Glycosaminoglycans: What Remains To Be Deciphered?

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics.

Intramolecular crankshaft-type rearrangement in a photoisomerised glycoconjugate.

High-resolution NMR spectroscopy revealed that a novel glycoconjugate, consisting of two ß-glucopyranoses attached to a quinazolinone-like structure, exhibited photoisomerization around the -N-N[double bond, length as m-dash] and [double bond, length as m-dash]CH-C- bonds of the -N-N[double bond, length as m-dash]CH-C- linkage in the same timeframe (the so-called "crankshaft rotation") upon exposure to UV light. Experimental NMR data combined with DFT calculations discovered that the attachment of carbohydrate residues to photoactive compounds significantly changed the isomerization process and intramolecular rearrangement compared to the unglycosylated system, while the overall molecular structure remained virtually unchanged.

Chemistry towards Biology.

Although it may not seem like it, chemical biology has existed for a long time from today's perspective [...].

Chemistry towards Biology-Instruct: Snapshot.

The knowledge of interactions between different molecules is undoubtedly the driving force of all contemporary biomedical and biological sciences. Chemical biology/biological chemistry has become an important multidisciplinary bridge connecting the perspectives of chemistry and biology to the study of small molecules/peptidomimetics and their interactions in biological systems. Advances in structural biology research, in particular linking atomic structure to molecular properties and cellular context, are essential for the sophisticated design of new medicines that exhibit a high degree of druggability and very importantly, druglikeness. The authors of this contribution are outstanding scientists in the field who provided a brief overview of their work, which is arranged from in silico investigation through the characterization of interactions of compounds with biomolecules to bioactive materials.

A study of the photochemical behaviour and relaxation mechanisms of anti-syn isomerisation around quinazolinone -N-N[double bond, length as m-dash] bonds.

High-resolution NMR experiments revealed that differently substituted quinazolinone-based Schiff bases undergo anti to syn isomerisation on exposure to ultraviolet light in DMSO solution. The degree anti to syn conversion varied significantly upon substitution (between 5% and 100%) and also showed two noteworthy features: that relaxation back to the anti-form goes far faster (by at least 3 orders of magnitude) when the C6 rings B and C have ortho-OH substituents, and that relaxation can also be significantly sped up by addition of acid. Two possible mechanisms explaining the differences in relaxation process have been proposed: (I) the interaction of the azomethine hydrogen with the carbonyl oxygen results in slower reversion to the anti-form and/or (II) suppression of conjugation of the N3 lone pair with the N[double bond, length as m-dash]CH double bond by protonation and/or internal H-bonding. Both of these mechanisms have been analysed theoretically.

Photochemical anti-syn isomerization around the -N-N[double bond, length as m-dash] bond in heterocyclic imines.

EPR and NMR experiments on a quinazolinone-based Schiff's base in DMSO solution showed that irradiation with UV light (365 nm) leads to photochemically-induced isomerization from the anti- to the higher-energy syn-form around the -N-N[double bond, length as m-dash] linkage. The anti- to syn-isomerization was relatively fast, and the maximum amount of conversion detected (25%) was reached within 10 min; thermodynamic equilibrium re-established itself in about 15 min. DFT calculations were performed on the investigated compound and small model systems, and reproduced the experimental fact of the anti-conformer being lower in energy than the syn. Theoretical analysis of excited states, including visualisation of natural transition orbitals, identified possible pathways for syn-anti isomerisation, although the details vary with π-system size, making the use of small models of limited utility. The investigated compound probably isomerises through the third singlet excited state (S3), a π-π* excitation, relaxing through S2, also a π-π* state.

Solution Conformation of Heparin Tetrasaccharide. DFT Analysis of Structure and Spin⁻Spin Coupling Constants.

Density functional theory (DFT) has provided detailed information on the molecular structure and spin⁻spin coupling constants of heparin tetrasaccharide (GlcNS,6S-IdoA2S-GlcNS,6S-IdoA2S-OMe) representing the predominant heparin repeating-sequence. The fully optimised molecular structures of two tetrasaccharide conformations (differing from each other in the conformational form of the sulphated iduronic acid residue⁻one ¹C4 and the other ²S0) were obtained using the B3LYP/6-311+G(d,p) level of theory and applying explicit water molecules to simulate the presence of a solvent. The theoretical data provided insight into variations of the bond lengths, bond angles and torsion angles, formations of intra- and intermolecular hydrogen bonds and ionic interactions. Optimised molecular structures indicated the formation of a complex hydrogen bond network, including interresidue and intraresidue bonds. The ionic interactions strongly influence the first hydration shell and, together with hydrogen bonds, play an important role in shaping the 3D tetrasaccharide structure. DFT-derived indirect three⁻bond proton⁻proton coupling constants (³JH-C-C-H) showed that the best agreement with experiment was obtained with a weighted average of 67:33 (¹C4:²S0) of the IdoA2S forms. Detailed analysis of Fermi-contact contributions to ³JH-C-C-H showed that important contributions arise from the oxygen lone pairs of neighbouring oxygen atoms. The analysis also showed that the magnitude of diamagnetic spin⁻orbit contributions are sufficiently large to determine the magnitude of some proton⁻proton coupling constants. The data highlight the need to use appropriate quantum-chemical calculations for a detailed understanding of the solution properties of heparin oligosaccharides.

Solution Structure of Heparin Pentasaccharide: NMR and DFT Analysis.

High-resolution NMR and density functional theory (DFT) calculations have been applied to analysis of heparin pentasaccharide 3D structure in aqueous solution. The fully optimized molecular geometry of two pentasaccharide conformations (differing from each other in the form, one (1)C4 and the other (2)S0, of the sulfated iduronic acid residue) were obtained using the B3LYP/6-311+G(d,p) level of theory in the presence of solvent, the latter included as explicit water molecules. The presented approach enabled insight into variations of the bond lengths, bond angles, and torsion angles, formations of intra- and intermolecular hydrogen bonds, and ionic interactions in the two pentasaccharide conformations. A rather complex hydrogen bond network is formed, including inter-residue and intraresidue bonds between the NH group in the GlcN,3,6S with oxygens linked to C-2 at the IdoA2S residue and the glycosidic O-1 and the neighboring OSO3(-) group linked to C-3 in the same residue. On the other hand, because the first hydration shell is strongly influenced by strong ion-ion and ion-dipole interactions between sodium ions, sulfates, carboxylates, and -OH groups, ionic interactions play an important role in the stabilization of the 3D structure. The DFT-computed three-bond proton-proton coupling constants also showed that best agreement with experiment was obtained with a weighted average of 15:85 ((1)C4/(2)S0) of the sulfated iduronic acid forms indicating that the ratio is even more shifted toward the (2)S0 form than previously supposed. The DFT-computed pentasaccharide conformation differs from the previously published data, with the main changes at the glycosidic linkages, namely, the ψ1 torsion angles and the ϕ3 angle. The comparison of the glycosidic linkage torsion angle values in solution with the antithrombin-pentasaccharide complex also indicates that the pentasaccharide conformation changes upon binding to antithrombin III. The data supports the assumption that the protein selects the more populated (2)S0 conformer of heparin pentasaccharide and, consequently, the binding process of heparin pentasaccharide with antithrombin III is energetically more favorable than formerly expected.

Cationization of heparin for film applications.

Trimethylammonium-2-hydroxypropyl-(TMAHP) spacer was introduced into heparin (H) and the prepared films were characterized by elemental analysis, NMR, SEC-MALS, TG/DTG/DTA, AFM and mechanical tester. When quaternized at the ratio of H/NaOH/alkylating agent/H2O=0.1-1/0-2/0.1-1/50-500 mmol, H was substituted at A6 and A3 positions. The formation of double-substituted structures by substitution of free hydroxyl group of the previously introduced TMAHP substituent is evident. In the absence of NaOH (H/GTMAC/H2O=1:1:500) the most drastic decrease of M(n) to 8.639 kg/mol and M(w)/M(n) at 1.48 was observed in comparison to H (Mn=9532 g/mol with M(w)/M(n)=1.38). The film mechanical properties were better on H (E=4030 MPa; σ(b)=65 MPa; ɛ(b)=4.6%) than on quaternized specimens (E=2500-3340 MPa; σ(b)=25-40 MPa; ɛ(b)=1.7-1.8%). The AFM images did not prove relation between mechanical properties and surface shape.

NMR and DFT analysis of trisaccharide from heparin repeating sequence.

NMR and density functional theory (DFT) have afforded detailed information on the molecular geometry and spin-spin coupling constants of a trisaccharide from the heparin repeating-sequence. The fully optimized molecular structures of two trisaccharide conformations (differing from each other in the form of the central iduronic acid residue) were obtained using the B3LYP/6-311+G(d,p) level of theory in the presence of solvent, the latter included as either explicit water molecules or via a continuum solvent model. NMR spin-spin coupling constants were also computed using various basis sets and functionals and then compared with measured experimental values. Optimized structures for both conformers showed differences in geometry at the glycosidic linkages and in the formation of intramolecular hydrogen bonds. Three-bond proton-proton coupling constants ((3)JH-C-C-H), based on fully optimized geometry computed using the B3LYP/6-311+G(d,p)/UFF level of theory and hydrated with 57 water molecules, showed that the best agreement with experiment was obtained with the 6-311+G(d,p) basis set and a weighted average of 55:45 ((1)C4:(2)S0) of the IdoA2S forms. Other basis sets, DGDZVP and TZVP, also gave acceptable data for most coupling constants, with DGDZVP outperforming the TZVP. Detailed analysis of Fermi-contact contributions to (3)JH-C-C-H showed that important contributions arise from oxygen at both glycosidic linkages, as well as from oxygen atoms on the neighboring monosaccharide units. Their contribution to the Fermi term cannot be neglected and must be taken into account for a correct description of coupling constants. The analysis also showed that the magnitude of paramagnetic (PSO) and diamagnetic (DSO) spin-orbit contributions is comparable to the magnitude of the Fermi-contact contribution in some coupling constants in the IdoA2S residue. Calculations of the localized molecular orbital contributions to the DSO terms from separate conformational residues showed that the contribution from adjacent residues is not negligible and can be important for the spin-spin coupling constants between protons located close to the geometrical center of the molecule. These contributions should be taken into account when interpreting DSO terms in spin-spin coupling constants especially in large molecules.

A new type of rearrangement in branched-chain carbohydrates: isomerization of 3-C-branched aldoses.

A new type of rearrangement is described for 3-C-branched chain aldoses. The studied transformation is based on the Mo(VI)-catalyzed isomerization of carbohydrate carbon skeleton and allows preparation of C-3 isomers of 3-C-branched aldoses in a simple way without formation of side products. This rearrangement at C-3 carbon differs from the previously described epimerization at C-2 of aldoses catalyzed by Mo(VI) ions, known as Bílik reaction. The potential of this new transformation is illustrated on the preparation of new, 3-C-methyl-D-glucose and 3-C-vinyl-D-glucose from 3-C-methyl-d-allose and 3-C-vinyl-D-allose, respectively.

Effect of solvent and counterions upon structure and NMR spin-spin coupling constants in heparin disaccharide.

Density functional theory (DFT) has been used to analyze structure and NMR spin-spin coupling constants in heparin disaccharide. Both B3LYP/6-311++G** and M05-2X/6-311++G** methods have been used for optimization of disaccharide geometry. Solvent effect was treated by use of explicit water molecules. Solvent-caused variations of DFT-computed indirect one-bond proton-carbon coupling constants up to 17 Hz between isolated and solvated states, however, had a limited influence upon magnitudes of proton-proton spin-spin coupling constants. Interatomic distances and bond and torsion angles indicated that the structure of the 2-O-sulfated iduronic acid residue affected the geometry of the N,6-sulfated glucosamine residue. Optimized disaccharide geometry showed that the change of counterion (Ca(2+) instead of Na(+)) influenced geometry of pyranose rings and the glycosidic linkage conformation. DFT-computed three-bond proton-proton spin-spin coupling constants agreed well with published experimental data and indicated that the population of the (1)C(4) chair form of the 2-O-sulfated iduronic acid residue increased in the presence of Ca(2+) ions compared to the presence of Na(+) ions. Analysis also showed that the Fermi contact term was not always dominant and that paramagnetic and diamagnetic contributions considerably influenced magnitudes of proton-proton spin-spin coupling constants.

Residual dipolar coupling investigation of a heparin tetrasaccharide confirms the limited effect of flexibility of the iduronic acid on the molecular shape of heparin.

The solution conformation of a fully sulfated heparin-derived tetrasaccharide, I, was studied in the presence of a 4-fold excess of Ca(2+). Proton-proton and proton-carbon residual dipolar couplings (RDCs) were measured in a neutral aligning medium. The order parameters of two rigid hexosamine rings of I were determined separately using singular value decomposition and ab initio structures of disaccharide fragments of I. The order parameters were very similar implying that a common order tensor can be used to analyze the structure of I. Using one order tensor, RDCs of both hexosamine rings were used as restraints in molecular dynamics simulations. RDCs of the inner iduronic acid were calculated for every point of the molecular dynamics trajectory. The fitting of the calculated RDCs of the two forms of the iduronic acid to the experimental values yielded a population of (1)C(4) and (2)S(o) conformers of iduronic acid that agreed well with the analysis based on proton-proton scalar coupling constants. The glycosidic linkage torsion angles in RDC-restrained molecular dynamics (MD) structures of I are consistent with the interglycosidic three-bond proton-carbon coupling constants. These structures also show that the shape of heparin is not affected dramatically by the conformational flexibility of the iduronic acid ring. This is in line with conclusions of previous studies based on MD simulations and the analysis of (1)H-(1)H NOEs. Our work therefore demonstrates the effectiveness of RDCs in the conformational analysis of glycosaminoglycans.

Minimum FGF2 binding structural requirements of heparin and heparan sulfate oligosaccharides as determined by NMR spectroscopy.

Heparin and heparan sulfate (HS) glycosaminoglycans (HSGAGs) are sulfated polysaccharidesthat play important roles in fundamental biological processes by binding to proteins. The prototypic exampleof HSGAG-protein interactions is that with the fibroblast growth factors (FGFs), specifically FGF1 andFGF2. Structural and biochemical studies have shown that the chain length, sulfation pattern, andconformation of HSGAGs play a critical role in FGF binding and activity. Previously, we showed that atetrasaccharide of the form ANS,6X-I2S-ANS,6X-I2S-OPr (where X is OH or O-sulfate and Pr is propyl) withat least one of the ANS,6X residues having a 6-O sulfate group was the minimum binding motif for FGF1[Guerrini, M., Agulles, T., Bisio, A., Hricovini, M., Lay, L., Naggi, A., Poletti, L., Sturiale, L., Torri, G.,and Casu, B. (2002) Biochem. Biophys. Res. Commun. 292, 222-230]. We report NMR structural analysisusing two-dimensional NOE spectroscopy (2D-NOESY) and transferred NOESY (trNOESY) on a non-6-O-sulfated synthetic tetrasaccharide TETRA (ANS-I2S-ANS-I2S-OPr) both in its free state and bound toFGF2. This tetrasaccharide comprises both the structural trisaccharide motif ANS-I2S-ANS that forms "kinks"in longer heparin chains induced by FGF binding [Raman, R., Venkataraman, G., Ernst, S., Sasisekharan,V., and Sasisekharan, R. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2357-2362] and the common bindingmotif I2S-ANS-I2S present in octasaccharides that exhibited strong FGF2 binding [Kreuger, J., Salmivirta,M., Sturiale, L., Gimenez-Gallego, G., and Lindahl, U. (2001) J. Biol. Chem. 276, 30744-30752]. Thesedata suggest that TETRA could be the shortest HSGAG oligosaccharide that binds to FGF2. Furthermore,our study confirms that both the IdoA residues in TETRA adopt the chair 1C4 conformation upon FGF2binding to provide the best molecular fit in contrast to an analogous 6-O-sulfated tetrasaccharide motifobserved in the FGF2-HSGAG cocrystal structure where one of the IdoAs adopts skew-boat 2SOconformation. Thus, our study highlights the fact that the conformational plurality of IdoA is able toaccommodate the changes in the sulfation pattern to provide the necessary specificity for protein binding.

Interaction of heparins with fibroblast growth factors: conformational aspects.

Heparin and heparin-like oligo- and polysaccharides bind to fibroblast growth factors (FGFs) and modulate their ability to form active ternary complexes with FGF receptors (FGFRs). Considerable efforts have been made in recent years to identify the minimal heparin and heparan sulfate (HS) sequences that bind and activate individual FGFs. Heparin sequences involved in interaction with FGFs invariably contain at least one residue of 2-O-sulfated iduronic acid (IdoA2S), which adopts either the (1)(4) chair conformation or the equi-energetic skew-boat (2)S(0). In solution and in the absence of a binding protein, both these conformations are present in a dynamic equilibrium. In oligosaccharide-protein co-crystals, the protein selects those conformers that provide optimal contacts. The crystalline structure of a heparin hexasaccharide/FGF complex exhibits one of the two IdoA2S residues in the active site of the growth factor in (1)C(4) conformation and the other (outside the active site) in (2)S(0) conformation. NMR studies suggest that active conformations of heparin/HS oligosaccharides in solution could be distinct from those adopted in crystals. Heparin tetrasaccharides in the presence of FGF1 and FGF2 have both their IdoA2S residues prevalently in the (1)C(4) form. Current NMR and molecular modelling studies are being extended to longer heparin oligosaccharides as well as to heparins with "glycol-split" residues along their chains.

A simple NMR analysis of the protonation equilibrium that accompanies aminoglycoside recognition: dramatic alterations in the neomycin-B protonation state upon binding to a 23-mer RNA aptamer.

A complete characterisation of the protonation equilibrium that accompanies the molecular recognition of neomycin-B by a specific RNA receptor has been achieved by employing simple NMR measurements.

B3LYP/6-311++G** study of structure and spin-spin coupling constant in methyl 2-O-sulfo-alpha-L-iduronate.

Structures of three most stable conformers ((1)C4, (4)C1, (2)S0) of methyl 2-O-sulfo-alpha-L-iduronate monosodium salt have been analyzed by DFT using the B3LYP/6-311++G** method. The optimized geometries confirmed the influence of both 2-O-sulfate and carboxylate groups upon the pyranose ring geometry. The computed energies showed that the chair (1)C4 form is the most stable one. Time-averaged DFT-calculated proton-proton and proton-carbon spin-spin coupling constants agree with the experimental data and indicate that only two chair forms contribute to the conformational equilibrium of methyl 2-O-sulfo-alpha-L-iduronate monosodium salt. The influence of the charged groups upon the magnitudes of spin-spin coupling constants is also discussed.

Dynamic properties of biologically active synthetic heparin-like hexasaccharides.

A complete study of the dynamics of two synthetic heparin-like hexasaccharides, D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (1) and -->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHAc-6-SO4-alpha-(1-->4)-L-IdoA-alpha-(1-->4)-D-GlcNHSO3-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (2), has been performed using 13C-nuclear magnetic resonance (NMR) relaxation parameters, T1, T2, and heteronuclear nuclear Overhauser effect (NOEs). Compound 1 is constituted from sequences corresponding to the major polysaccharide heparin region, while compound 2 contains a sequence never found in natural heparin. They differ from each other only in sulphation patterns, and are capable of stimulating fibroblast growth factors (FGFs)-1 induced mitogenesis. Both oligosaccharides exhibit a remarkable anisotropic overall motion in solution as revealed by their anisotropic ratios (tau /tau||), 4.0 and 3.0 respectively. This is a characteristic behaviour of natural glycosaminoglycans (GAG) which has also been observed for the antithrombin (AT) binding pentasaccharide D-GlcNHSO3-6-SO4-alpha-(1-->4)-D-GlcA-beta-(1-->4)-D-GlcNHSO3-(3,6-SO4)-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-1-->Me (3) (Hricovíni, M., Guerrini, M., Torri, G., Piani, S., and Ungarelli, F. (1995) Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study. Carbohydr. Res., 277, 11-23). The motional properties observed for 1 and 2 provide additional support to the suitability of these compounds as heparin models in agreement with previous structural (de Paz, J.L., Angulo, J., Lassaletta, J.M., Nieto, P.M., Redondo-Horcajo, M., Lozano, R.M., Jiménez-Gallego, G., and Martín-Lomas, M. (2001) The activation of fibroblast growth factors by heparin: synthesis, structure and biological activity of heparin-like oligosaccharides. Chembiochem, 2, 673-685; Ojeda, R., Angulo, J., Nieto, P.M., and Martin-Lomas. M. (2002) The activation of fibroblast growth factors by heparin: synthesis and structural study of rationally modified heparin-like oligosaccharides. Can. J. Chem,. 80, 917-936; Lucas, R., Angulo, J., Nieto, P.M., and Martin-Lomas, M. (2003) Synthesis and structural studies of two new heparin-like hexasaccharides. Org. Biomol. Chem., 1, 2253-2266) and biological data (Angulo, J., Ojeda, R., de Paz, J.L., Lucas, R., Nieto, P.M., Lozano, R.M., Redondo-Horcajo, M., Giménez-Gallego, G., and Martín-Lomas, M. (2004) The activation of fibroblast growth factors (FGFs) by glycosaminoglycans: influence of the sulphation pattern on the biological activity of FGF-1. Chembiochem, 5, 55-61). Fast internal motions observed for the less sulphated compound 2, as compared with 1, may be related to their different behavior in stimulating FGF1-induced mitogenic activity.

Structural aspects of carbohydrates and the relation with their biological properties.

Structural diversity of carbohydrates plays a crucial role in their large variety of roles in biological systems. This paper focuses on aspects of structure and biological functions of three classes of carbohydrates, N-linked oligosaccharides, blood group oligosaccharides and glycosaminoglycans. Conformations and dynamics in solution, as well as structure of protein-carbohydrate complexes are discussed. A short overview also describes theoretical and experimental methodologies that are used in current glycobiological research, particularly high-resolution NMR spectroscopy, X-ray crystallography and methods of computational chemistry.