Conformational Studies of Oligosaccharides

Abstract The conformation of a molecule strongly affects its function, as demonstrated for peptides and nucleic acids. This correlation is much less established for carbohydrates, the most abundant organic materials in nature. Recent advances in synthetic and analytical techniques have enabled the study of carbohydrates at the molecular level. Recurrent structural features were identified as responsible for particular biological activities or material properties. In this Minireview, recent achievements in the structural characterization of carbohydrates, enabled by systematic studies of chemically defined oligosaccharides, are discussed. These findings can guide the development of more potent glycomimetics. Synthetic carbohydrate materials by design can be envisioned.


Introduction
The function of am olecule is strongly connected to its three dimensionals hape. [1] Structurals tudies of proteins and nucleic acids were fueled by synthetic and gene expression technologies. [2] Well-defined syntheticm aterials serve as standards to establish definitive structure-function correlations. [3] Analytical techniques, such as X-ray crystallography [4] and cryogenic electron microscopy (cryo-EM), [5] have enabled subnanometer resolution images. In addition, spectroscopict echniques like circular dichroism (CD) and NMR have made structurals tudies routine in most chemistry laboratories. [6] These results permitted the development and validation of highly accurate computational tools that can predict and suggest the fabrication of materials by design (e.g.,DNA origami and de novo proteins). [7] In contrast, structural studies of carbohydrates,t he most abundant organic materials in nature,a re rare. [8] Carbohydrates are mainly extracted from natural sources, resulting in ill-defined mixtureso fc ompounds. [9] Chemical synthesis offers a valid alternative to isolation, but requiresahuge synthetic effort. The advent of one-pot synthesis and automated techniques has allowedf or the access to collections of related compounds, as ideal probesf or structurals tudies. [10] Oligosaccharidesw ith defined composition, length, ands ubstitution are now available for structural elucidation. Importantly,t he insertion of specific modifications, such as NMR active nuclei (i.e., 13 Ca nd 19 F) can be easily achieved, offering at remendous advantage during analysis. [10b, 11] Unnatural functionalities able to lock particular conformations or to disrupt particular geometries can be imagined and used for the creation of glycomi-metics. [12] Even thoughs uch strategies remain limitedb ys ynthetic challenges, examples of structurally designed glycomimeticsw ith high affinity for at arget protein suggest the potentialo ft his approach. [13] An additional bottleneck in the structurala nalysis of carbohydrates is that standard characterization techniques are often not applicable. [8b] Carbohydrates' high flexibility prevent the formation of singlec rystals suitable for X-ray analysis, sensitivity to electron beam makest hem poor candidate to EM characterization,a nd lack of chromophores prevents standard CD analysis. NMR remains the most useful characterization technique;h owever,t he analysisi sh indered by severe chemical shift degeneracy, often requiring special pulse sequences or the insertion of labels. [14] Duet ot he lack of validating standards, computationalt ools are far less developeda sc ompared to peptides and nucleic acids. [15] To date, the combination of chemicals ynthesis, NMR analysis, and molecular dynamics (MD) simulations permitted to identify recurrent structural features commonf or some glycan classes. [13a, 16] Here, we review the recent resultso btained in the field of glycanc onformational analysis.W ed iscuss four classes of glycans and their structural features. Particular focus is given to the synthetic strategies that were employedt oh elpt he characterization.

Glycans Conformation
When considering conformation of glycans, the monosaccharide unit is generally treated as ar igid block. [15a] The geometry of the glycosidic bond is of fundamental importance and it is identified by using standard descriptors (Figure1). Twot orsion angles define the relative orientation of the two monosaccharides connected through the glycosidic bond: . For 1,6-linkages, the additional torsion angle w (O 6 -C 6 -C 5 -O 5 )i sr equired.
Several factors can affect theset orsion angles (Figure 1). [15a] Hyperconjugation between the exocyclic oxygen lone electron pair andt he antibonding orbital (s*) of the endocyclic CÀO bond stabilizes the exo-syn(F)c onformation (exo-anomeric effect). [17] Steric interactions mostly affect the Y dihedral,favoring the anti(Y)c onformer. [18] Electronic effects can promote particular w geometries (gauche effect). [19] Hydrogenb onds between hydroxyl groups can stabilize particular conformational states. [20] Water has ah uge effect on the conformational freedom of glycans. Indeed, water can easily disrupt intermolecular hydrogen bonds, resulting in highly flexible conformations, and plays am ajor role in glycan-protein interactions. Modeling the process of solvation remains am ajor challenge for computationalchemists, limiting our ability to reliably predict glycans' conformations, and their recognition processes. [21] Moreover, severalg lycans bear ionic functionalities that further complicate the description.
Upon interaction with ap rotein, additional parameters can come into play.H ydrogen bonding and coordinationt oc alcium ions can contributet oc arbohydrate binding. [22] Moreover, despite the high hydrophilicity of glycans, carbohydrate-aromatic interactions are often involved in carbohydrate recognition. [23] These noncovalent interactions between two or three CH groups of the pyranose unit and the p electron density of the aromatic ring are able to stabilizep articular conformations and favor binding. Stereoelectronic effects also play ar ole in glycan recognition. [24] All thesef actorsn eed to be considered to develop reliable tools to predict glycans' conformations. Force fields, specifically optimized for carbohydrates, are available and MD simulations have become substantially more accurate. [15a, 25] Still, the computational predictions requirec onstant validation with synthetic standards. To date, NMR analysis haso ffered the best solution with scalar J-couplings and residual dipolarc ouplings (RDCs) being relativelye asy measurements and extremely informative. [14a] However,g lycans' intrinsic flexibilityo ften leads to an averaged 3D structure, merging the contributionsf rom multiple conformational states.
N-Linked-glycans N-Glycans are oligosaccharidesc ovalently linked to secreted and membrane-bound proteins througha nN -glycosidic bond. They play central roles in the folding, sorting, and transport of proteins as well as mediating cell-cell interactions. [26] The N-glycans chemicals tructure features ap entasaccharidec ore motif, consisting of ac hitobiose (GlcNAcb1!4GlcNAc) and three mannoseu nits. Depending on the residues attached to this core structure, N-glycans are classified into three main categories:o ligomannose, complex, and hybrid ( Figure 2). Due to their importantb iological roles, much research has been devoted to their structuralanalysis.
NMR spectroscopy is the most frequently used methodt o get insight into the shape and dynamics of N-glycans in solution. Still, the complexityo ft hese oligomers poses as evere bottleneck fors tructurals tudies. In ap ioneeringw ork, the three-dimensional structure of the high mannose-type Nglycan domain (-(N-acetylglucosamine) 2 -(mannose) 5-8 )d ecorating the glycoprotein CD2 was elucidated. [28] The sample was extracted from Chineseh amster ovary cells. The geometricr e-  straintsi dentified through NOE signals suggested as tructural model with one of the glycan arms folded towardt he GlcNAc1-GlcNAc2-Man3 trisaccharide core.T he comparison of 13 CNMR peak widths shed light on the N-glycan flexibility, upon interaction with ap rotein. These results indicated that the N-glycan does not directly mediate the binding of CD2 to its counterreceptor CD58, since the N-glycan is oriented in the opposite direction to the binding site of CD2. Instead, the main role of this N-glycan is to stabilize the folding of CD2, counterbalancing the energetically unfavorable cluster of positive charges arising from five lysine residues.
Due to the heterogeneity of N-glycans in biological systems, extracted N-glycans exists as am ixture of compounds, which leads to ambiguity in NMR peak assignment. This lack of pure and well-definedg lycansh inders detaileds tructure-functionality correlations.G enetic engineering provides tools to facilitate glycan structurals tudies. The biosynthesis of high-mannose Nglycansi ny east takes place in the endoplasmic reticulum, where an undecasaccharide Man 9 GlcNAc 2 (M9) is constructed. Subsequently,a na-mannosidasey ield the decasaccharide Man 8 GlcNAc 2 (M8B), whichi st hen transported to the Golgi apparatusf or further structuralm odifications. By knocking out the genes encoding for specific enzymesi nt his route, M9 and M8B were overexpressed and isolated ( Figure 3). Moreover,b y feeding 13 C-labeled glucose to the yeast, isotopic labeling can be achieved,w hich greatlyh elps the NMR analysis. Following this approach, it wasc onfirmed that the mannose outer branch (D2 and D3) folds back toward the core chain, similarly to what observed for the above mentioned high mannosetype glycans in CD2. Notably,c ompared to M9, as ignificantly different NOE network and enhanced back-folding was observed for M8B ( Figure 3). [29] Further difficulties in the NMR analysiso fN -glycans arise from the multiantennaryp seudo-symmetry that leads to signal overlapping. In addition, NOEs and scalar couplings only afford local structural information (up to 5 ). For N-glycansw ith sizes of severaln anometers, analyzingm ethods able to detect long-range interactions are required. Paramagneticl anthanide ions can generate strong chemical shift variations, pseudo-contact shifts (PCS), and provide globalg eometrici nformation.A lanthanide-binding tag was attached at the reducing end of a complex-type N-glycan ( Figure 4a). Thes hift of the NMR signals caused by the paramagnetic ion resultedi n3 4 1 HNMR PCS, that can be interpreted into atom-atomd istances in a range of 30 .B yc omputing the obtained conformational information,aT-shaped rotamer at the Mana1-6Man portion   Lanthanide-tagged N-glycansf or structural analysis. AT-shaped rotamer at the Mana1-6Man portion (red bond)was identified as the major conformer.The monosaccharides are represented following the symbol nomenclature for glycans (SNFG). [27] (Figure 4a,r ed bond) was revealed as the major conformer among the five suggestedb yM D. [30] This approach was then appliedtor evisit the structure of oligomannose-type N-glycans (Figure 4b), [31] supporting the previouso bservations with a more quantitative description of the glycan dynamics and flexibility in solution.
While it is cleart hat N-glycans exercise their biological functions through interaction with proteins, the structural basis of this process is not yet solved. Due to the synthetic anda nalytical difficulties, most studies on glycan-protein interactions are based on fragments of natural glycans. Still, the assumption that the binding behavior of natural N-glycans can be extrapolated from glycan fragments can be misleading. With the recent advancementi nb oth chemical synthesis and NMR technologies, the binding pattern of natural complex-type N-glycans to differentl ectins can be carefullys tudied. Ac ollection of synthetic N-glycans, including severals maller fragments, was synthesized. [32] Saturation-transfer difference NMR (STD-NMR) analysisshowedt hat the interaction between glycan and protein highly depends on the chemical nature of both components and cannot be predicted from simplified monodomain models. Lanthanide tags were also employed for the structural study of glycan-protein recognition. [33] With this method, the four antennae of the complex-type N-glycan can be discriminated and thusi nformation with unprecedented resolution was obtained. The involvement of each individual branch of the N-glycan in the recognition was described based on PCS ( Figure 5). The use of paramagnetic NMR was also applied to the study of the conformationo fm ultiantennaN -glycans and their interaction with HK/68 hemagglutinin from influenzav iruses.T his study was enabled by chemoenzymatic synthesis of long-chain N-glycans containing poly-LacNAc and Neu5Acr esidues,f ollowed by conjugation with al anthanide binding tag. [34] An alternative method to break the chemical shift degeneracy observed for multiantennary N-glycans relies on the introductiono fu nnatural and NMR active nuclei. The chemical shift of 19 Fi sv ery sensitive to subtle changes in the chemical environmenta nd spans from approximately À60 to around À220 ppm. Therefore, the substitution of ah ydroxyl group with af luorine atom enables the use of fluorine-basedN MR methods for epitope mapping, as exemplified for the trimannoside core structure (Figure6). In order to minimizet he impact of the deoxifluorination on binding to Pisum sativum agglutinin, the modification was installed at the C2 of the mannoser ings, following the consideration that the 2-OH does not participate in this recognition event.2 DN OESY-TOCSYreF experiments revealed two binding modes in which either mannose Io rm annoseIIa re involved. In both modes, the mannose III residue contributes to the binding through collateral effect. [35] Histo-blood group antigens Histo-blood group antigens( HBGAs) are af amily of oligosaccharides found on the surfaceo fr ed blood and tissuec ells or as soluble antigens. [36] HBGAs are biosynthesizedf rom two pre-cursors (type 1a nd 2c hains) by glycosyltransferases andc an be classifieda sA BH or Lewis antigens (Figure 7). ABH antigens determine the blood phenotype( A, B, AB, or O) of humans and recent studies suggest that they also affect the susceptibility to bacterial and viral infection. [37] The abnormale xpression of HBGAs might contribute to the increased mobility of tumor cells, resulting in poor prognosis. [38] Structural studies of HBGAs and HBGAs-protein complex are crucial tou nravel the molecular basis of the HBGAsrecognition by pathogens. [8a, 39] Among the Lewis antigens, sialyl Lewis X( sLe x )i st he most intensively studied structure due to its vital role in cell-cell communication, upon interaction with selectins. Thec ore structure, Lewis X( Le X ), possesses at risaccharide motif that adopts ad efined conformation in solutiona sr evealed by NMR [40] and molecular dynamics. [40] Such conformation is stabilized by the exo-anomerice ffect, steric compression, and hydrophobic interactions. In 1996, the first crystal structure of Le X was reported, showing the dense network of hydrogen bonds that stabilizei ts conformation. [41] To better understand the solution phase conformation, the Le X trisaccharide was synthesized and covalently linked to an isotopically labelled bacterial protein. [42] This strategy permitted to slow down the oligosaccharide tumbling and, therefore, favored the detection of NOEs. Nine inter-residue NOEs were detected, while only three could be visualized for the free Le X .N oN OEs were detected between Le X andt he protein, confirming thatt hese results are representative of the conformation of free Le X .T hiss tudy provided important structural information on the torsion angles of the glycosidicb onds and the overall shape of Le X .I np articular, an onconventional CÀH···O H-bonding between the fucosea nd the galactose residue was identifieda nd confirmed by the downfield chemical shift of the H5 of the fucose residue (Figure 8a). Such interaction stabilizes the "closed" conformation of the Le X trisaccharide motif. The existence of this unconventional CÀH···O H-bonding was later confirmed in sLe x with ex-tensiveN MR study. [43] As ystematic analysiso fs everalf ucose containing oligosaccharides showedt hat this particular CÀH···O H-bonding is ac ommon secondary structural element in a wide range of bacterial and mammalian oligosaccharides and can be generalized with the X-b1,4-[Fuca1,3]-Y and X-b1,3-[Fuca1,4]-Y description (Figure 8b). [16a] Upon bindingt om ost lectins, the "closed" conformation of Le X is preserved, as confirmed by crystallographic and NMR analysis. [44] In these cases,f ucosylation seems to be responsible for the HBGAs high binding affinity to lectins, by promoting a "pre-organization" that eliminates the step of conformational selection.T os ystematically prove this hypothesis, A-and Bblood-group tetrasaccharides (type II), as well as their non-fucosylated analogues weres ynthesized ( Figure 9). [45] STD-NMR suggested that the b-Gal residue (Figure 9, dashed boxes) directly participates in the binding to galectin-3, while the fucose residue does not interact with the lectin. This proved that Fuc-containing glycanss hare the same bindingm ode with the non-fucosylated analogues,e ven though exert higher affinity to the lectin. Thermodynamic and kinetic parameters suggest that the fucoser esidue contributes indirectly to the binding, reducing the conformational flexibility and so minimizing the entropic penalty of binding.  . Classification of histo-blood groupa ntigens. The monosaccharides are represented following the symbol nomenclaturef or glycans (SNFG). [27] Figure 8. Nonconventional CÀH···O H-bonding identified by NMR(a). This is ar ecurrent secondarystructural element in aw ide range of fucose containing oligosaccharides (b). Figure 8a is reprinted with permissionf rom Ref. [42].Copyright 2013, American Chemical Society.
Similarly,alarge entropy contribution drivest he binding between sLe X and E-selectin. Commonly,t he interaction between glycansa nd lectins is entropically unfavorable, but driven by the favorable binding enthalpy. [46] However,i nc ase of sLe X -Eselectin interaction, the pre-organization of sLe X serves as a surrogate for the water cluster present in the binding site. [47] Upon binding, water is released from the binding site, resulting in an entropic benefit for the overall process. In addition, the pre-organization of sLe X offers an array of directed Hbonds increasing the specificity of the binding.
In contrast, upon bindingw ith Ralstonia solanacearum lectin, distortion of the Le X "closed" conformationw as observed. [48] Several" open" conformers, in which the fucose residue forms H-bonds with the lectin, were identified by using highly detailed MD simulations of the glycan in water and its interaction with the lectin. This deformation releases the steric hindranceb etween the galactose residue and aT rp residue of the lectin. Such an adaptive conformation of Le X can be chemically tuned for the design of high affinity glycomimetics. For example, the substitution of the terminal galactose with a mannoseunit was shown to disrupt the conformational rigidity of Le X and stabilizet he "open" conformation required for binding ( Figure 10). [13b] The reduced steric hindrance of this unnatural analoguer esulted in a1 7times higher affinity fort he lectin than the native counterpart.

Bacterialglycans
Glycans in bacteria are mostly present as glycoconjugates, such as glycolipids and peptidoglycans. [49] These glycoconjugates play key roles in the protection of the bacteria from the host immune system and control cellular permeability. [50] Compared to mammalian glycans, bacterial glycans exhibit am uch greater diversity, especially in terms of monosaccharide composition. [51] These "uncommon"m onosaccharides play important roles in the local conformationso fb acterial glycans. For example, l-rhamnose is absenti nm ost mammals, but widely distributes in lipopolysaccharides (LPS) of Gram-negative bacteria and in capsular polysaccharides of Gram-positive bacteria. The conformational preference of rhamnose-containing glycans wass tudiedw ith ad isaccharide model( a-l-Rhap-(1-2)-al-Rhap-OMe, Figure 11,t op). [52] NMRa nd computational analy-sis permitted the identification of two preferred conformations in water,e xisting in a3:2 ratio. Another sugar motif commonly found in bacterial glycans is the 3-amino-3,6-dideoxy-a-d-galactopyranose ( Figure 11,b ottom). [53] Particular attention was paid to the conformation of its N-formyl and N-acetyl derivatives. The N-acetyl derivativee xhibits ah igherp reference (DG8 %À2.5 kcal mol À1 )f or the trans conformationc ompared to its N-formyl counterpart (DG8 %À0.8 kcal mol À1 ), with ac alculated transition energy barrierofaround20kcal mol À1 .Quantum mechanics energy calculations suggest intramolecular Hbonds between the oxygen of the amide and the axial OH4 or the equatorial OH2.
The great varietyo fm onosaccharide composition,s ubstitution, and connectivity observed in bacterial glycansi sr eflected in great structural diversity that can trigger particular immunological responses. LPS on the surface of Gram-negative bacteria are based on repetitive polysaccharides that can adopt different shapes. [54] Due to the structural flexibility,m ultiple models are generally employed to represent the low energy conformations of these polysaccharides, as in the case of the O-antigen polysaccharides of Escherichia coli O5ac and O5ab. [55] These structures share the same tetrasaccharide repeating unit connected via different linkages. The conformational preference of   these two polysaccharides was studied with NMR methods, including 1 H, 1 H-NOESY,a nd NOE build-up curves, showing the co-existenceo fs everal different conformers. This structural flexibility could explain the cross-reactivity of the O5ac and the O5ab antigensini mmunological assays.
Similarly,g roup B Streptococcus serotypes Ia and Ib capsular polysaccharides (CPS) sharet he same monosaccharide constituents with the only differencebeing one of the glycosydic linkage (GlcNAcb1-3Gal vs. GlcNAcb1-4Gal, respectively). To compare the conformation adopted by these two capsularp olysaccharides,t he pentasaccharide repeating units were synthesized. [56] Conformational studies revealed that the difference in GlcNAc-Gall inkage mainly affects the orientation of the Neu5Aca2-3Gal branching,w ith the Neu5Aca2-3Gal linkages adopting the exo-anti(F) conformation for Ia and the exosyn(F) for Ib ( Figure 12). This different conformational preference might justify the different immunological activity of Ia and Ib capsular polysaccharides.
Much work has been devotedt oc orrelate the glycan structure to an immunological response. [57] Zwitterionic polysaccharides (ZPs) from pathogenic bacteria can elicit T-cell proliferation, [58] whereas carbohydrates are generally poor T-cell stimulator. [59] Conformational studies on extracted ZPs revealed an extendedr ight-handed helix with positive and negative charges alternately distributed on the molecular surface. This three dimensionals tructure features ar egularly distributed groove, which servesa sp rimary binding domain. [60] To confirmt he correlationb etween the helical structure and the interaction with antibodies, ac ollection of zwitterionic Streptococcusp neumoniae serotype1 oligosaccharides was chemically synthetized. [13a] Structures with lengths of 3t o1 2m onosaccharide units were prepared, following ap reglycosylation-oxdidation strategy.Astrongc orrelation between the length of sugar chain and the antibody binding affinity was demonstrated, with the highesta ffinity for the nonasaccharide, able to adopt af ull helical turn ( Figure 13).
As imilarl ength-dependent immunological activity was observed for the Haemophilus influenzae b antigens. [61] Chemically synthesized glycoconjugates containing oligoribosyl-ribitolphosphate (PRP) with 4t o1 0r epeatingu nits show different immunogenicities, with the tetramer and octamera ble to elicit the highest antibody levels. This chain length dependence could be the result of ap articular three dimensional structure, best adopted withacertainn umber of repeating units (i.e.,4 and 8r epeating units).

Carbohydrate materials
Polysaccharides serve as important biomaterials in naturea nd are attractive resource of raw material for textile, food, paper, energy,a nd pharmaceutical industries. [62] Their primary structure determines their conformational preference and aggregation patterns, which eventually influence the material property. Still, these correlationsare far from being established.
Cellulose, the most abundant biomass in Nature, consists of repeating glucoseu nits, connected through b-1,4-glycosidic bonds. [63] Conformational studies based on natural cellulose have yieldedc onsiderable knowledge on its 3D structures owing to its high crystallinity,w hich allows for substantial Xray diffraction analysis. [64] To date, four types of cellulose crystalline forms based on different H-bonging patterns have been characterized (Cellulose I-IV). [65]   Chemical modificationsc an alter cellulose crystallinity and modulate cellulose properties. [9,66] However,the lack of regioselective modification strategies yields ill-defined compounds that preventd etailed structural studies. Automated glycan assembly (AGA) enabled rapid access to cellulose analogues with defined lengths. [67] A set of "unnatural" monosaccharide building blocks permitted the AGA of chemically modified analogues. With this approach, methylated, deoxygenated, deoxyfluorinated, as well as carboxymethylated cellulose analogues are synthesized with full control over the length (six or twelve units) and modification pattern (Figure 14). The modifications were designed to precisely tune the network of H-bonds, the steric bulk, and the electronic properties of cellulose.
The powder XRD profile of the non-modified analogues is identicalt on ative cellulose (Cellulose II), indicating that short oligomers (i.e.,h exasaccharides) adopt the same arrangement as the polysaccharide counterpart ( Figure 15). MD simulations revealed that modifications result in an increased conformational flexibility,w hich is reflected in an increased water solubility.N otably,m ethylated analogues with the same degree but different pattern of modification show drastic conformational differences. The analogue with evenly distributedm ethylation displays aq uasi-linear structure, whereas more compacted geometries are observed with ab lock-wisem odification pattern (Figure15). This also affect the solid state arrangement, with ah igher" cellulose character" observedf or the evenly methylated analogue and atotallyamorphous character for the block oligomer.T his synthetic approach wast hen extended to ionic cellulose analogues,b earing amino groups and/or carboxylic acids. [68] Structural analysisr eveals how the charge pattern affects glycanconformation. Different classes of oligo-and polysaccharides resembling naturala nd unnatural structures were synthesized with AGA.
[10b] MD simulations andN MR analysis indicate that each oligomer presents ad ifferent geometry and flexibility. For example,aa-1,6-oligomannoside adopts af lexible linear structure in water,w hile the analogue b-1,6-oligoglucoside displays am ore compact helical structure ( Figure 16). The synthetic approach permitted the synthesis of 13 C-labeled analogues,e nabling NMR analysis. A 13 C 6 -labeled glucoseu nit was inserted in specific position of the hexasaccharide chain, allowing for the measurement of J-couplings that confirmed the MD model. Glycosaminoglycans (GAGs) are an important class of structural materials, with vital biological roles in mammals. [69] GAGs are negatively charged polysaccharides composed of disaccharide repeating units. Hyaluronate (HA), chondroitin sulphate (CS), dermatan sulphate (DS), keratan sulphate (KS), heparan sulphate( HS) and heparin are the most commonG AGs. Their structurald iversity and conformational flexibility hampers their structurala nalysis. Chemical strategies to access well-defined structures are stillv ery labor demanding, [70] and big collections of related synthetic GAGs are not yet available. In addition, their denselyd istributed charges drasticallyi nfluence their interaction with water and metal ions, often resulting in the formation of gels. [71] Most GAGs are calculated to exhibit lefthandedh elices, except for chondroitin and dermatan sulfate, which display ar ight-handed helical structure. Fragmentso f  chondroitin andh yaluronanw ere used as models to identify the highly dynamic intramolecular H-bonds responsible for their conformation. [72] Most structural studies on GAGs oligomers are focusedo nl ocal conformational changes. While most monosaccharides maintain af ixed chair conformation (i.e., 4 C 1 and 1 C 4 ), l-iduronic acid, found in heparin and heparin sulphate, adoptss everal conformations. NMR analysis of as ynthetic pentasaccharide identified an unusual 2 S 0 skew-boat conformer as the major conformer of l-iduronic acid. [73] To understandt he biological meaning of this abnormal conformation, three pentasaccharides were synthesized with the l-iduronic residue locked into the 4 C 1 , 1 C 4 ,o r 2 S 0 conformations ( Figure 17). [74] Only the 2 S 0 -locked pentasaccharide strongly binds to antithrombina nd potentiates the inhibition of the blood coagulation protease factor.T his unambiguously demonstrates that the 2 S 0 skew-boat geometry is necessary for the control of bloodcoagulation. The importance of the 2 S 0 conformer was further investigated by using NMR experiments, crystallography,a nd computational methods, ultimately resulting in the visualization of the 2 S 0 conformer by X-ray diffraction. [75] The sulfation pattern also plays an important role in GAGs conformation and dynamics. Fragments of hyaluronic acids with different sulfation patterns (sHA) were synthesized and covalently linked to as urface. [76] Electrochemical impedance spectroscopy (EIS) suggested that the interaction between GAGs and metal ions is governedb yt he sulfation pattern rather than by the glycanc ore.

Summary and Outlook
For years, the complexity and inherent flexibility of glycansh as hampered their conformational analysis. As ar esult,o ur knowledge of carbohydrate structures dwarf in comparison to that of peptides and proteins.R ecent discoveries have given an ew impetust ot he structural analysiso fg lycans, showing that glycans can adopt defined secondary structures. [10b, 16a] Chemical synthesis has produced well-defined materials to simplify the analysis. [11] Several automated techniques are now available for the quick production of collections or related oligomers, as ideal probes for systematic structurals tudies. [10a, 77] Powerful NMR spectrometers have enabled detailed analysis, requiring a minute amount of compounds. [14a] These data is vital for the developments of reliable computationalm ethods. [15a] Still, structurala nalysiso fc arbohydrates is far from being routine in most synthetic laboratories.
Collaborativee fforts between synthetic anda nalytical experts have provent hat even relatively short oligosaccharides can adopt defined conformations.T hese structural features play an important role in protein recognition and can be exploited for the design of more potent glycomimetics. [12] To this end, chemical strategies able to disrupt or stabilizep articular conformations need to be developed. The conformational space accessible by an oligosaccharide define its aggregation, strongly influencing the carbohydrate material properties. [67] A better understanding of these interactions could drive the creation of carbohydrate materials by design.
To date, most analytical techniques produce ensemble-averaged resultsa nd might neglect importants tructuralf eatures responsible for ap articulara ctivity.N ovel single-molecule imaging techniques can overcome this limitation and allow for definitive structure-function correlations. [78] Ac ooperative effort between synthetic, analytical andc omputational chemists is required to ultimately understand glycans at the molecular level.