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Publicly Available Published by De Gruyter June 14, 2019

Conformational changes in common monosaccharides caused by per-O-sulfation

  • Alexey G. Gerbst , Vadim B. Krylov and Nikolay E. Nifantiev ORCID logo EMAIL logo

Abstract

Polysulfated carbohydrates play an important role in many biological processes because of their ability to bind to various protein receptors such as different growth factors, blood coagulation factors, adhesion lectins etc. Precise information about spatial organization of sulfated derivatives is of high demand for molecular modelling of such interactions as well as for understanding of the mechanism of pyranoside-into-furanoside rearrangement. In this review we summarize the changes recently revealed for the conformations of common pyranosides and furanosides upon total O-sulfation which were studied by means of NMR spectroscopy as well as molecular modelling. It was found that pentoses, being more flexible, undergo complete conformational chair inversion. Meanwhile, for hexoses the situation strongly depends on the monosaccharide configuration. Conformational changes are most pronounced in gluco-compounds though quantum chemical calculations helped to establish that no complete chair inversion occurred. In furanosides distortions of two types were observed: either the ring conformation or the conformation of the side chain changed. The presented data may be used for the analysis of chemical, physical and biological properties of sulfated carbohydrates.

Introduction

Sulfated derivatives of carbohydrates represent significant biological interest due to their ability to exhibit different types of physiological activity including anticoagulant, anti-inflammatory, antiangiogenic [1], [2], [3], [4]. The mechanism of these actions is based on specific interactions with certain protein receptors [5], [6]. For example, heparan sulfate interacts with fibroblast growth factors [7], chemokines [8], antithrombin-III [9], etc. Remarkably, conformational flexibility of a certain residue within the polysaccharide chain (iduronic acid) plays a key role in binding and biological properties of the whole polysaccharide [10]. Moreover, it was shown that conformational behaviour of iduronic acid, and thus the profile of biological activity of heparan sulfate fragments, is regulated by the sulfation pattern [11], [12].

Additionally, recently discovered pyranoside-into-furanoside rearrangement [13], [14] and the method for preparation of highly sulfated oligosaccharides [15], [16] open new ways for utilization of exhaustively sulfated derivatives as precursors of valuable synthetic blocks for oligosaccharide synthesis [17], [18], [19], [20], [21]. Investigation of mechanism of pyranoside-into-furanoside rearrangement [13], [14], [22] and understanding of its driving force [23] required precise data about spatial organization of highly sulfated derivatives.

Conformations of common hexoses are thought, as a rule, to be 4C1 or 1C4 depending on the monosaccharide configuration (D or L). However, modification of carbohydrates with bulky and negatively charged sulfate groups is expected to lead to changes of their conformations, which should be taken into account within in silico modelling of carbohydrate-protein interactions or mechanistic investigations of rearrangements of sulfated derivatives. Such changes were not studied in detail until recently. In this review we summarise the recent works devoted to changes in the conformation behaviour of common pyranosides and furanosides upon their total O-sulfation.

Conformational behaviour of totally O-sulfated pyranosides

During our studies of the newly discovered pyranoside-into-furanoside (PIF) rearrangement that proceeds via totally sulfated monosaccharides we came across pronounced changes in NMR spectra of these compounds as compared to the parent non-sulfated structures. Thus, sometimes 1H-1H coupling constants in the sulfated derivatives differed drastically from those in the free sugars.

In our work [24] several hexoses and pentoses were investigated. Particularly these were α- and β-isomers of arabino-, xylo-, gluco-, galacto- and manno-monosaccharides taken as methyl glycosides (Fig. 1). The analysis of their NMR spectra allowed for the following conclusions. First, monosaccharides with α-configuration of the anomeric center аs well as β-galactose and β-mannose did not undergo significant conformation changes caused by introduction of sulfates. On the other hand β-xylose completely inverted from 4C1 into 1C4 upon the O-sulfation. This was clear both from changes in inter-proton coupling constants and quantum chemical calculations. Pronounced changes occurred in the β-glucoside. Although the latter fact had been already mentioned in 1995 by Wessel [25], [26], he did not provide sufficient data to come to a certain conclusion about what transformations took place and just postulated that they probably resulted from 4C1 to 1C4 chair inversion. However, in work [24] with the use of nuclear Overhauser effect it was shown that dominant conformations of β-glucoside were skew-boats.

Fig. 1: Dominant conformations of common monosaccharides in totally O-sulfated and non-sulfated form.
Fig. 1:

Dominant conformations of common monosaccharides in totally O-sulfated and non-sulfated form.

To gain further insight into this problem we conducted a detailed study [27] of the β-glucuronic acid propyl glycoside that contained an additional charged group. This monosaccharide is widely present in natural biopolymers, namely, chondroitin sulfates (Fig. 2).

Fig. 2: Studied β-glucuronic acid propyl glycosides.
Fig. 2:

Studied β-glucuronic acid propyl glycosides.

Using quantum chemistry methods (HF/6-311++G** level of theory) and NMR experiments it was found that the distortion in the pyranoside ring of the glucuronic acid upon exhaustive sulfation is primarily explained by presence of two skew-boat conformers, OS2 and 3S1 (Fig. 3) [27]. Appearance of the fully inverted conformer 1C4 (suggested by several investigators before) is unlikely due to its higher relative energy and is not confirmed by NOE experiments. This also explains the unusual downfield shift of the anomeric proton upon sulfation. In methanol the proportion of the skewed conformers additionally increases due to its lower polarity. Considering that glucuronic acid is of important constituting blocks in such biologically significant polysaccharides as chondroitin sulfates, this knowledge may be very useful for correct modelling of interactions between CS or their fragments and protein targets.

Fig. 3: Schematic view of skew-boat conformers OS2 and 3S1 in per-O-sulfated β-glucuronic acid propyl glycosides. In OS2 conformer sulfates at C-2 and C-3 are located in trans orientation, however in 3S1 conformer sulfates at C-3 and C-4 are trans orientated.
Fig. 3:

Schematic view of skew-boat conformers OS2 and 3S1 in per-O-sulfated β-glucuronic acid propyl glycosides. In OS2 conformer sulfates at C-2 and C-3 are located in trans orientation, however in 3S1 conformer sulfates at C-3 and C-4 are trans orientated.

Two sets of NMR characteristics had to be evaluated during this work [27]: 1H-1H coupling constants and 1H chemical shifts (Tables 1 and 2, respectively). Chemical shifts for the two skewed conformers (OS2 and 3S1) and for the both chair conformations (4C1 and 1C4) of sulfated monosaccharides 2a,b were calculated and compared to the experimental data (Table 1). These data shows, that the computed chemical shifts exhibit sufficiently good correlation with the experimental ones. It was found that, OS2 and 1C4 conformers showed significant downfield shift of the anomeric proton. Although the 1C4 conformer energy of was considerably higher than for the skew conformers. Additional NOE experiments were undertaken to finally establish if the inverted 1C4 conformer played any role. These results strongly support the idea that the skew-boat conformers make large contribution to the conformational equilibrium of the persulfated glucuronide.

Table 1:

Experimental and calculated chemical shifts for the conformers of structures 2a,b.

CompoundConformerH-1H-2H-3H-4H-5
2aExperimental4.864.464.905.054.33
OS24.824.434.874.574.08
3S14.464.415.415.644.24
4C14.343.794.744.533.83
1C44.834.265.775.224.50
2bExperimental4.914.615.025.294.52
OS24.753.844.525.693.72
3S14.734.134.715.974.28
4C14.313.294.244.253.67
Table 2:

Experimental and calculated coupling constants for the conformers of structures 2a.

ConformerJ1,2J2,3J3,4J4,5
Experimental6.32.94.73.6
OS20.23.70.68.9
3S16.20.64.20.8
4C17.510.49.79.5
  1. For the calculated values no counter-ions were considered.

Calculated 1H-1H coupling constants are presented in Table 2. As expected, they do not change very much for the same conformer upon the solvent change as they primarily depend on the values of torsional angles. OS2 and 3S1 conformers are complimentary to each other in the sense that their JH1-H2, JH2-H3 and JH3-H4 constant values alternate between them. Thus a combination of these two conformers should be sufficient to describe the whole experimental picture where JH2-H3 and JH3-H4 are almost equally small and JH1-H2 is a bit larger though significantly smaller than in the non-sulfated compound 1.

Conformational behaviour of totally O-sulfated furanosides

The furanosides naturally have greater conformational flexibility than pyranosides and thus the effect of O-sulfation in them should be more pronounced. Indeed, the pattern of J-coupling constants significantly differed for non-sulfated and per-O-sulfated furanosides which allowed for a conclusion that their conformations changed after the sulfation [28]. To rationalize these changes, we undertook theoretical conformational analysis of monosaccharides 3–5 and 3s–5s (Fig. 4). This analysis included both studies of the conformation of the furanoside ring and conformation of the side chain at C(4). The conformation of the furanoside ring can be one of ten envelopes (E) or ten twist (T) forms while the conformation of the exocyclic chain (i.e. rotation of C4−C5 and C5−C6 bonds) is described by corresponding torsion angles.

Fig. 4: Studied propyl furanosides 3–5 and 3s-5s.
Fig. 4:

Studied propyl furanosides 3–5 and 3s-5s.

Geometry optimization of all possible furanoside pseudo-rotamers for all the studied monosaccharides, both in non-sulfated (3–5) and sulfated (3s–5s) forms, tended to produce one or two low-energy conformers which differed from each other by less than 2 kcal/mol, while the other found conformations had considerably higher energies. For all the structures examined changes in the ring conformation upon the introduction of sulfates are observed. Particularly, in the mannoside, preference for ring conformers changes: while in the free form the calculations predict it to exist preferably in C3-endo conformation (Fig. 5), in the sulfated form C1-exo conformer becomes dominant. In the glucoside, the conformation of the furanoside ring in the conformer with minimal energy remains approximately the same C2-exo. In case of the galactosides the low energy C3-exo conformers which should dominate in the non-sulfated form disappear after introduction of O-sulfates and conformational shift towards C1-endo occurs [28].

Fig. 5: Conformation of furanoside ring in low-energy conformers for non-sulfated and per-O-sulfated propyl furanosides.
Fig. 5:

Conformation of furanoside ring in low-energy conformers for non-sulfated and per-O-sulfated propyl furanosides.

In case of the mannoside 3 and glucoside 4, in the absence of sulfates, the preferable conformation of the C4-C5 bond is characterized with trans-orientation of H4 and H5 protons. However, for their sulfated derivatives 3s, 4s gauche-rotamer is dominant (Fig. 6). This observation is obviously connected with introduction of sulfate at O-5. Thus, in non-sulfated form the most bulky group at C-5 is CH2OH which prefers to take the trans-orientation to C3-atom of the ring. However, in sulfated derivatives SO3-group at O-5, due to its size, is tended it locate in trans- orientation to C(3) (see Fig. 6). In the case of galactose 5, however, the situation is more complex. This saccharide in its furanoside form supposedly has increased conformational flexibility, because for its lowest energy conformer all three rotamers around C4-C5 bond do not have great energy difference between each other [28].

Fig. 6: Changes in preferable conformation of the C(4)-C(5) bond caused by per-O-sulfation propyl furanosides.
Fig. 6:

Changes in preferable conformation of the C(4)-C(5) bond caused by per-O-sulfation propyl furanosides.

All the mentioned changes certainly affect the values of the 1H-1H coupling constants. To study this influence in detail, DFT/B3LYP/pcJ-1 calculation of the constants for low-energy conformers was performed (Table 3). The first thing to note is that for non-sulfated α-propyl mannofuranoside 3 in the lowest-energy conformer (HF/6-311++G** level of theory) all the computed intra-ring constants, and, to some extent, 3J4,5 reproduce the experimental values. According to the calculations the drastic decrease of the experimentally measured H1-H2 coupling constant in the mannofuranoside upon sulfation arises from the change of the conformational preference towards C1-exo in the sulfated saccharide. Good agreement between the theoretical and experimental data was obtained also in the case of the sulfated compounds 4s and 5s [28].

Table 3:

Experimental 1H-1H coupling constants (Hz) and those calculated for different conformers (Hz) for furanosides 3–5 and 3s–5s.

EntryCompoundFuranoside ring conformation (H4-C4-C5-H5 dihedral)Relative energy, kcal/molJ1,2J2,3J3,4J4,5
13Experimental4.64.62.98.8
2C3-endo (+179°)0.04.94.42.69.6
3C1-exo (+178°)1.60.35.58.39.8
43sExperimental1.25.67.02.8
5C1-exo (+70°)0.00.36.99.91.8
6C3-endo (+84°)1.25.65.02.70.5
74Experimental-<11.24.59.0
8C2-exo (+174°)0.00.10.55.410.0
104sExperimental<10.74.84.9
11C2-exo (+84°)0.00.10.85.90.5
125Experimental2.44.46.84.0
13C3-exo (−57°)0.04.88.59.41.6
14C3-exo (+53°)1.05.28.19.56.3
15C3-exo (+173°)1.54.87.29.68.4
16O4-exo (−59°)1.20.31.46.96.4
17O4-exo (+52°)1.80.31.36.51.7
185sExperimental<1<14.62.4
19C1-endo (−63°)0.00.10.43.91.4

Conclusions

Thus, we can conclude that introduction of sulfate groups into different monosaccharide residues results in serious changes of their conformational behavior. The origin of such effects lies in the repulsive interactions between the bulky and charged sulfate groups. In case of pyranosides, the most pronounced effect was found in the case of compounds bearing all equatorial substituents – β-xylose, β-glucose and β-glucuronic acid glycosides. The experimental pattern of spin-spin coupling constants and ab initio calculations suggest inverted chair conformations for β-xylose instead of the usual 4C1 conformation. In case of β-glucose and glucuronic acid a complex distorted conformation of the sugar ring was assumed which was confirmed by NOE experiments and quantum chemistry calculations. In furanosides distortions of two types were observed: either the ring conformation or the conformation of the side chain changed. The presented data may be used for the analysis of chemical, physical and biological properties of sulfated carbohydrate, as well as for mechanistic investigation of pyranoside-into-furanoside rearrangement and for understanding of its driving force.


Article note

A collection of invited papers based on presentations at the 29th International Carbohydrate Symposium (ICS-29), held in the University of Lisbon, Portugal, 14–19 July 2018.


Award Identifier / Grant number: 14-23-00199

Funding statement: This work was supported by the Russian Science Foundation, Funder Id: http://dx.doi.org/10.13039/501100006769, (grant 14-23-00199).

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Published Online: 2019-06-14
Published in Print: 2019-07-26

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