Elsevier

Carbohydrate Research

Volume 341, Issue 18, 29 December 2006, Pages 2912-2920
Carbohydrate Research

Investigations into the role of oxacarbenium ions in glycosylation reactions by ab initio molecular dynamics

https://doi.org/10.1016/j.carres.2006.09.027Get rights and content

Abstract

We present a constrained ab initio molecular dynamics method that allows the modeling of the conformational interconversions of glycopyranosyl oxacarbenium ions. The model was successfully tested by estimating the barriers to ring inversion for two 4-substituted tetrahydropyranosyl oxacarbenium ions. The model was further extended to predict the pathways that connect the 4H3 half-chair conformation of 2,3,4,6-tetra-O-methyl-d-glucopyranosyl cation to its inverted 5S1 conformation and the 4H3 half-chair conformation of 2,3,4,6-tetra-O-methyl-d-mannopyranosyl cation to its inverted 3E conformation. The modeled interconversion pathways reconcile a large body of experimental work on the acid-catalyzed hydrolysis of glycosides and the mechanisms of a number of glucosidases and mannosidases.

Introduction

The X-ray crystal structure of the glycosidase lysozyme led to the first proposal for an enzyme mechanism based on a protein structure.1 This mechanism has as a central feature, a glycopyranosyl oxacarbenium ion intermediate with a 4H3 half-chair conformation. Subsequent studies have proposed a second mechanism whereby a neutral enzyme-bound intermediate is separated from the reactants and products by two transition states (TS) with considerable oxacarbenium character.2 This second mechanism must involve conformational changes of the glycopyranose ring to accommodate the planarity about the C-5–O-5–C-1–C-2 dihedral angle in the oxacarbenium ions. The oxacarbenium ion intermediates have very short lifetimes3 and consequently their conformations have been primarily deduced from kinetic isotope effect (KIE) studies4 or from biophysical studies of enzyme inhibitors combined with modeling.5 The critical role of modeling for these studies naturally arises from the paucity of the experimental data. However, to date, no successful computational strategies to model the ring transformations of glycopyranoses, especially pseudorotation, have been developed. Typical approaches involve modeling snapshots along proposed pathways.6 Such studies rely on a priori assumptions and hence can easily miss important features.

In the present work we show trajectories based on ab initio molecular dynamics (AIMD) studies of substituted glycopyranosyl oxacarbenium ions. AIMD trajectories naturally follow the free-energy surface,7 and so the lowest energy ring conformational changes are found without a priori assumptions.8 We have mapped our trajectories on to a sphere based on our description of the six-membered ring conformations.9 We anticipate that our method can be applied to the modeling of any glycosyl-processing enzyme whose mechanism is thought to proceed through an oxacarbenium intermediate or TS. Such enzymes include most glycosidases10 and most glycosyltransferases.11 Our study is further anticipated to assist in the design and development of inhibitors for such enzymes.12 The list of disease states and industrial processes to which such studies could be applied includes antivirals such as the neuramidase inhibitors like Tamiflu; bioethanol production from agricultural byproducts, and potential cancer drugs based on inhibitors of core 2 O-linked glycoprotein or 1,6-branched N-linked glycoprotein forming glycosyltransferases.13

Chemical glycosylation reactions are also thought to proceed through oxacarbenium ion intermediates.14 Furthermore, it is thought that the conformational properties of these ions, at least in part, control the stereoselectivity of this important class of reactions.15 One classic example that bears directly on the studies to be presented here was reported by Lemieux more than 50 years ago.16 His research showed that the α/β isomeric 3,4,6-tri-O-acetyl-d-glucopyranosyl chlorides underwent solvolysis with a high degree of stereoselectivity. That is the α-chloride gave the β-acetate, and the β-chloride gave the α-acetate. Such stereoselectivity suggested an associative SN2-like mechanism. Detailed kinetic studies for both isomers were entirely consistent with a SN1-like mechanism. In order to rationalize this apparent anomaly, Lemieux invoked the possibility that the α-chloride ionized to a glucopyranosyl oxacarbenium with a 4H3 ring conformation, and the β-chloride formed a similar ion, but with a 3H4 ring conformation (Scheme 1). Further, he invoked that the two different ring conformations led to opposite facial selectivity leading to the observed products. To the best of our knowledge Lemieux was the first to propose multiple conformations of glycopyranosyl oxacarbenium ions. Some of these ideas have been recently revived to account for the stereoselectivity of C-glycosylation in which the assumption was made that the two oxacarbenium ion conformers rapidly interconvert and that stereoselectivity is determined by the facial selectivity of nucleophilic attack.17

Our group’s previous study of the conformations of glycopyranosyl oxacarbenium ions by density functional theory (DFT) calculations including those for 2,3,4,6-tetra-O-methyl-d-glucopyranosyl (1) and 2,3,4,6-tetra-O-methyl-d-mannopyranosyl (2) oxacarbenium ions has consistently found at least two minima.18 For (1), a 4H3 half chair and a 5S1 twist boat pair, whereas for (2), a 4H3 half chair and a 3E envelope pair were found (Scheme 2). If the ring conformations of these minima are represented on a sphere, as originally suggested by Hendrickson,19 they are found on opposite hemispheres and on opposite (2) or nearly opposite faces (1) (see below). That is, interconversion in this representation, requires net movement about the equator (changing faces) and movement perpendicular to the equator (changing hemispheres). Our study seeks to find possible interconversion pathways between these minima. In particular we wish to assess if the barriers to interconversion are low enough to allow rapid equilibration.

We have developed a mathematical model that describes all pyranose conformations uniquely in terms of one chair, one boat, and one skew-boat conformation. Further, we have derived quantitative expressions for the characterization of the pyranose and other six-membered ring conformations. For details, see Supplementary data. The three internal coordinates (chair (q1), boat (q2), and skew boat (q3)) defined in our previous papers can be applied as constraints in AIMD studies20 of any six-membered ring inversion (q1) or pseudorotation (q2 and q3).21 Dynamical density functional theory (DFT) calculations were carried out with the projector augmented-wave (PAW) method of Blöchl, which is an implementation of the Car–Parrinello AIMD.22 AIMD calculations were used to simulate the inversion/pseudorotation trajectories and to determine the free-energy difference along the reaction coordinate by thermodynamic integration of the average force on the constraint at a given temperature. The reported static calculations were carried out with the gaussian 98 program package. The static calculations were employed as a complement for the dynamical simulations and to derive the enthalpies of reaction, which are sometimes available from experimental data. Further computational details are presented in Supplementary data available in the electronic version of this paper.

Section snippets

Results

Before engaging in a detailed study of 1 and 2, we wanted to validate our methodology by studying an example with a firm experimental basis.

A recent publication by the Woerpel group convincingly demonstrated that both the 4-methyltetrahydropyranosyl (3) and 4-benzyloxytetrahydropyranosyl (4) oxacarbenium ions can undergo ring inversion under their reaction conditions before nucleophilic attack.23 Furthermore, both 3 and 4 exhibit facial selectivity based on a preferred 4-methyl pseudoequatorial

Discussion

Hydrolysis of glycosides, whether chemically catalyzed by acid or by acid catalysis induced by glycosidases, as discussed above, are thought to proceed through glycopyranosyl oxacarbenium ion TSs. Similarly, many variants of the reverse glycosylation reaction are thought to proceed through oxacarbenium ion intermediates or TSs. In the case of six-membered rings in particular, there are two mechanistic extremes for how the conformation of the ion can be formed. In one mechanism the neutral

Conclusions

The goal of this paper was to investigate the conformational interconversions of glycopyranosyl oxacarbenium ions in both dynamical and static calculations. We have combined the constrained method with ab initio molecular dynamics simulations to fully sample the potential energy surface of two 4-substituted tetrahydropyranosyl oxacarbenium ions and 2,3,4,6-tetra-O-methyl-d-gluco- and mannopyranosyl cations. The inversion process was conducted via q1, q2, and q3 constraints. The AIMD inversion

Acknowledgment

This work was supported by the High-Performance Computing initiative of the NRC Canada. This is NRC paper #42511.

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