lines with altered carbohydrate traits have the potential to increase rumen nutrient availability to ruminants. The altered carbohydrate conformation of hulless barley affected hourly ED ratios, thus affecting rumen nitrogen to energy synchronization.

Enantiomeric Selection Properties of β‐homoDNA: Enhanced Pairing for Heterochiral Complexes

The analysis of the physicochemical properties of sugarmodified nucleic acids is currently at the core of intense multidisciplinary investigations including chemistry, biology, biotechnology, and medicine. On one side, synthetic polymers acting as RNA/DNA mimics have extensively been devised for applications in therapy, diagnostics, and synthetic biology. On the other side, the construction of alternative pairing systems has been explored either to consider their use as orthogonal nucleic acid candidates or with the aim to potentially yield insights into the chemical evolution criteria ultimately leading to the current genetic system. In all cases, structural changes of natural (deoxy)ribose cores have been established to determine profound consequences in the pairing potential of the resulting artificial nucleic acids. In some noteworthy examples, oligonucleotide systems endowed with six-membered sugars in the backbone have been observed to hold the singular property (unique of its kind) of pairing with homochiral complements having opposite sense of chirality. Relevant to etiology-oriented investigations on nucleic acid structure, these findings could suggest the existence of a relationship between nature of the sugar backbone and chiral-selection properties of nucleic acids, thereby providing insights to enrich our understanding of the structural prerequisites for base pairing. From a comparative analysis of the pairing behavior of six-membered nucleic acids we perceived that, despite the large structural differences, oligonucleotide systems capable of isoand heterochiral hybridization (Figure 1) shared preorganized carbohydrate conformations with equatorially-oriented nucleobases. This observation took us to wonder if such an arrangement of the aglycon moiety, especially whereas inducing strong backbone-base inclination or even enabling formation of quasilinear oligomeric structures, could lead sugar chirality not to be crucial in hybridization processes. In view of systematic investigations aimed at addressing this question, we herein considered the chiral selection properties of the well-known pairing system composed of (6’!4’)-linked b-erythro-hexopyranosyl nucleotides (b-homoDNA; Figure 1). Based on above assumptions and early experimental data, we reasoned that the strongly inclined complexes provided by the “allequatorial” pyranose backbone of b-homoDNA could make the latter an interesting candidate displaying potential for heterochiral hybridization. An investigation into the enantioselectivity of the hybridization processes of b-homoDNA required access to oligomeric sequences in both enantiomeric forms (b-dand b-lhomoDNA). From a synthetic standpoint, while access to d-hexopyranosyl nucleosides was easily obtained by a carbohydrate-based route, the synthesis of the corresponding lenantiomers under the same reaction conditions was hampered by the limited commercial availability of almost all lhexoses. In an alternative path, our long studied de novo approach to l-monosaccharides and other structurallyrelated compounds was recently exploited for the preparation of the l-nucleosides 2a,b (T and A acting as model nucleobases) from the homologating agent 1 (Scheme 1). Figure 1. Sugar-modified nucleic acids displaying pairing aptitude for homochiral complements of opposite chirality.

Mass Spectrometric Characterization of Protein:Carbohydrate Non Covalent Complexes: What is Fact and What is Fiction

This seminar will present Ion Mobility Mass Spectrometry data showing separation and detection of isomeric glycosaminoglycans (GAG) both with and without protein complexation. Chemokine proteins are involved in inflammation and as such complex with GAG during the activation step of chemotaxis. Comparison of different chemokine conformations suggests that the GAG will complex to different areas of the protein and that sulfation pattern may play a part in complexation. A relatively new modeling program, RAPTOR, is used for determining theoretical cross sections and overall conformation of proteins for which there is no crystal or NMR structure. Both RAPTOR and IM provide valuable information on isomeric separation, identification and conformation of both carbohydrate alone, and with its' protein partner.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Exploration of conformational flexibility and hydrogen bonding of xylosides in different solvents, as a model system for enzyme active site interactions

The predominantly populated conformation of carbohydrates in solution does not necessarily represent the biologically active species; rather, any conformer accessible without too large an energy penalty may be present in a biological pathway. Thus, the conformational preferences of a naphthyl xyloside, which initiates in vivo synthesis of antiproliferative glycosaminoglycans, have been studied by using NMR spectroscopy in a variety of solvents. Equilibria comprising the conformations (4)C1, (2)SO and (1)C4 were found, with a strong dependence on the hydrogen bonding ability of the solvent. Studies of fluorinated analogues revealed a direct hydrogen bond from the hydroxyl group at C2 to the fluorine atom at C4 by a (1h)JF4,HO2 coupling. Hydrogen bond directionality was further established via comparisons of fluorinated levoglucosan molecules.

Review: use of residual dipolar couplings to determine the structure of carbohydrates

Solution nuclear magnetic resonance spectroscopy is especially useful in the carbohydrate field. The measurement of residual dipolar couplings provides long-range structural information, a valuable complement for the structural study of carbohydrates either in its free form or in the bound state to proteins. They permit to deduce the geometry and the flexibility of the glycosidic linkages, which have a major influence on the conformation of carbohydrates and their overall shape. This article reviews the current application of the residual dipolar couplings methodology to carbohydrates.

Performance of the SCC-DFTB Model for Description of Five-Membered Ring Carbohydrate Conformations: Comparison to Force Fields, High-Level Electronic Structure Methods, and Experiment.

The solution conformations of biologically important mono- and di-α-d-arabinofuranosides were investigated using the dispersion-corrected self-consistent charge density functional tight binding (SCC-DFTB) and the AMBER/GLYCAM models. Simulations were performed using both long dynamics and umbrella sampling simulations. Angular distributions about the exocyclic C-C bonds and puckering distributions of the rings obtained from the SCC-DFTB model were quite different from those obtained with the AMBER/GLYCAM approach. The joint probability distribution of rotamer and ring puckering parameters reveals further discrepancies, while both methods predict weak correlation between exocyclic torsions and ring puckering. To assess the reliability of the simulations, ensemble-averaged vicinal proton-proton coupling constants ((3)JH,H) were calculated and compared directly to experimental NMR coupling constants. It is found that the (3)JH,H values obtained from the AMBER/GLYCAM simulations agree with experiment, while those obtained from the SCC-DFTB method, in most cases, differ from experimental (3)JH,H values. Potential energy surfaces (PES) along the exocyclic torsion obtained from ab initio and DFT calculations differ significantly from those obtained with the SCC-DFTB method. This study establishes that a high-quality all-atom force field would be more suitable than one of the superior semiempirical methods, SCC-DFTB, for investigation of rotamer populations and ring puckering in floppy ring systems such as furanosides.