Abstract
Methyl 3-O-α-d-glucopyranosyl 2-acetamido-2-deoxy-α-d-galactopyranoside as a monohydrate, C15H27NO11·H2O, crystallizes in space group P212121, with four molecules in the unit cell. It constitutes the methyl glycoside of the carbohydrate part of the teichoic acid type polysaccharide from Micrococcus sp. A1, in which the disaccharides are joined through phosphodiester linkages. The conformation of the disaccharide is described by the glycosidic torsion angles ϕ = − 31° and ψ = + 1°, and the hydroxymethyl groups of the constituent monosaccharides are present in the gg and gt conformations for the sugar residues having the gluco- and galacto-configuration, respectively. For the N-acetyl group at C2 of the galactosamine residue the torsion angle τH = 147°, i.e., the amide proton has an antiperiplanar relationship to H2 of the sugar ring. The structure shows extensive hydrogen bonding along the a-direction, including the water molecule, and forms sheets with hydrophilic interactions within the sheets as a result of hydrogen bonding between disaccharides as well as hydrophobic interactions between the sheets, in particular, amongst methyl groups of the N-acetyl group of the α-d-GalpNAc residue in the disaccharides.
Graphical Abstract
The structure of the title disaccharide as a monohydrate, C15H27NO11·H2O, which shows extensive hydrogen bonding as well as hydrophobic interactions, was determined from minute crystals using synchrotron radiation and verified by solid state DFT calculations using plane waves.
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Introduction
The structural diversity of carbohydrates and glycoconjugates is very large and alterations of the oligosaccharide part in molecules presented on cell surfaces are often the result of changes in the metabolism of the cell [1]. Moreover, the anti-inflammatory activity of the monomeric immunoglobulin G is highly dependent of precise structure of the carbohydrate portion that is linked to the antibody, thereby stressing the importance of structure–function relationships [2, 3]. Ten different monosaccharides are used to build glycan structures in humans [4, 5], whereas in bacteria additional monosaccharides are present [6] and the number of different monosaccharides described is considerably larger [7]. Many of these monomers are aminosugars, of which most are N-acetylated. In hexopyranoses the amino group is often linked to C2, but also to C3 or C4, e.g., in 2-amino-2-deoxy-d-glucose, 3-amino-3,6-dideoxy-d-galactose or 4-amino-4,6-dideoxy-d-mannose.
The crystal structure of methyl 3-O-α-d-glucopyranosyl 2-acetamido-2-deoxy-α-d-galactopyranoside monohydrate (I) is presented herein (Fig. 1) and represents a model for part of the microbial cell wall teichoic acid polymer from Micrococcus sp. A1, which contains the disaccharide entity α-d-Glcp-(1 → 3)-α-d-GalpNAc connected by phosphodiester linkages at O6 of the glucose residue and O1 of the galactosamine residue [8].
Experimental
Synthesis and Crystallization
The synthesis of methyl 3-O-α-d-glucopyranosyl 2-acetamido-2-deoxy-α-d-galactopyranoside was described by Weinz [9], in which the constituent monosaccharides have the d absolute configuration and was subsequently characterized by 1H and 13C NMR spectroscopy [10]. The compound was crystallized by slow evaporation of a mixture of water and ethanol (1:1) at a temperature of 20–25 °C.
Single Crystal Diffraction
The tiny crystals of the title compound where mounted with epoxy glue on a thin glass fiber and mounted on a MAR platform equipped with a 165 mm MARCCD detector at beam line I911/5 in Maxlab-II, Lund, Sweden. The crystal to detector distance was 35 mm and the rotation range for each frame was 1°; 200 frames were collected in a single φ scan. Data reduction was done with the Crysalis software package [11].
The structure of 3-O-α-d-glucopyranosyl 2-acetamido-2-deoxy-α-d-galactopyranoside hydrate (I) was solved by direct methods with SHELXS [12]. Most of the non-hydrogen atoms were located in the initial electron density map and the rest of them in subsequent difference Fourier maps. All hydrogen atoms except the two on the hydrate water molecules were geometrically placed and allowed to ride on the carbon or oxygen atom to which they were connected; those in the water molecule were located from difference-density maps and restricted with a bond distance restraint as well as a bond distance restraint between the two hydrogen atoms in the water molecule, in order to maintain the known geometry of the water molecule. The model was refined by full-matrix least-square calculations with SHELXL [13]. The Flack parameter [14] was inconclusive but the absolute configuration could be assigned by reference to an unchanging chiral center. Anomalous dispersion corrections for the used wavelength were calculated with “CROMER for Windows” based on the Cromer-Liberman method using a Kissel-Pratt correction [15]. Anisotropic displacement parameters were used for all atoms in combination with an ISOR-restraint because of the small amount of significant data. A summary of the crystallographic data is found in Table 1.
Even with the high intensity radiation from the Max-II synchrotron the diffraction data above 1 Å resolution show very low signal-to-noise ratios (Fig. 2); thus, the R-values including all data were quite high. However, the R-values decreased considerably when less data were included in the refinements, essentially without any effect on the geometrical parameters. As a result of the small crystal size (50 × 20 × 10 μm3) and possibly poor crystallinity, the final figure of merit at different resolutions is rather high (cf. Table 2). The reconstruction of the 0kl plane shown in Fig. 2 shows the limited resolution of the data set. Up to 1 Å resolution the data are quite significant but further out in diffraction space the reflection data is much less significant, thus mainly contributing to the large R-values but not much to change the structure model.
Results and Discussion
The crystal structure of 3-O-α-d-glucopyranosyl 2-acetamido-2-deoxy-α-d-galactopyranoside hydrate (I) has the two hexopyranosides residues arranged in a syn conformation at the central glycosidic linkage with the hydrogen atoms H1′ and H3 on the same side of the plane perpendicular to the C1′–H1′ vector (Fig. 3). The two hexopyranosides both reside in the 4C1 conformation as θ values are close to zero degrees; the Cremer & Pople puckering parameters [16], calculated by PLATON [17] are given by: Q = 0.52(2) Å, θ = 3(2)° and φ = 71(23)° for the ring O5 → C5 and Q = 0.57(2) Å, θ = 2(2)° and φ = 198(41)° for the O5′ → C5′ ring.
Crystal structure of 3-O-α-d-glucopyranosyl 2-acetamido-2-deoxy-α-d-galactopyranoside hydrate (I) with atomic labels. Anisotropic displacement ellipsoids are drawn at 30% probability level [25]
Torsion angles related to major degrees of freedom are compiled in Table 3 from which it can be observed that the ϕ torsion angles have the exo-anomeric conformation, most commonly observed for glycans and the ψ torsion angle has an eclipsed conformation with a value close to zero degrees. The ω torsion angle has a gt conformation for the sugar with galacto-configuration whereas the ω′ torsion angle has a gg conformation for the sugar with gluco-configuration, both of which being one of the two preferred conformations of hydroxymethyl groups in hexopyranoses [18]. The torsion at the amide bond of the N-acetyl group has a trans conformation, which is almost exclusively observed in saccharides in solution [19, 20]. Interestingly, the N-acetyl group is tilted as the torsion angle τH = 146° and deviates from an idealized antiperiplanar orientation. It is quite similar to the corresponding torsion in d-GlcNAc as a constituent of the pentasaccharide LNF-1 then having an average value for τH = 157° based on molecular dynamics (MD) simulations [21] and to N-acetyl groups in LNF-1 with τ = 60° (± 20) and in the hexasaccharide LND-1 with τ = 90° (± 30) deduced from 1H,1H-NOESY NMR experiments [22] as compared to τ = 85° in disaccharide I. The recent investigation of conformational preferences of the N-acetyl groups in e.g. methyl N,N′-diacetyl-chitobioside based on NMR scalar coupling constants revealed only small deviations from an antiperiplanar orientation of H2 and HN, with τ = 115° and τ′ = 110°, whereas in α-d-GlcNAc-OMe it differed somewhat more with τ = 106°, but that good agreement with MD simulation in aqueous solution was observed [20]. Thus, it is surmised that the additional tilting in disaccharide I, as well as that for the milk oligosaccharides, is due to the fact that the N-acetyl containing sugar residue has a glycosyl group at O3 vicinal to its N-acetyl group.
The N-acetyl group at position 2 of the d-galactosamine residue makes the pyranose sugar bulkier as the substituent protrudes compared to d-galactopyranose. The structural entity α-d-Hexp-(1 → 3)-α-d-Galp where Hexp corresponds to a hexose sugar residue having the pyranose ring form is, like the title compound, present in a B blood group trisaccharide, specifically in α-l-Fucp-(1 → 2)[α-d-Galp-(1 → 3)]-β-d-Galp-OMe, for which the torsion angles at the (1 → 3)-linkage are ϕ = − 63° and ψ = − 55° in the crystal structure (Cambridge Structural Database code LOKDIY) [23]. In the trisaccharide the fucosyl residue is linked to position 2 of the disubstituted d-galactopyranoside, which in effect results in a change of, in particular, the ψ torsion angle relative to the sterically less crowded disaccharide that upon potential energy minimization using the HSEA (hard sphere exo anomeric) force field showed ϕ = − 49° and ψ = − 32° [23]. Geometry optimization of 3-O-α-d-glucopyranosyl 2-acetamido-2-deoxy-α-d-galactopyranoside by density functional theory (DFT) calculations at the theory level B3LYP/6-31G* using NWChem [24] resulted in ϕ = − 55° and ψ = − 44°. Thus, the presence of the N-acetyl group may have an influence such that the ψ torsion angle in the disaccharide on its own (in vacuo) becomes similar to that of the trisaccharide carrying a fucosyl residue in the corresponding position. Moreover, the computational results of the title disaccharide as a single molecule whose ψ torsion angle differs by ~ 45° to that in the solid state indicates that intermolecular forces (crystal packing) affect the conformation of the molecule significantly in the crystal.
Packing of disaccharides and water molecules in the bc-plane is depicted in Fig. 4. Extensive N–H…O(C) hydrogen bonding takes place along the a-direction and the hydrate water molecules mediate additional hydrogen bonding along the a-direction O–H…O3′ as well as between molecules via O–H…O(C) along the b-direction (Table 4 and Fig. 5).
Packing of the disaccharide molecules in the bc-plane and hydrogen bonding scheme; the water molecule has been illustrated as an oxygen atom with larger radii and light blue color [25]
Part of the hydrogen bonding scheme along the a-direction. The left structure is the model from the X-ray data and the right one is from the DFT model [24]
The structure model was further investigated by solid state DFT calculations with plane waves and pseudopotentials using NWChem [24], describing both the heavy atom framework as well as the hydrogen bond geometry very well. The differences between the X-ray model and the model derived from DFT calculations are minor, with slightly longer C–H, N–H and O–H distances in the DFT model, thus supporting the structure of the X-ray model even though the high angle diffraction data were quite insignificant. A visual comparison of hydrogen bond geometries from X-ray and DFT models is shown in Fig. 5, as well as a comparison between selected torsion angles from the X-ray and DFT models given in Table 3. Note that there are four generated individual values in the DFT model for each torsion angle defined by the X-ray model, due to the fact that the DFT optimizations were done in space group P1.
Data Availability
Data available within the article and from CCDC with deposition number 2213803.
References
Prien JM, Huysentruyt LC, Ashline DJ et al (2008) Differentiating N-linked glycan structural isomers in metastatic and nonmetastatic tumor cells using sequential mass spectrometry. Glycobiology 18:353–366. https://doi.org/10.1093/glycob/cwn010
Anthony RM, Nimmerjahn F, Ashline DJ et al (2008) Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320:373–376. https://doi.org/10.1126/science.1154315
Anthony RM, Wermeling F, Karlsson MCI, Ravetch JV (2008) Identification of a receptor required for the anti-inflammatory activity of IVIG. Proc Natl Acad Sci 105:19571–19578. https://doi.org/10.1073/pnas.0810163105
Schnaar RL (2016) Glycobiology simplified: diverse roles of glycan recognition in inflammation. J Leukoc Biol 99:825–838. https://doi.org/10.1189/jlb.3ri0116-021r
Lebrilla CB, Liu J, Widmalm G, Prestegard JH (2022) Oligosaccharides and polysaccharides. In: Varki A, Cummings RD, Esko JD et al (eds) Essentials of glycobiology, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 33–42
Liu B, Furevi A, Perepelov AV et al (2020) Structure and genetics of Escherichia coli O antigens. FEMS Microbiol Rev 44:655–683. https://doi.org/10.1093/femsre/fuz028
Adibekian A, Stallforth P, Hecht M-L et al (2011) Comparative bioinformatics analysis of the mammalian and bacterial glycomes. Chem Sci 2:337–344. https://doi.org/10.1039/C0SC00322K
Partridge MD, Davison AL, Baddiley J (1971) A polymer of glucose and N-acetylgalactosamine 1-phosphate in the wall of Micrococcus sp. A1. Biochem J 121:695–700
Weinz B (1989) Syntes, NMR och konformationberäkningar av (1–3)-bundna disackarider. Stockholm University
Roslund MU, Säwén E, Landström J et al (2011) Complete 1H and 13C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program CASPER. Carbohydr Res 346:1311–1319. https://doi.org/10.1016/j.carres.2011.04.033
Rigaku Oxford Diffraction (2019) CrysAlis PRO Software system version 1.171.40.53. Rigaku Oxford Diffraction
Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr A 64:112–122. https://doi.org/10.1107/S0108767307043930
Sheldrick GM (2015) Crystal structure refinement with SHELXL. Acta Crystallogr C 71:3–8. https://doi.org/10.1107/S2053229614024218
Flack HD (1983) On enantiomorph-polarity estimation. Acta Cryst A39:876–881
Kissel L, Pratt RH (1990) Corrections to tabulated anomalous-scattering factors. Acta Crystallogr A 46:170–175. https://doi.org/10.1107/S0108767389010718
Cremer D, Pople JA (1975) General definition of ring puckering coordinates. J Am Chem Soc 97:1354–1358. https://doi.org/10.1021/ja00839a011
Spek AL (2009) Structure validation in chemical crystallography. Acta Crystallogr D 65:148–155. https://doi.org/10.1107/S090744490804362X
Widmalm G (2013) A perspective on the primary and three-dimensional structures of carbohydrates. Carbohydr Res 378:123–132. https://doi.org/10.1016/j.carres.2013.02.005
Engström O, Mobarak H, Ståhle J, Widmalm G (2017) 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. J Phys Chem B 121:9487–9497. https://doi.org/10.1021/acs.jpcb.7b05611
Meredith RJ, Tetrault T, Yoon M-K et al (2022) N-acetyl side-chain conformation in saccharides: solution models obtained from MA’AT analysis. J Org Chem 87:8368–8379. https://doi.org/10.1021/acs.joc.2c00189
Säwén E, Stevensson B, Östervall J et al (2011) Molecular conformations in the pentasaccharide LNF-1 derived from NMR spectroscopy and molecular dynamics simulations. J Phys Chem B 115:7109–7121. https://doi.org/10.1021/jp2017105
Cagas P, Kaluarachchi K, Bush CA (1991) 2D NOESY simulations of amide protons in acetamido sugars. J Am Chem Soc 113:6815–6822. https://doi.org/10.1021/ja00018a016
Otter A, Lemieux RU, Ball RG et al (1999) Crystal state and solution conformation of the B blood group trisaccharide α-L-Fucp-(1→2)-[α-D-Galp]-(1→3)]-β-D-Galp-OCH3. Eur J Biochem 259:295–303. https://doi.org/10.1046/j.1432-1327.1999.00036.x
Aprà E, Bylaska EJ, de Jong WA et al (2020) NWChem: past, present, and future. J Chem Phys 152:184102. https://doi.org/10.1063/5.0004997
Brandenburg K (2001) DIAMOND 2.1e Crystal Impact GbR
Acknowledgements
The DFT computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at HPC2N (UMU), Sweden. This work has received financial support from the Swedish Research Council (2017-03703).
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G.W. crystallized the sample L.E. solved the crystal structure. G.W. and L.E. wrote the manuscript together.
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Eriksson, L., Widmalm, G. Crystal Structure of Methyl 3-O-α-d-Glucopyranosyl 2-Acetamido-2-Deoxy-α-d-Galactopyranoside Hydrate. J Chem Crystallogr 53, 400–406 (2023). https://doi.org/10.1007/s10870-023-00979-z
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DOI: https://doi.org/10.1007/s10870-023-00979-z