Stereocontrolled Synthesis of α-Xylofuranosides Using a Conformationally Restricted Donor.

A number of biologically relevant glycoconjugates possess 1,2- cis-furanosidic linkages, a class of glycosidic bond that remains challenging to introduce with high stereoselectivity. In this paper, we report an approach to one family of such linkages, α-xylofuranosides, via the use of thioglycoside donors possessing a conformationally restricting xylylene protecting group. The method was shown to provide the desired targets in good to excellent yield and stereoselectivity. Computational investigations support the proposal that the protecting group locks the electrophilic intermediate in these reactions into a conformation that leads to the high selectivity. The power of the methodology was demonstrated through the synthesis of a complex hexasaccharide motif from lipoarabinomannan, an immunomodulatory polysaccharide from mycobacteria.

Specificity profiling of dual specificity phosphatase vaccinia VH1-related (VHR) reveals two distinct substrate binding modes.

Vaccinia VH1-related (VHR) is a dual specificity phosphatase that consists of only a single catalytic domain. Although several protein substrates have been identified for VHR, the elements that control the in vivo substrate specificity of this enzyme remain unclear. In this work, the in vitro substrate specificity of VHR was systematically profiled by screening combinatorial peptide libraries. VHR exhibits more stringent substrate specificity than classical protein-tyrosine phosphatases and recognizes two distinct classes of Tyr(P) peptides. The class I substrates are similar to the Tyr(P) motifs derived from the VHR protein substrates, having sequences of (D/E/ϕ)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) or (D/E/ϕ)(T/S)(D/E)pY(G/A/S/Q) (where ϕ is a hydrophobic amino acid and pY is phosphotyrosine). The class II substrates have the consensus sequence of (V/A)P(I/L/M/V/F)X1-6pY (where X is any amino acid) with V/A preferably at the N terminus of the peptide. Site-directed mutagenesis and molecular modeling studies suggest that the class II peptides bind to VHR in an opposite orientation relative to the canonical binding mode of the class I substrates. In this alternative binding mode, the Tyr(P) side chain binds to the active site pocket, but the N terminus of the peptide interacts with the carboxylate side chain of Asp(164), which normally interacts with the Tyr(P) + 3 residue of a class I substrate. Proteins containing the class II motifs are efficient VHR substrates in vitro, suggesting that VHR may act on a novel class of yet unidentified Tyr(P) proteins in vivo.

On the mechanism of action of gated molecular baskets: The synchronicity of the revolving motion of gates and in/out trafficking of guests.

We used dynamic (1)H NMR spectroscopic methods to examine the kinetics and thermodynamics of CH(3)CCl(3) (2) entering and leaving the gated molecular basket 1. We found that the encapsulation is first-order in basket 1 and guest 2, while the decomplexation is zeroth-order in the guest. Importantly, the interchange mechanism in which a molecule of CH(3)CCl(3) directly displaces the entrapped CH(3)CCl(3) was not observed. Furthermore, the examination of the additivity of free energies characterizing the encapsulation process led to us to deduce that the revolving motion of the gates and in/out trafficking of guests is synchronized, yet still a function of the affinity of the guest for occupying the basket: Specifically, the greater the affinity of the guest for occupying the basket, the less effective the gates are in "sweeping" the guest as the gates undergo their revolving motion.

2,3-Anhydrosugars in glycoside bond synthesis: mechanism of 2-deoxy-2-thioaryl glycoside formation.

A series of investigations probing the mechanism of the 2,3-anhydrosugar migration-glycosylation reaction were performed using a thioglycoside with the D-lyxo stereochemistry as the substrate. Among the work reported are the results of quantum mechanical calculations, NMR studies, the measurement of alpha-deuterium kinetic isotope effects, and the synthesis of a series of substrate analogues. All studies point to a consistent finding: that the reaction proceeds through an oxocarbenium ion intermediate, not an episulfonium ion as previously suggested. It is proposed that the high stereoselectivity of the reaction arises from a preferred "inside attack" of the nucleophile onto the oxocarbenium ion intermediate.

The prospect of selective recognition of nerve agents with modular basket-like hosts. A structure-activity study of the entrapment of a series of organophosphonates in aqueous media.

We designed, prepared, and characterized three cup-shaped cavitands 1-3 for trapping organophosphonates (O═PR(OR')2, 118-197 Å(3)) whose shape and size correspond to G-type chemical warfare agents (132-186 Å(3)). With the assistance of computational (molecular dynamics) and experimental ((1)H NMR spectroscopy) methods, we found that host [1-H3](3+) orients its protonated histamine residues at the rim outside the cavity, in bulk water. In this unfolded form, the cavitand traps a series of organophosphonates 5-13 (K(app) = 87 ± 1 to 321 ± 6 M(-1) at 298.0 K), thereby placing the P-CH3 functional group in the inner space of the host. A comparison of experimental and computed (1)H NMR chemical shifts of both hosts and guests allowed us to derive structure-activity relationships and deduce that, upon the complexation, the more sizable P-OR functional groups in guests drive organophosphonates to the northern portion of the basket [1-H3](3+). This, in turn, causes a displacement of the guest's P-CH3 group and a contraction of the cup-shaped scaffold. The proposed induced-fit model of the recognition is important for turning these modular hosts into useful receptors capable of a selective detection/degradation of organophosphorus nerve agents.

Theoretical Investigations on the Conformation of the ß-d-Arabinofuranoside Ring.

A method for the conformational analysis of furanose rings that involves the prediction of (3)JH,H that can be compared directly to experimental values is investigated. This method, which differs from the traditional PSEUROT approach for conformational studies of furanose rings, was previously applied to a number of α-d-arabinofuranosides and enabled the direct comparison of (3)JH,H values to those obtained from NMR spectroscopy. In this paper, the use of this approach to study the conformational preferences of oligosaccharides containing ß-linked arabinofuranose residues is reported. Density functional theory (DFT) calculations were carried out to derive Karplus relationships that are specifically tailored for these ring systems. In addition, probability distributions obtained from GLYCAM/AMBER molecular dynamics simulations were employed to calculate (3)JH,H values from these Karplus relationships. However, unlike the results obtained with α-arabinofuranosides, the (3)JH,H values computed for ß-arabinofuranosides agreed poorly with experimental values. This prompted the exploration of other methodologies including reevaluation and optimization of the initial MD protocol, use of various force field models, and recalculation of the DFT-derived coupling profiles using an optimized basis set. After extensive investigations, we established that the conformer distributions obtained from MD simulations with the GLYCAM force fields and the furanoside-specific CHARMM force field in combination with the DFT Karplus equations, determined using an augmented basis set (B3LYP/aug-cc-pVTZ-J), produced the best agreement compared to experimental (3)JH,H values. Using these protocols, there is relatively good agreement in (3)JH,H for all coupling pathways with the exception of (3)J2,3 and (3)J3,4, which are underestimated.

Conformational Analysis of Oligoarabinofuranosides: Overcoming Torsional Barriers with Umbrella Sampling.

In this report, the conformations of a series of mono- and oligoarabinofuranosides were probed through the use of umbrella sampling simulations with the AMBER force field and the GLYCAM carbohydrate parameter set. The rotamer population distribution about the exocyclic C4-C5 bonds and the puckering distributions of the rings obtained from these umbrella sampling simulations were found to be in excellent agreement with those obtained from conventional long MD simulations for small monosaccharide fragments. For larger systems, the conventional MD approach becomes impractical, and we propose the use of umbrella sampling to circumvent poor sampling of certain conformations. The same umbrella sampling simulations were used to calculate the distributions about the vicinal protons and ensemble-averaged vicinal proton-proton coupling constants ((3)JH,H). The distributions about the vicinal protons of a monomer, methyl-α-l-arabinofuranoside (1), were found to be very similar to those obtained from direct umbrella sampling simulations about the vicinal protons. We calculated (3)JH,H based on DFT-based Karplus-like relationships for l-arabinofuranosides. The (3)JH,H values were found to be very similar to those obtained with the conventional MD simulations. For 1, the (3)JH,H values obtained with the DFT-based Karplus equations agree very well with experimental results; the agreement is, however, not as good for the larger oligomers. An approach to determine the experimental rotamer populations from the simulations is also discussed.

Conformational Analysis of Arabinofuranosides: Prediction of (3)JH,H Using MD Simulations with DFT-Derived Spin-Spin Coupling Profiles.

A molecular dynamics (MD) investigation on a series of oligo-α-arabinofuranosides (1-8) using the AMBER force field and the GLYCAM carbohydrate parameter set is reported. The validation of the method was carried out by direct comparison of experimental vicinal proton-proton coupling constants ((3)JH,H) with those obtained by using an empirically determined Karplus equation and density functional theory (DFT)-derived relationships specifically tailored for α-arabinofuranosyl systems. A simple code was developed to implement the determination of (3)JH,H by applying these relationships to the probability distributions of rotamers and ring conformations displayed by the simulations. The empirical Karplus relationship and the DFT-derived equations yielded, in most cases, the same trend as experiment for intra-ring (3)JH,H values. This direct comparison circumvents additional sources of errors that may arise from the assumptions introduced by the deconvolution procedures often used to calculate population of rotamers and ring conformations from experimental (3)JH,H.

Conformational Studies of Methyl ß-d-Arabinofuranoside Using the AMBER/GLYCAM Approach.

Furanosides are important constituents of a number of glycoconjugates from many microorganisms. The highly flexible nature of these furanosyl moieties is believed to contribute significantly to their role in biological processes. Therefore, an understanding of the conformational preferences of these molecules is an important area of research. As part of a larger program involved in the conformational analysis of mycobacterial oligofuranosides, molecular dynamics simulations on methyl ß-d-arabinofuranoside (3) have been carried out using the AMBER forcefield and the GLYCAM carbohydrate parameter set. This approach was used to predict the rotamer population distribution about the hydroxymethyl group (C4-C5 bond) as well as the ring puckering of this flexible ring system. Comparison of the conformer distributions obtained during the simulation of 3 using the TIP3P water model with those obtained by analysis of (1)H-(1)H coupling constant data indicated that this water model was insufficient to describe the solvation of this system. However, the use of the TIP4P and TIP5P models led to improved agreement with conformer populations obtained from NMR data.