Letter pubs.acs.org/JPCL
Influence of the Anomeric Conformation in the Intermolecular Interactions of Glucose Imanol Usabiaga, Jorge González, Iker León, Pedro F. Arnaiz, Emilio J. Cocinero, and José A. Fernández* Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940 Leioa, Spain S Supporting Information *
ABSTRACT: Carbohydrates are, together with amino acids, DNA bases, and lipids, the building blocks of living beings. They play a central role in basic functions such as immunity and signaling, which are governed by noncovalent interactions between sugar units and other biomolecules. To get insights into such interactions between monosaccharide units, we used a combination of mass-resolved laser spectroscopy in supersonic expansions and molecular structure calculations. The results obtained clearly demonstrate that the small stability difference between the α/β anomers of glucopyranose derivatives is reversed and amplified during molecular aggregation, making the complexes of the β-anomers significantly more stable. The amplification mechanism seems to be formation of extensive hydrogen-bond networks extending through the two interacting molecules. The same mechanism must be at play in the interactions of biological and synthetic receptors with glycans, which exhibit, in general, a higher affinity for a specific anomer, usually the beta anomer.
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mechanism that somehow amplifies the small changes in the orientations of the hydroxyl units of a sugar to result in very different interaction with other (biological and synthetic) molecules. To get insights into such mechanisms of amplification and the preference for a given anomer, we formed molecular aggregates (dimers) of glucose. More precisely, we formed dimers of methyl-α/β-D-glucopyranoside (α/β-MeGlc) with phenyl-β-D-glucopyranoside (β-PhGlc, Scheme 1 and Scheme S4 of the Supporting Information) using a laser desorption system and a pulsed supersonic expansion inside the ionization
rom the conformational point of view, carbohydrates are a versatile family of molecules.1 They may appear in linear and cyclic conformations (usually as five- and six-membered rings), and they may even switch between them. Furthermore, they present several hydroxyl substituents that may be in axial or equatorial orientations, leading to different epimers. Sugar monomers aggregate in living beings to form polysaccharides that may be found isolated or attached to lipids and proteins, frequently decorating the extracellular side of the plasmatic membrane and playing a central role in the immune system of multicellular organisms.2 Those polysaccharides contain a variable number of units, which may also exhibit modifications such as acetylations or silylations and a different degree of branching. The number of possible combinations is enormous and, according to some authors, exceeds those of amino acids or DNA bases, as the number of “letters” in the alphabet of sugars is several orders of magnitude larger than in the other two alphabets.3 The immune system makes extensive use of such variability to work in a molecular-recognition basis, in which a protein binds to a glycan, thanks to the complementarity between the sequence of amino acids in the protein’s active center and the composition and spatial arrangement of the sugar units of the glycan.4 The specificity is so high that a seemingly minor change in the orientation of a single substituent may suffice to elicit or not an immune response. Such is the case of the blood groups.2 Likewise, interaction of sugars with synthetic receptors is very selective: In most cases, the artificial receptor attaches preferentially to an anomer (α or β) of a given sugar.3 The reason behind such preference is not known, and it may be different in each case, but these examples point to a concerted © XXXX American Chemical Society
Scheme 1. (a) Phenyl-β-D-glucopyranoside (β-PhGlc), (b) Methyl-β-D-glucopyranoside (β-MeGlc), and (c) Methyl-α-Dglucopyranoside (α-MeGlc)
Received: January 20, 2017 Accepted: February 23, 2017 Published: February 23, 2017 1147
DOI: 10.1021/acs.jpclett.7b00151 J. Phys. Chem. Lett. 2017, 8, 1147−1151
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The Journal of Physical Chemistry Letters chamber of a mass spectrometer. (See the Supporting Information for a thorough description of the experimental system.) Collisions during the expansion cool the molecules and favor the formation of molecular aggregates that may afterward be studied using mass-resolved spectroscopic techniques. Figure 1
Figure 2. Mass-resolved IR/UV spectra of: (a) β-PhGlc···α-MeGlc and (b) β-PhGlc···β-MeGlc. Simulated spectra for the assigned structures are also presented for comparison. Correction factors of 0.9385 in the OH region and of 0.9525 in the CH region were used to account for anharmonicity. The structures, computed at the M06-2X/ 6-311++G(d,p) level, may be found in Figure 3.
Figure 1. One-color REMPI spectra of (a) β-PhGlc, (b) β-PhGlc···αMeGlc, and (c) β-PhGlc···β-MeGlc.
shows the resonance-enhanced multiphoton ionization (REMPI) spectrum of β-PhGlc (a) and of its aggregates with α-MeGlc (b) and β-MeGlc (c). The spectrum of β-PhGlc presents several well-resolved bands that, as demonstrated in previous works by Simons’ group,5 correspond to at least three isomers that differ in the relative orientation of the exocyclic hydroxymethyl group. (See also Figure S9 of the Supporting Information.) Because of the appearance of low-frequency intermolecular modes, structural changes upon electronic excitation, or changes in the excited-state lifetime, the formation of complexes with α/β-MeGlc results in broad REMPI spectra, shifted to the red from the origin band of β-PhGlc, especially in the case of β-PhGlc···α-MeGlc, which may indicate a direct interaction of α-MeGlc with the chromophore of β-PhGlc. Both spectra present several discrete features, built on top of a broad absorption. The spectra in Figure 1 are not very informative but allowed us to record the mass-resolved IR spectra of the complexes using a modified version of the technique of IR/UV double resonance. To improve the S/N ratio and to eliminate the variations in signal intensity due to the fluctuations on the ablation process, we introduced an additional laser to probe in real time the amount of material desorbed by each laser shot. (See the description of the methods in Supporting Information.) Figure 2 shows the mass-resolved IR/UV traces obtained for β-PhGlc···α-MeGlc (a) and β-PhGlc···β-MeGlc (b) in the stretching region, covering from the C−H (∼2800−3000 cm−1) to the O−H stretches (3400−3700 cm−1). Several bands appear on both regions of the spectrum, a consequence of the 20 C−H and the eight O−H bonds present in the aggregates. The bands corresponding to the C−H stretches usually appear as a group of transitions with several combination bands, congesting the spectrum and precluding the extraction of structural information. Conversely, the OH stretching region is usually more informative and renders important structural information because the stretching vibrations are very sensitive to the formation of hydrogen bonds. Three well-resolved bands appear at the blue end of the spectrum of β-PhGlc···α-
MeGlc, together with three broad absorptions. The complex has eight OH groups; therefore, the observed transitions may hide contribution from several vibrations. In previous publications,6−8 it was demonstrated that the hydroxyl groups of sugars are usually found with cooperative hydrogen-bond networks that hamper (but do not completely block) their interaction with other molecules.9,10 Therefore, the hydroxyl groups may be involved in inter/intramolecular interactions of different strength, which result in a collection of shifts in the spectrum. Interpretation of the experimental spectra in Figure 2 requires the guidance of predictions from quantum mechanics. Thus we explored the conformational landscape for the interaction between the two molecules using a combination of molecular mechanics and DFT methods, a strategy already used by several authors in systems of similar complexity.9,11,12 As demonstrated elsewhere,13,14 the interaction between carbohydrates is a balance of changes in enthalpy and entropy; therefore, both have to be taken into account when analyzing the energetic order of the calculated structures. (See the description of calculation procedure and Figures S3, S7, S12, S14, S17, and S18 in the Supporting Information.) The final assignment of β-PhGlc···α-MeGlc is shown in the upper panel of Figure 2. No single isomer was able to reproduce all of the features in the spectrum; a combination of two species was required. Recording of UV/UV hole-burning experiments in this system in a search for additional conformers was unsuccessful due to the broad nature of the spectrum, but IR/UV traces obtained probing different wavenumbers of the REMPI spectrum (see Supporting Information Figure S2) point to the existence of at least two isomers contributing to the spectrum. Complete separation of their spectra was not possible. The structure of the two assigned isomers (Figure 3) demonstrates that they correspond to very different interacting geometries. In isomer 1 the O4H group of α-MeGlc incorporates into the hydrogen-bond network of β-PhGlc (O4−H···O2′−H···O3), and at the same time there is a direct interaction with the aromatic ring. In isomer 2, the contact 1148
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interaction between the substituents of the anomeric carbons add extra stabilization to the complex thanks to C−H···π interactions. Such an elegant structure presents a significantly high binding energy of 67 kJ/mol, according to the calculations at M06-2X/6-311++G(d,p) level, a value that is substantially larger than the binding energy found for the isomers of βPhGlc···α-MeGlc. It is well known6 that the small structural difference between α and β anomers, just the equatorial/axial position of the hydroxyl group, results in a small extra stabilization of the αanomers. However, our results indicate that the intermolecular interactions involving these groups dramatically changed the conformational landscape. Clearly, the symmetric and energetically favorable structure adopted by the most stable isomer βPhGlc···β-MeGlc cannot be reached in the β-PhGlc···α-MeGlc complex. In this structure both O1 act as proton acceptors and take part in the H-bond network, which acts as a multiplier, resulting in the observed increase in aggregation energy. An equatorial-to-axial (β-to-α) change significantly alters the morphology of the hydrogen-bond network. To characterize the stronger interaction in the three possible combinations of anomers (α−α, β−α, and β−β), we explored the formation of the following dimers, α-MeGlc···α-MeGlc, βMeGlc···α-MeGlc, β-MeGlc···β-MeGlc, β-PhGlc···β-Glc, βPhGlc···α-Glc, and α-PhGlc···α-MeGlc, using the same calculation level at which the experimental results shown in Figures 1 and 2 were reproduced. The results are summarized in Figure 4, where the relative Gibbs binding free energy of the most stable structure for each
Figure 3. Structure of the two β-PhGlc···α-MeGlc and the single βPhGlc···β-MeGlc conformers assigned to the experimental spectra.
between the two molecules takes place away from the aromatic ring through O2−H···O4′−H···O1 + O3−H···O6′ interactions. In this second isomer, several intermolecular hydrogen-bond networks are formed. The free-energy difference between the two structures is 10 kJ/mol larger than that of the global minimum during the whole interval from room temperature to 0 K. The existence of a single isomer of the βPhGlc···β-MeGlc complex despite the existence of three isomers of isolated β-PhGlc in the expansion is due to its ability to form hydrogen bonds of the exocyclic hydroxymethyl group, which acts as a hook in both glucopyranosides. The ability of this “hook” to form cooperative hydrogen bonds was already observed in previous works in both gas phase10 and Xray crystal structures,15 and it is due to its flexibility that allows it to accommodate its orientation so as to maximize the intermolecular interactions. In addition, there is a significant reduction of the isomerization barrier that connects the three bare molecule conformers upon the formation of the aggregate from the bare molecule (β-MeGlc ∼23 kJ/mol) to the complex (∼10 kJ/mol). Under the conditions of our expansion, which allow the jet to reach mach 2, collisions with Ar atoms (∼10 kJ/ mol; see Figures S9 and S10 of the Supporting Information) should be energetic enough to surmount the isomerization barrier in the complex but not in the bare molecule. Thus the assigned isomer presents a structure in which the hydroxymethyl substituent of each molecule is integrated into the hydrogen-bond network of the partner molecule through O2−H···O6′−H···O1 interactions, embracing one each other and putting the hydrophobic CH groups in contact. The
Figure 4. Relative Gibbs binding free energy of the most stable structures of: (A) β-MeGlc···β-MeGlc, (B) β-PhGlc···β-Glc, (C) βPhGlc···β-MeGlc, (D) β-PhGlc···α-Glc (isomer β-PhGlc·α-Glc, 4), (E) β-Ph/MeGlc···α-MeGlc (isomer β-Ph/MeGlc·α-MeGlc-08), (F) α-PhGlc···α-MeGlc, and (G) α-MeGlc···α-MeGlc. The most stable isomer without interaction with the chromophore was considered.
system is plotted between 0 and 400 K. It is clear that the β−β complexes (blue lines) always present the highest interaction energy, well-separated from α−α (red) and β−α (green) systems. As a final test, we introduced in the expansion a mixture of βPhGlc and D-glucopyranose (Glc) to form the corresponding aggregates. In this case, the absence of a substituent in the anomeric carbon resulted in a mixture of α and β anomers, as they can interconvert through the linear form of the sugar. The resulting REMPI and IR/UV spectra may be found in the Supporting Information (Figures S16−S18), together with the quantum-mechanical predictions. The existence of both anomers in the expansion resulted in the formation of complexes of β-PhGlc with both species, largely complicating 1149
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the spectroscopy of the system. Actually, comparison between the experimental spectrum and the predicted traces showed that at least three species, one with the α and two with the β anomer, may be contributing to the experimental spectrum. The structure of these species is very close to those presented in Figure 3, and they also follow the same trend in stability, where the complexes with the β-anomer are significantly more stable than those with the α-anomer. All of the above points to a high degree of selectivity in the intermolecular interactions of sugars. There is a small energy difference between anomers of glucopyranose, but such difference is reversed and amplified by the intermolecular interactions, resulting in molecular aggregates of very different stability, depending on the anomeric conformation of their constituents. This is a striking observation because the ability of the anomeric substituent to take part in hydrogen bonds is limited to an acceptor role due to the phenyl and methyl substituents in α/β-PhGlc and α/β-MeGlc, respectively. However, the orientation of the anomeric substituent forces the complexes of β-PhGlc with α/β-MeGlc to adopt completely different structures. Translated into the biological world, this means that the design of a receptor for a sugar molecule has to take into account the anomer that the receptor is expected to bind to. Or, put in other words, it would not be very difficult for a receptor to distinguish between anomers of a given sugar molecule, as it will interact in completely different ways with each anomer due to the specific morphology of their hydrogen bond networks. This is certainly the base for the high specificity that the biological receptors exhibit for a given glycan. A similar observation was also reported for synthetic receptors.3 Most of them exhibit different affinity for α/β anomers, and very often they have a higher affinity for the β form. The observed difference in stability in the systems studied here also correlates with macroscopic properties of sugars. Both cellulose and starch are formed by D-glucopyranose units, but while starch is formed by α-anomers, cellulose is formed exclusively by β-anomers. The superior aggregation abilities of the latter may hinder to a larger extent the attack of water molecules to the biopolymer and may be in part responsible for the difference in solubility between starch and cellulose. If such is the case, then the present system would be a beautiful example of how a molecular mechanism is amplified to cause a macroscopic effect.
AUTHOR INFORMATION
Corresponding Author
*Phone: +34946015387. Fax: +34946013500. E-mail: josea.
[email protected]. ORCID
José A. Fernández: 0000-0002-7315-2326 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research leading to these results has received funding from the Spanish MINECO (CTQ-2014-54464-R, CTQ-201568148), FEDER. E.J.C. acknowledges a ‘“Ramón y Cajal”’ contract. I.U. thanks the Basque Government for a predoctoral fellowship. J.G. thanks the UPV/EHU for a predoctoral fellowship. I.L. thanks the MINECO for a Juan de la Cierva postdoctoral fellowship. Computational resources from the SGI/IZO-SGIker network were used for this work. We acknowledge the technical support from the personnel from the UPV/EHU laser facility.
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EXPERIMENTAL SECTION See the Supporting Information for a detailed description of the experimental setup.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.7b00151. Detailed description of the experimental and computational methods, together with diagrams, annotated REMPI spectra, IR/UV hole-burning traces, IR/UV spectra recorded probing different wavenumbers of each aggregate, relative ΔG diagrams, list of all computed structures for each system and ball-and-stick models of the most relevant ones, computations on the height of the barriers for isomerization, and comparison between simulated and experimental IR/UV spectra. (PDF) 1150
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The Journal of Physical Chemistry Letters Quantitative Structure-Stability Relationships for Carbohydrate/ Aromatic Complexes. Chem. Sci. 2015, 6, 6076−6085. (14) Santana, A. G.; Jiménez-Moreno, E.; Gómez, A. M.; Corzana, F.; González, C.; Jiménez-Oses, G.; Jiménez-Barbero, J.; Asensio, J. L. A Dynamic Combinatorial Approach for the Analysis of Weak Carbohydrate/Aromatic Complexes: Dissecting Facial Selectivity in CH/p Stacking Interactions. J. Am. Chem. Soc. 2013, 135, 3347−3350. (15) Harrop, S. J.; Helliwell, J. R.; Wan, T. C. M.; Kalb (Gilboa), A. J.; Tong, L.; Yariv, J. Structure Solution of a Cubic Crystal of Concanavalin A Complexed with methyl-α-d-glucopyranoside. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1996, 52, 143−155.
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