Monika Poláková

Synthetic esters recognized by glucuronoyl esterase from Schizophyllum commune

Glucuronoyl esterase is a novel carbohydrate esterase recently discovered in the cellulolytic system of the wood-rotting fungus Schizophyllum commune on the basis of its ability to hydrolyze methyl ester of 4-O-methyl-d-glucuronic acid. This substrate was not fully corresponding to the anticipated function of the enzyme to hydrolyze esters between xylan-bound 4-O-methyl-d-glucuronic acid and lignin alcohols occurring in plant cell walls. In this work we showed that the enzyme was capable of hydrolyzing two synthetic compounds that mimic the ester linkages described in lignin-carbohydrate complexes, esters of 4-O-methyl-d-glucuronic and d-glucuronic acid with 3-(4-methoxyphenyl)propyl alcohol. A comparison of kinetics of hydrolysis of methyl and 3-(4-methoxyphenyl)propyl esters indicated that the glucuronoyl esterase recognizes the uronic acid part of the substrates better than the alcohol type. The catalytic efficiency of the enzyme was much higher with the ester of 4-O-methyl-d-glucuronic acid than with that of d-glucuronic acid. Examination of the action of glucuronoyl esterase on a series of methyl esters of 4-O-methyl-d-glucopyranuronosyl residues α-1,2-linked to xylose and several xylooligosaccharides suggested that the rate of deesterification is independent of the character of the carbohydrate part glycosylated by the 4-O-methyl-d-glucuronic acid.

α-d-Mannose derivatives as models designed for selective inhibition of Golgi α-mannosidase II

Human Golgi α-mannosidase II (hGM) is a pharmaceutical target for the design of inhibitors with anti-tumor activity. Nanomolar inhibitors of hGM exhibit unwanted co-inhibition of the human lysosomal α-mannosidase (hLM). Hence, improving specificity of the inhibitors directed toward hGM is desired in order to use them in cancer chemotherapy. We report on the rapid synthesis of D-mannose derivatives having one of the RS-, R(SO)- or R(SO(2))- groups at the α-anomeric position. Inhibitory properties of thirteen synthesized α-D-mannopyranosides were tested against the recombinant enzyme Drosophila melanogaster homolog of hGM (dGMIIb) and hLM (dLM408). Derivatives with the sulfonyl [R(SO(2))-] group exhibited inhibitory activities at the mM level toward both dGMIIb (IC(50) = 1.5-2.5 mM) and dLM408 (IC(50) = 1.0-2.0 mM). Among synthesized, only the benzylsulfonyl derivative showed selectivity toward dGMIIb. Its inhibitory activity was explained based on structural analysis of the built 3-D complexes of the enzyme with the docked compounds.

Synthesis of alkyl and cycloalkyl α-d-mannopyranosides and derivatives thereof and their evaluation in the mycobacterial mannosyltransferase assay

The synthesis of a series of alkyl (having from C6 to C20 aglycones), cyclohexyl, and cyclohexylalkyl alpha-d-mannopyranosides, 6-deoxygenated analogs, thioglycosides, and sulfones derived thereof, is reported. Here, under the in vitro assay conditions used, none of the 15 tested compounds acted as an inhibitor of the mannose transfer catalyzed by the enzymes present in mycobacterial membrane and cell wall fractions. Mannopyranosides comprising shorter aliphatic, up to 8 carbon atoms long linear, or cyclic aglycone served as the acceptor substrates in the mycobacterial mannosyltransferase reaction. The thioglycosides exhibited similar behavior, in contrast to the sulfones, which were essentially not recognized by the mycobacterial enzymes. 6-Deoxygenated glycosides were not processed by the enzymes, suggesting that the mannose transfer occurs at position 6 of the acceptors.

Synthesis and cytotoxicity of some D-mannose click conjugates with aminobenzoic acid derivatives.

Two sets of new conjugates obtained from d-mannose derivatives and o-, m-, and p-substituted benzoic acid esters interconnected through a triazole ring were synthesized by Cu(I) catalyzed azide-alkyne cycloaddition. All synthesized compounds were tested for their in vitro cytotoxic activity against seven cancer cell lines with/without multidrug resistance phenotype as well as non-tumor MRC-5 and BJ fibroblasts. Butyl ester of 4-aminobenzoic acid 6c showed the highest activity among all tested compounds, however, it was active only against K562 myeloid leukemia cells. N-Glycosyltriazole conjugates, both acetylated and nonacetylated at mannose moiety, were almost completely inactive. In contrast, some of the acetylated O-glycosyl conjugates showed cytotoxic activity which was cell line dependent and strongly affected by position of benzoic acid substitution as well as a length of its ester alkyl chain; the most potent compound was acetylated mannoside conjugated with octyl ester of m-substituted benzoic acid. However, deacetylation resulting in hydrophilicity increase of the glycosides almost completely abolished their cytotoxic potency.

'Click chemistry' synthesis of 1-(α-D-mannopyranosyl)-1,2,3-triazoles for inhibition of α-mannosidases.

Three new triazole conjugates derived from d-mannose were synthesized and assayed in in vitro assays to investigate their ability to inhibit α-mannosidase enzymes from the glycoside hydrolase (GH) families 38 and 47. The triazole conjugates were more selective for a GH47 α-mannosidase (Aspergillus saitoi α1,2-mannosidase), showing inhibition at the micromolar level (IC50 values of 50-250 μM), and less potent towards GH38 mannosidases (IC50 values in the range of 0.5-6 mM towards jack bean α-mannosidase or Drosophila melanogaster lysosomal and Golgi α-mannosidases). The highest selectivity ratio [IC50(GH38)/IC50(GH47)] of 100 was exhibited by the phenyltriazole conjugate. To understand structure-activity properties of synthesized compounds, 3-D complexes of inhibitors with α-mannosidases were built using molecular docking calculations.

Novel synthetic (1 → 6)-α-D-mannodisaccharide substrates support processive mannosylation catalysed by the mycobacterial cell envelope enzyme fraction

Three new (1 → 6)-α-D-mannodisaccharides with cyclohexylalkyl or octylsulfonyl function like aglycone were synthesized and screened in the mycobacterial mannosyltransferase assay. 2-Cyclohexylethyl (1 → 6)-α-D-Man2 acted as the best acceptor substrate, whereas the sulfonyl group significantly reduced the ability of the mannodisaccharide to serve as the acceptor. Despite these differences, mass spectrometric analysis confirmed the capability of all synthetic mannodisacharides to accept up to ten additional mannose units, i.e. the transfer was not affected by the type of aglycone. The results reported here suggest that the enzyme responsible for the consecutive mannose attachment is the processive α-mannopyranosyltransferase present in the cell-free system of the mycobacterial cell envelope.

Synthesis of modified D-mannose core derivatives and their impact on GH38 α-mannosidases

Nine new compounds having five- and modified six-member carbohydrate core derived from D-lyxose or D-mannose, and non-hydrolysable aglycones (benzylsulfonyl or aryl(alkyl)triazolyl) were synthesised to investigate their ability to inhibit the recombinant Drosophila melanogaster homologs of two human GH38 family enzymes: Golgi mannosidase II (dGMIIb) and lysosomal mannosidase (dLMII). Two compounds were weak selective dGMIIb inhibitors showing IC50 at mM level. Moreover, it was found that another GH38 enzyme, commercial jack bean α-mannosidase, was inhibited by triazole conjugates regardless of the carbohydrate core while the corresponding sulfones were inactive.

N-Benzyl Substitution of Polyhydroxypyrrolidines: The Way to Selective Inhibitors of Golgi α-Mannosidase II

Inhibition of the biosynthesis of complex N‐glycans in the Golgi apparatus influences progress of tumor growth and metastasis. Golgi α‐mannosidase II (GMII) has become a therapeutic target for drugs with anticancer activities. One critical task for successful application of GMII drugs in medical treatments is to decrease their unwanted co‐inhibition of lysosomal α‐mannosidase (LMan), a weakness of all known potent GMII inhibitors. A series of novel N‐substituted polyhydroxypyrrolidines was synthesized and tested with modeled GH38 α‐mannosidases from Drosophila melanogaster (GMIIb and LManII). The most potent structures inhibited GMIIb (Ki=50–76 μm, as determined by enzyme assays) with a significant selectivity index of IC50(LManII)/IC50(GMIIb) >100. These compounds also showed inhibitory activities in in vitro assays with cancer cell lines (leukemia, IC50=92–200 μm) and low cytotoxic activities in normal fibroblast cell lines (IC50>200 μm). In addition, they did not show any significant inhibitory activity toward GH47 Aspergillus saitoiα1,2‐mannosidase. An appropriate stereo configuration of hydroxymethyl and benzyl functional groups on the pyrrolidine ring of the inhibitor may lead to an inhibitor with the required selectivity for the active site of a target α‐mannosidase.

Synthesis of 1,4-imino-L-lyxitols modified at C-5 and their evaluation as inhibitors of GH38 α-mannosidases

A synthetic approach to 1,4-imino-L-lyxitols with various modifications at the C-5 position is reported. These imino-L-lyxitol cores were used for the preparation of a series of N-(4-halobenzyl)polyhydroxypyrrolidines. An impact of the C-5 modification on the inhibition and selectivity against GH38 α-mannosidases from Drosophila melanogaster, the Golgi (GMIIb) and lysosomal (LManII) mannosidases and commercial jack bean α-mannosidase from Canavalia ensiformis was evaluated. The modification at C-5 affected their inhibitory activity against the target GMIIb enzyme. In contrast, no inhibition effect of the pyrrolidines against LManII was observed. The modification of the imino-L-lyxitol core is therefore a suitable motif for the design of inhibitors with desired selectivity against the target GMIIb enzyme.

Alkyl glycosides as potential anti-Candida albicans growth agents

Candida infections are becoming increasingly prevalent and many clinical isolates are resistant to common azole derivatives treatment. Accordingly, the capacity of a series of 19 alkyl glycosides, mainly mannosides and glucosides but also a cellobioside with aglycone chain-length from C-6 to C-20, to inhibit the growth of laboratory and clinically isolated strains of Candida albicans, was investigated. The study showed that only glycosides with the C-10 and C-12 aglycones were effective growth inhibitors of both types of Candida, strains, whose metabolic activity was also significantly reduced as revealed by an XTT assay.