Sugars are crucial components in biosystems and industrial applications. In aqueous environments, the natural state of short saccharides or charged glycosaminoglycans is floating and wiggling in solution. Therefore, tools to characterize their structure in a native aqueous environment are crucial but not always available. Here, we show that a combination of Raman/ROA and, on occasions, NMR experiments with Molecular Dynamics (MD) and Quantum Mechanics (QM) is a viable method to gain insights into structural features of sugars in solutions. Combining these methods provides information about accessible ring puckering conformers and their proportions. It also provides information about the conformation of the linkage between the sugar monomers, i.e., glycosidic bonds, allowing for identifying significantly accessible conformers and their relative abundance. For mixtures of sugar moieties, this method enables the deconvolution of the Raman/ROA spectra to find the actual amounts of its molecular constituents, serving as an effective analytical technique. For example, it allows calculating anomeric ratios for reducing sugars and analyzing more complex sugar mixtures to elucidate their real content. Altogether, we show that combining Raman/ROA spectroscopies with simulations is a versatile method applicable to saccharides. It allows for accessing many features with precision comparable to other methods routinely used for this task, making it a viable alternative. Furthermore, we prove that the proposed technique can scale up by studying the complicated raffinose trisaccharide, and therefore, we expect its wide adoption to characterize sugar structural features in solution.

Department of Chemistry University of Antwerp Antwerp Belgium

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Vuorio J, Škerlová J, Fábry M, Veverka V, Vattulainen I, Řezáčová P, et al.. N-glycosylation can selectively block or foster different receptor–ligand binding modes. Scientific Reports. 2021;11(1). doi: 10.1038/s41598-021-84569-z PubMed DOI PMC

Tarbell JM, Cancel LM. The glycocalyx and its significance in human medicine. Journal of Internal Medicine. 2016;280(1):97–113. doi: 10.1111/joim.12465 PubMed DOI

Tang Y, Cheng F, Feng Z, Jia G, Li C. Stereostructural elucidation of glucose phosphorylation by Raman optical activity. The Journal of Physical Chemistry B. 2019;123(37):7794–7800. doi: 10.1021/acs.jpcb.9b05968 PubMed DOI

Sodhi H, Panitch A. Glycosaminoglycans in tissue engineering: A review. Biomolecules. 2020;11(1):29. doi: 10.3390/biom11010029 PubMed DOI PMC

Imberty A, Pérez S. Structure, conformation, and dynamics of bioactive oligosaccharides: theoretical approaches and experimental validations. Chemical Reviews. 2000;100(12):4567–4588. doi: 10.1021/cr990343j PubMed DOI

Wormald MR, Petrescu AJ, Pao YL, Glithero A, Elliott T, Dwek RA. Conformational studies of oligosaccharides and glycopeptides: Complementarity of NMR, X-ray crystallography, and molecular modelling. Chemical Reviews. 2002;102(2):371–386. doi: 10.1021/cr990368i PubMed DOI

Rudolph MG, Speir JA, Brunmark A, Mattsson N, Jackson MR, Peterson PA, et al.. The crystal structures of Kbm1 and Kbm8 reveal that subtle changes in the peptide environment impact thermostability and alloreactivity. Immunity. 2001;14(3):231–242. doi: 10.1016/S1074-7613(01)00105-4 PubMed DOI

Peréz S, Mouhous-Riou N, Nifant’ev NE, Tsvetkov YE, Bachet B, Imberty A. Crystal and molecular structure of a histo-blood group antigen involved in cell adhesion: the Lewis x trisaccharide. Glycobiology. 1996;6(5):537–542. doi: 10.1093/glycob/6.5.537 PubMed DOI

Barron LD, Bogaard P, Buckingham AD. Raman scattering of circularly polarized light by optically active molecules. Journal of the American Chemical Society. 1973;95(2):603–605. doi: 10.1021/ja00783a058 DOI

Palivec V, Kopecký V, Jungwirth P, Bouř P, Kaminský J, Martinez-Seara H. Simulation of Raman and Raman optical activity of saccharides in solution. Physical Chemistry Chemical Physics. 2020;22(4). doi: 10.1039/C9CP05682C PubMed DOI

Palivec V, Michal P, Kapitán J, Martinez-Seara H, Bouř P. Raman optical activity of glucose and sorbose in extended wavenumber range. ChemPhysChem. 2020;21(12). doi: 10.1002/cphc.202000261 PubMed DOI

Mutter ST, Zielinski F, Johannessen C, Popelier PLA, Blanch EW. Distinguishing epimers through Raman optical activity. Journal of Physical Chemistry A. 2016;120(11):1908–1916. doi: 10.1021/acs.jpca.6b00358 PubMed DOI

Melcrová A, Kessler J, Bouř P, Kaminský J. Simulation of Raman optical activity of multi-component monosaccharide samples. Physical Chemistry Chemical Physics. 2016;18(3):2130–2142. doi: 10.1039/C5CP04111B PubMed DOI

Kaminský J, Kapitán J, Baumruk V, Bednárová L, Bouř P. Interpretation of Raman and Raman optical activity spectra of a flexible sugar derivative, the gluconic acid anion. Journal of Physical Chemistry A. 2009;113(15):3594–3601. doi: 10.1021/jp809210n PubMed DOI

Rüther A, Forget A, Roy A, Carballo C, Mießmer F, Dukor RK, et al.. Unravelling a direct role for polysaccharide β-Strands in the higher order structure of physical hydrogels. Angewandte Chemie International Edition. 2017;56(16):4603–4607. doi: 10.1002/anie.201701019 PubMed DOI

Pendrill R, Mutter ST, Mensch C, Barron LD, Blanch EW, Popelier PLA, et al.. Solution structure of mannobioses unravelled by means of Raman optical activity. ChemPhysChem. 2019;20(5):695–705. doi: 10.1002/cphc.201801172 PubMed DOI

Brehm M, Thomas M. Computing Bulk Phase Raman Optical Activity Spectra from ab initio Molecular Dynamics Simulations. Journal of Physical Chemistry Letters. 2017;8(14):3409–3414. doi: 10.1021/acs.jpclett.7b01616 PubMed DOI

Cheeseman JR, Shaik MS, Popelier PLA, Blanch EW. Calculation of Raman optical activity spectra of methyl-β-d-glucose incorporating a full molecular dynamics simulation of hydration effects. Journal of the American Chemical Society. 2011;133(13):4991–4997. doi: 10.1021/ja110825z PubMed DOI

Zielinski F, Mutter ST, Johannessen C, Blanch EW, Popelier PLA. The Raman optical activity of β-d-xylose: where experiment and computation meet. Physical chemistry chemical physics: PCCP. 2015;17(34):21799–809. doi: 10.1039/C5CP02969D PubMed DOI

Ghidinelli S, Abbate S, Koshoubu J, Araki Y, Wada T, Longhi G. Solvent Effects and Aggregation Phenomena Studied by Vibrational Optical Activity and Molecular Dynamics: The Case of Pantolactone. Journal of Physical Chemistry B. 2020;124(22):4512–4526. doi: 10.1021/acs.jpcb.0c01483 PubMed DOI PMC

Perera AS, Thomas J, Poopari MR, Xu Y. The clusters-in-a-liquid approach for solvation: New insights from the conformer specific gas phase spectroscopy and vibrational optical activity spectroscopy. Frontiers in Chemistry. 2016;4(FEB):9. doi: 10.3389/fchem.2016.00009 PubMed DOI PMC

Perera AS, Cheramy J, Merten C, Thomas J, Xu Y. IR, Raman, and Vibrational Optical Activity Spectra of Methyl Glycidate in Chloroform and Water: The Clusters-in-a-liquid Solvation Model. ChemPhysChem. 2018;19(17):2234–2242. doi: 10.1002/cphc.201800309 PubMed DOI

Cheeseman JR, Frisch MJ. Basis set dependence of vibrational Raman and Raman optical activity intensities. Journal of Chemical Theory and Computation. 2011;7(10):3323–3334. doi: 10.1021/ct200507e PubMed DOI

Zuber G, Hug W. Rarefied Basis Sets for the calculation of Optical Tensors. 1. The Importance of gradients on hydrogen atoms for the Raman scattering tensor. The Journal of Physical Chemistry A. 2004;108(11):2108–2118. doi: 10.1021/jp031284n DOI

Bouř P, Keiderling TA. Partial optimization of molecular geometry in normal coordinates and use as a tool for simulation of vibrational spectra. The Journal of Chemical Physics. 2002;117(9):4126–4132. doi: 10.1063/1.1498468 DOI

Cremer D, Pople AJ. General definition of ring puckering coordinates. Journal of the American Chemical Society. 1975;97(6):1354–1358. doi: 10.1021/ja00839a011 DOI

ACDLabs. ChemSketch 2015.2.5.

Inkscape. http://www.inkscape.org/; 2020.

Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, et al.. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1-2:19–25. doi: 10.1016/j.softx.2015.06.001 DOI

Bonomi M, Bussi G, Camilloni C, Tribello GA, Banáš P, Barducci A, et al.. Promoting transparency and reproducibility in enhanced molecular simulations. Nature Methods. 2019;16(8):670–673. doi: 10.1038/s41592-019-0506-8 PubMed DOI

Kirschner KN, Yongye AB, Tschampel SM, González-Outeiriño J, Daniels CR, Foley BL, et al.. GLYCAM06: A generalizable biomolecular force field. Carbohydrates. Journal of Computational Chemistry. 2008;29(4):622–655. doi: 10.1002/jcc.20820 PubMed DOI PMC

Izadi S, Onufriev AV. Accuracy limit of rigid 3-point water models. The Journal of Chemical Physics. 2016;145(7):074501. doi: 10.1063/1.4960175 PubMed DOI PMC

Barducci A, Bussi G, Parrinello M. Well-tempered metadynamics: A smoothly converging and tunable free-energy method. Physical Review Letters. 2008;100(2):020603. doi: 10.1103/PhysRevLett.100.020603 PubMed DOI

Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al. Gaussian16 Revision B.01; 2016.

Orozco M, Marchn I, Soteras I, Vreven T, Morokuma K, Mikkelsen KV, et al.. Beyond the continuum approach. In: Continuum solvation models in chemical physics. Chichester, UK: John Wiley & Sons, Ltd; 2007. p. 499–605. Available from: http://doi.wiley.com/10.1002/9780470515235.ch4. DOI

Dapprich S, Komáromi I, Byun KS, Morokuma K, Frisch MJ. A new ONIOM implementation in Gaussian98. Part I. The calculation of energies, gradients, vibrational frequencies and electric field derivatives. Journal of Molecular Structure: THEOCHEM. 1999;461-462:1–21. doi: 10.1016/S0166-1280(98)00475-8 DOI

Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics. 1983;79(2):926–935. doi: 10.1063/1.445869 DOI

Nafie L. Vibrational optical activity: Principles and applications. Chichester: Wiley; 2011.

Ruud K, Helgaker T, Bouř P. Gauge-origin independent density-functional theory calculations of vibrational Raman optical activity. Journal of Physical Chemistry A. 2002;106(32):7448–7455. doi: 10.1021/jp026037i DOI

Polavarapu PL, Covington CL. Comparison of Experimental and Calculated Chiroptical Spectra for Chiral Molecular Structure Determination. Chirality. 2014;26(9):539–552. doi: 10.1002/chir.22316 PubMed DOI

Säwén E, Massad T, Landersjö C, Damberg P, Widmalm G. Population distribution of flexible molecules from maximum entropy analysis using different priors as background information: Application to the, ψ-conformational space of the α-(1→2)-linked mannose disaccharide present in N- and O-linked glycoproteins. Organic and Biomolecular Chemistry. 2010;8(16):3684–3695. PubMed

Jensen F. Basis set convergence of nuclear magnetic shielding constants calculated by density functional methods. Journal of Chemical Theory and Computation. 2008;4(5):719–727. doi: 10.1021/ct800013z PubMed DOI

Sattelle BM, Hansen SU, Gardiner J, Almond A. Free energy landscapes of iduronic acid and related monosaccharides. Journal of the American Chemical Society. 2010;132(38):13132–13134. doi: 10.1021/ja1054143 PubMed DOI

Mikkelsen LM, Hernáiz MJ, Martín-Pastor M, Skrydstrup T, Jiménez-Barbero J. Conformation of Glycomimetics in the Free and Protein-Bound State: Structural and Binding Features of the C-glycosyl Analogue of the Core Trisaccharide α-d-Man-(1→3)-[α-d-Man-(1→6)]-d-Man. Journal of the American Chemical Society. 2002;124(50):14940–14951. doi: 10.1021/ja020468x PubMed DOI

Raich I, Lövyová Z, Trnka L, Parkan K, Kessler J, Pohl R, et al.. Limitations in the description of conformational preferences of C-disaccharides: The (1→3)-C-mannobiose case. Carbohydrate research. 2017;451:42–50. doi: 10.1016/j.carres.2017.09.006 PubMed DOI

Bouckaert J, Hamelryck TW, Wyns L, Loris R. The crystal structures of Man(alpha1-3)Man(alpha1-O)Me and Man(alpha1-6)Man(alpha1-O)Me in complex with concanavalin A. The Journal of biological chemistry. 1999;274(41):29188–29195. doi: 10.1074/jbc.274.41.29188 PubMed DOI

Brisson JR, Carver JP. Solution conformation of asparagine-linked oligosaccharides: .alpha.(1-2)-, .alpha.(1-3)-, .beta.(1-2)-, and .beta.(1-4)-linked units. Biochemistry. 2002;22(15):3671–3680. doi: 10.1021/bi00284a021 PubMed DOI

Kim HM, Choi YJ, Lee JH, Jeong KJ, Jung SH. Conformational analysis of trimannoside and bisected trimannoside using aqueous molecular dynamics simulations. Bulletin of the Korean Chemical Society. 2009;30(11):2723–2728. doi: 10.5012/bkcs.2009.30.11.2723 DOI

Kan Z, Yan X, Ma J. Conformation dynamics and polarization effect of α,α-trehalose in a vacuum and in aqueous and salt solutions. The journal of physical chemistry A. 2015;119(9):1573–1589. doi: 10.1021/jp507692h PubMed DOI

Bock K, Defaye J, Driguez H, Bar-Guilloux E. Conformations in solution of α,α-trehalose, α-d-glucopyranosyl α-d-mannopyranoside, and their 1-thioglycosyl analogs, and a tentative correlation of their behaviour with respect to the enzyme trehalase. European journal of biochemistry. 1983;131(3):595–600. doi: 10.1111/j.1432-1033.1983.tb07304.x PubMed DOI

Choi Y, Cho KW, Jeong K, Jung S. Molecular dynamics simulations of trehalose as a’dynamic reducer’ for solvent water molecules in the hydration shell. Carbohydrate Research. 2006;341(8):1020–1028. doi: 10.1016/j.carres.2006.02.032 PubMed DOI

Olsson U, Säwén E, Stenutz R, Widmalm G. Conformational flexibility and dynamics of two (1→6)-linked disaccharides related to an oligosaccharide epitope expressed on malignant tumour cells. Chemistry—A European Journal. 2009;15(35):8886–8894. doi: 10.1002/chem.200900507 PubMed DOI

Zhu Y, Zajicek J, Serianni AS. Acyclic forms of [1-13C]aldohexoses in aqueous solution: Quantitation by 13C NMR and deuterium isotope effects on tautomeric equilibria. Journal of Organic Chemistry. 2001;66(19):6244–6251. doi: 10.1021/jo010541m PubMed DOI

Bubb WA. NMR spectroscopy in the study of carbohydrates: Characterizing the structural complexity. Concepts in Magnetic Resonance. 2003;19A(1):1–19. doi: 10.1002/cmr.a.10080 DOI

Franks F, Lillford PJ, Robinson G. Isomeric equilibria of monosaccharides in solution. Influence of solvent and temperature. Journal of the Chemical Society, Faraday Transactions 1. 1989;85(8):2417. doi: 10.1039/f19898502417 DOI

Bezabeh T, Ijare OB, Albiin N, Arnelo U, Lindberg B, Smith ICP. Detection and quantification of d-glucuronic acid in human bile using 1H NMR spectroscopy: relevance to the diagnosis of pancreatic cancer. Magnetic Resonance Materials in Physics, Biology and Medicine. 2009;22(5):267–275. doi: 10.1007/s10334-009-0171-5 PubMed DOI

Moreau B, Lognay G, Blecker C, Destain J, Gerbaux P, Chéry F, et al.. Chromatographic, spectrometric and NMR characterization of a new Set of glucuronic acid esters synthesized by lipase. Biotechnol Agron Soc Environ. 2007;11(1):9–17.

Liu FC, Su CR, Wu TY, Su SG, Yang HL, Lin JHY, et al.. Efficient 1H-NMR quantitation and investigation of N-Acetyl-d-glucosamine (GlcNAc) and N,N’-diacetylchitobiose (GlcNAc)2 from chitin. International Journal of Molecular Sciences. 2011;12(9):5828–5843. doi: 10.3390/ijms12095828 PubMed DOI PMC

Espinosa JF, Bruix M, Jarreton O, Skrydstrup T, Beau JM, Jiménez-Barbero J. Conformational differences between C- and O-glycosides: The α-C-mannobiose/α-O-mannobiose case. Chemistry—A European Journal. 1999;5(2):442–448. doi: 10.1002/(SICI)1521-3765(19990201)5:2<442::AID-CHEM442>3.0.CO;2-1 DOI

Naidoo KJ, Denysyk D, Brady JW. Molecular dynamics simulations of the N-linked oligosaccharide of the lectin from Erythrina corallodendron. Protein Engineering Design and Selection. 1997;10(11):1249–1261. doi: 10.1093/protein/10.11.1249 PubMed DOI

Zhang W, Turney T, Meredith R, Pan Q, Sernau L, Wang X, et al.. Conformational Populations of β-(1→4) O-glycosidic linkages using redundant NMR J-couplings and circular statistics. Journal of Physical Chemistry B. 2017;121(14):3042–3058. doi: 10.1021/acs.jpcb.7b02252 PubMed DOI PMC

Zhang W, Meredith R, Pan Q, Wang X, Woods RJ, Carmichael I, et al.. Use of circular statistics to model αMan-(1→2)-αMan and αMan-(1→3)-α/βMan O-glycosidic linkage conformation in 13C-labeled disaccharides and high-mannose oligosaccharides. Biochemistry. 2019;58(6):546–560. doi: 10.1021/acs.biochem.8b01050 PubMed DOI

Alonso-Gil S, Parkan K, Kaminský J, Pohl R, Miyazaki T, Alonso-Gil S, et al. Unlocking the hydrolytic mechanism of GH92 α-1,2-mannosidases: computation inspires using C-glycosides as Michaelis complex mimics; 2021. Available from: https://chemrxiv.org/engage/chemrxiv/article-details/618a3df7e04a8ec2a42cbd84. PubMed PMC

Dudek M, Zajac G, Szafraniec E, Wiercigroch E, Tott S, Malek K, et al.. Raman Optical Activity and Raman spectroscopy of carbohydrates in solution. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2019;206:597–612. doi: 10.1016/j.saa.2018.08.017 PubMed DOI

Profant V, Johannessen C, Blanch EW, Bouř P, Baumruk V. Effects of sulfation and the environment on the structure of chondroitin sulfate studied via Raman optical activity. Physical Chemistry Chemical Physics. 2019;21(14):7367–7377. doi: 10.1039/C9CP00472F PubMed DOI

Bogaerts J, Desmet F, Aerts R, Bultinck P, Herrebout W, Johannessen C. A combined Raman optical activity and vibrational circular dichroism study on artemisinin-type products. Physical Chemistry Chemical Physics. 2020;22(32):18014–18024. doi: 10.1039/D0CP03257C PubMed DOI

Koenis MAJ, Tiekink EH, van Raamsdonk DME, Joosten NU, Gooijer SA, Nicu VP, et al.. Analytical chemistry on many-center chiral compounds based on vibrational circular dichroism: Absolute configuration assignments and determination of contaminant levels. Analytica Chimica Acta. 2019;1090:100–105. doi: 10.1016/j.aca.2019.09.021 PubMed DOI

Yamamoto S, Kaminský J, Bouř P. Structure and vibrational motion of insulin from Raman optical activity spectra. Analytical Chemistry. 2012;84(5):2440–2451. doi: 10.1021/ac2032436 PubMed DOI

Kaminský J, Horáčková F, Biačková N, Hubáčková T, Socha O, Kubelka J. Double hydrogen bonding dimerization propensity of aqueous hydroxy acids investigated using vibrational optical activity. The Journal of Physical Chemistry B. 2021;125:11350–11363. doi: 10.1021/acs.jpcb.1c05480 PubMed DOI