Synthesis of a sulfonium ion analogue of the glycosidase inhibitor swainsonine

The synthesis of a bicyclic sulfonium ion analogue of a naturally occurring indolizidine alkaloid, swainsonine, in which the bridgehead nitrogen atom is replaced by a sulfonium ion, has been achieved by a multistep synthesis starting from (2S,3S,4R)-2,3-dibenzyloxy-4-formaldehyde-thiolane. The synthetic strategy relies on the intramolecular displacement of a leaving group on a pendant acyclic chain by a cyclic thioether. This bicyclic sulfonium salt provides a candidate with which to further probe the hypothesis that a sulfonium salt carrying a permanent positive charge would be an effective glycosidase inhibitor.     

Total synthesis of hyacinthacine A1, a glycosidase inhibitor

A practical and enantioselective total synthesis of hyacinthacine A1 is achieved involving syn allylic epoxide opening with retention using Pd catalysis and "domino" hydrogenation (five steps in one pot) sequences.     

Quantum-chemical study on the bioactive conformation of epothilones

Herein, I report a DFT study on the bioactive conformation of epothilone A based on the analysis of 92 stable conformations of free and bound epothilone to a reduced model of tubulin receptor. The equilibrium structures and relative energies were studied using B3LYP and X3LYP functionals and the 6-31G(d) standard basis set, which was considered appropriate for the size of the systems under study. Calculated relative energies of free and bound epothilones led me to propose a new model for the bioactive conformation of epothilone A, which accounts for several structure-activity data.     

Synthesis of selenium analogues of the naturally occurring glycosidase inhibitor salacinol and their evaluation as glycosidase inhibitors

The syntheses of two selenium analogues (10 and 11) of the naturally occurring sulfonium ion, salacinol (3), are described. Salacinol is one of the active principles in the aqueous extracts of Salacia reticulata that are traditionally used in Sri Lanka and India for the treatment of diabetes. The synthetic strategy relies on the nucleophilic attack of a 2,3,5-tri-O-benzyl-1,4-anhydro-4-seleno-D-arabinitol at the least hindered carbon of benzyl- or benzylidene-protected D- or L-erythritol-1,3-cyclic sulfate. The use of 1,1,1,3,3,3-hexafluoro-2-propanol as a solvent in the coupling reaction proves to be beneficial. Enzyme inhibition assays indicate that 10 is a better inhibitor (K(i) = 0.72 mM) of glucoamylase than 3, which has a K(i) value of 1.7 mM. In contrast, 11 showed no significant inhibition of glucoamylase. Compounds 10 and 11 showed no significant inhibition of barley-alpha-amylase or porcine pancreatic-alpha-amylase.     

Stereoselective synthesis of the glycosidase inhibitor australine through a one-pot, double-cyclization strategy

[reaction: see text] A stereocontrolled, convergent synthesis of the alkaloid australine, a glycosidase inhibitor of the pyrrolizidine class, is described. The chiral starting materials were ketone 3, derived from L-erythrulose, and alpha-alkoxy aldehyde 4, prepared from L-malic acid. A key step of the synthesis was the highly stereoselective aldol reaction between 4 and a Z boron enolate derived from 3. Another key step was the one-pot construction of the bicyclic pyrrolizidine system by means of a three-step sequence of SN2 displacements induced by benzylamine on a trimesylate precursor.     

Retention of bioactive ligand conformation in a gaseous protein-trisaccharide complex

Arrhenius parameters, obtained with the blackbody infrared radiative dissociation technique, are reported for the dissociation of gaseous protonated complexes of a single-chain variable fragment (scFv) of the monoclonal antibody Se155-4 with structurally constrained trisaccharide ligands that resemble the bioactive conformer. The similarity in the dissociation activation energies measured for the +10 charge-state complexes of the constrained ligands and the native trisaccharide is evidence that the bioactive conformation of the native ligand is retained in the gas phase.     

Novel synthesis of the glycosidase inhibitor deoxymannojirimycin and of a synthetic precursor D-lyxo-hexos-5-ulose

[reaction: see text]. The synthesis of D-lyxo-hexos-5-ulose (5-ketomannose, 1,5-dicarbonyl sugar), a synthetic precursor to the glycoprocessing inhibitor deoxymannojirimycin, was carried out by an in situ epoxidation and hydrolysis of a trimethylsilyl-protected 6-deoxyhex-5-enopyranoside followed by facile removal of the protecting groups. A novel nine-step synthesis of deoxymannojirimycin has also been achieved from methyl alpha-D-mannopyranoside; this involved methanolysis of epoxides derived from an acetylated 1-azido-6-deoxyhex-5-enopyranoside followed by deprotection and catalytic hydrogenation.     

Efficient synthesis of the glucosidase inhibitor blintol, the selenium analogue of the naturally occurring glycosidase inhibitor salacinol

An efficient synthesis of blintol, the selenium congener of the naturally occurring glycosidase inhibitor salacinol, and a potent glucosidase inhibitor itself, is described. Unlike our previously reported synthesis, this improved route makes use of p-methoxybenzyl ether protecting groups in the synthesis of one of the two key intermediates, 2,3,5-tri-O-p-methoxybenzyl-1,4-anhydro-4-seleno-D-arabinitol, from L-xylose. The other key intermediate, 2,4-O-benzylidene-L-erythritol-1,3-cyclic sulfate, was successfully prepared from D-glucose instead of the expensive L-glucose. All protecting groups in the resulting adducts were removed with trifluoroacetic acid to yield a mixture of stereoisomers, thereby obviating the problematic deprotection of benzyl ethers by hydrogenolysis. The major stereoisomer, blintol, was then obtained by fractional crystallization.     

Toward accurate relative energy predictions of the bioactive conformation of drugs

Quantifying the relative energy of a ligand in its target-bound state (i.e. the bioactive conformation) is essential to understand the process of molecular recognition, to optimize the potency of bioactive molecules and to increase the accuracy of structure-based drug design methods. This is, nevertheless, seriously hampered by two interrelated issues, namely the difficulty in carrying out an exhaustive sampling of the conformational space and the shortcomings of the energy functions, usually based on parametric methods of limited accuracy. Matters are further complicated by the experimental uncertainty on the atomic coordinates, which precludes a univocal definition of the bioactive conformation. In this article we investigate the relative energy of bioactive conformations introducing two major improvements over previous studies: the use sophisticated QM-based methods to take into account both the internal energy of the ligand and the solvation effect, and the application of physically meaningful constraints to refine the bioactive conformation. On a set of 99 drug-like molecules, we find that, contrary to previous observations, two thirds of bioactive conformations lie within 0.5 kcal mol(-1) of a local minimum, with penalties above 2.0 kcal mol(-1) being generally attributable to structural determination inaccuracies. The methodology herein described opens the door to obtain quantitative estimates of the energy of bioactive conformations and can be used both as an aid in refining crystallographic structures and as a tool in drug discovery.     

NMR determination of the bioactive conformation of peloruside A bound to microtubules

We report here on the determination of the conformation of Peloruside A bound to biochemically stabilized microtubules, by using TR-NOESY NMR experiments. As a previous step, the conformation of the free molecule in water solution has also been deduced. Despite the large size of the ring, Peloruside A mainly adopts two conformations in water solution. A conformational selection process takes place, and the microtubules-bound conformer is one of those present in the water solution, different than that existing in chloroform medium. A model of the binding mode to tubulin has also been proposed, by docking the bioactive conformation of peloruside, which involves the alpha-tubulin monomer, in contrast with taxol, which binds to the beta-monomer.     

New chain-extended analogues of salacinol and blintol and their glycosidase inhibitory activities. Mapping the active-site requirements of human maltase glucoamylase

The synthesis of new chain-extended sulfonium and selenonium salts of 1,4-anhydro-4-thio-(or 4-seleno)-d-arabinitol, analogues of the naturally occurring glycosidase inhibitor salacinol, is described. Nucleophilic attack at the least hindered carbon atom of 4,6-O-benzylidene-2,5-di-O-p-methoxybenzyl-d-mannitol-1,3-cyclic sulfate by 2,3,5-tri-O-p-methoxybenzyl-1,4-anhydro-4-thio-(or 4-seleno)-d-arabinitol gave the sulfonium and selenonium sulfates, respectively. Subsequent deprotection with trifluoroacetic acid yielded the target compounds. In these analogues, an extended polyhydroxylated aliphatic side chain has been incorporated while maintaining the stereochemistry of C-2' and C-3' of salacinol or blintol. These compounds were designed to probe the premise that they would bind with higher affinity to glucosidases than salacinol because the extra hydroxyl groups in the acyclic chain would make favorable polar contacts within the active site. Both target compounds inhibited recombinant human maltase glucoamylase, one of the key intestinal enzymes involved in the breakdown of glucose oligosaccharides in the small intestine, with Ki values in the low micromolar range. Comparison of these values to those of related compounds synthesized in previous studies has provided a better understanding of structure-activity relationships and the optimal stereochemistry at the different stereogenic centers required of an inhibitor of this enzyme. With respect to chain extension, the configurations at C-2' and C-4' are critical for activity, the configuration at C-3', bearing the sulfate moiety, being unimportant. The desired configuration at C-5' is also specified. However, comparison of the activities of the chain-extended analogues with those of salacinol and blintol indicates that there is no particular advantage of the chain-extension relative to salacinol or blintol. These results are similar to those reported earlier for kotalanol, a 7-carbon-extended derivative, versus salacinol against rat intestinal maltase, sucrase, and isomaltase.     

Direct observation of the protonation state of an imino sugar glycosidase inhibitor upon binding

Glycosidases are some of the most ubiquitous enzyme in nature. Their biological significance, coupled to their enormous catalytic prowess derived from tight binding of the transition state, is reflected in their importance as therapeutic targets. Many glycosidase inhibitors are known. Imino sugars are often potent inhibitors, yet many facets of their mode of action, such as their degree, if any, of transition-state "mimicry" and their protonation state when bound to the target glycosidase remain unclear. Atomic resolution analysis of the endoglucanase, Cel5A, in complex with a cellobio-derived isofagomine in conjunction with the pH dependence of Ki and kcat/KM reveals that this compound binds as a protonated sugar. Surprisingly, both the enzymatic nucleophile and the acid/base are unprotonated in the complex.     

Synthesis and evaluation of isourea-type glycomimetics related to the indolizidine and trehazolin glycosidase inhibitor families

A practical synthesis of reducing isourea-derived azasugar glycomimetics related to the indolizidine and trehazolin glycosidase inhibitor families with different pK(a) values is disclosed. The polyhydroxylated bicyclic system was built from readily accessible hexofuranose derivatives through a synthetic scheme that involves the preparation of a 5-deoxy-5-carbodiimido adduct by triphenylphosphine-mediated tandem Staudinger--aza-Wittig-type coupling of azide and isothiocyanate precursors, intramolecular cyclization of a transient vic-hydroxycarbodiimide derivative, and nucleophilic addition of the endocyclic nitrogen atom of the generated 2-amino-2-oxazoline intermediate, with a pseudo-C-nucleoside structure, to the masked aldehyde group of the monosaccharide. The last step is pH-dependent so that the final compounds can pivot between the furanose and the 2-oxaindolizidine forms. Nevertheless, the indolizidine tautomer having the R configuration at the aminoacetalic center, fitting the anomeric effect, was the only species detected in solution at neutral or slightly acidic pH when starting from solutions at basic pH. Glycosidase inhibition tests (K(i) values down to 1.9 microM) showed a marked dependence of the selectivity and potency toward alpha- and beta-glucosidases upon the nature of the substituent at the exocyclic isourea nitrogen, shifting from alpha- to beta-selectivity when going from hydrophilic to hydrophobic substituents. Enzyme inhibition is also pH dependent, supporting a dominant role for the uncharged form of the polyhydroxyiminoindolizidine system in the inhibition of beta-glucosidases.     

Trihydroxy-2-thiaquinolizidine derivatives as a new class of bicyclic glycosidase inhibitors

Trihydroxy-2-thiaquinolizidines, a new class of bicyclic dideoxy-iminohexitol glycosidase inhibitor derivatives with nominally the d-gluco, l-ido, d-manno and l-gulo configurations were synthesized. X-ray analyses indicated that the preferred conformation for d-gluco and d-manno derivatives was a flat trans-fused system. Unlike deoxynojirimycin, the compound with d-gluco configuration was selective for α-glucosidases (yeast and rice) and showed no inhibitory activity towards β-glucosidase (almond), α-galactosidase (green coffee beans), α-galactosidase (E. coli) and α-mannosidase (jack bean), while the l-ido derivative was specific for β-glucosidase (almond).     

A computational analysis of binding modes and conformation changes of MDM2 induced by p53 and inhibitor bindings

Molecular dynamics (MD) simulations followed by principal component analysis were performed to study the conformational change of MDM2 induced by p53 and two inhibitor (P4 and MI63a) bindings. The results show that the hydrophobic cleft of MDM2 is very flexible and adaptive to different structural binding partners. The cleft tends to become wider and more stable as MDM2 binds to the three binding partners, while unbound MDM2 shows a narrower and pretty flexible cleft, which agrees with recent experimental data and theoretical studies. It was also found that the binding of P4 and p53 stabilizes the motion of the loop L2 linking the helix α2 and β strand (β3), but the presence of MI63a makes the motion of L2 disordered. In addition, the binding free energies of the three partners to MDM2 were calculated using molecular mechanics generalized Born surface area to explain the binding modes of these three partners to MDM2. This study will be helpful not only for better understanding the functional, concerted motion of MDM2, but also for the rational design of potent anticancer drugs targeting the p53–MDM2 interaction.     

Oct-2-yn-4-enoyl-CoA as a multifunctional enzyme inhibitor in fatty acid oxidation

Oct-2-yn-4-enoyl-CoA was found to be a multifunctional irreversible enzyme inhibitor in fatty acid oxidation mainly targeting mitochondrial trifunctional protein beta-subunit. It can also inactivate enoyl-CoA hydratase 2 and medium-chain acyl-CoA dehydrogenase. This study increased our understanding for the effect of acetylenic acids on fatty acid oxidation.     

Synthesis and conformational analysis of bicyclic sulfonium salts. Structures related to the glycosidase inhibitor australine

The syntheses of eight sulfonium compounds with structures related to the naturally occurring pyrrolizidine alkaloid, australine, in which the bridgehead nitrogen atom is replaced by a sulfonium ion, are described. The synthetic strategy relies on the intramolecular attack of a cyclic thioether across a terminal double bond in the presence of a suitable electrophile. We postulate that these compounds, having a permanent positive charge on the sulfur atom, will mimic the highly unstable oxacarbenium ion transition state in a glycosidase-catalyzed hydrolysis reaction. The conformational preferences of these compounds, based on analysis of 1H-1H vicinal coupling constants and 1D-NOESY data, are attributed to both steric and electrostatic interactions. These compounds will be used in the study of structure-activity relationships with glycosidase enzymes.     

Crystal structure and molecular conformation of E-64, a cysteine protease inhibitor

In order to elucidate the conformational characteristics of cysteine protease inhibitors contributing to their inhibitory activities, the conformation of E-64 (N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine), a potent inhibitor of papain, was determined by X-ray crystal structure analysis. The molecules were packed in the crystal through electrostatic forces and hydrogen bonding between the oppositely charged terminal groups and between the amide groups. Two crystallographically independent E-64 molecules both took a flattened and slightly curved structure, which is similar to that of loxistatin, a related cysteine protease inhibitor. Based on the present results, a possible inhibitory mechanism of E-64 is proposed, with reference to the binding mode observed in the crystal structure of papain-substrate analogue complex.