Saturation transfer difference 1D-TOCSY experiments to map the topography of oligosaccharides recognized by a monoclonal antibody directed against the cell-wall polysaccharide of group A streptococcus

A new saturation transfer difference 1D-TOCSY NMR experiment that allows the investigation of complex ligands interacting with proteins and its application in the mapping of which portions of oligosaccharide ligands (epitope) interact with a complementary antibody are described. The interaction between trisaccharide and hexasaccharide ligands, corresponding to fragments of the cell-wall polysaccharide of Streptococcus Group A, and a monoclonal antibody directed against the polysaccharide is investigated at the molecular level. The polysaccharide consists of alternating alpha-(1-->2) and alpha-(1-->3) linked L-rhamnopyranose (Rha) residues with branching N-acetyl-D-glucopyranosylamine (GlcNAc) residues linked beta-(1-->3) to alternate rhamnopyranose rings. The epitope is proven to consist not only of the immunodominant GlcNAc sugar but also of an entire branched trisaccharide repeating unit. The experimental NMR data serve to check and validate the computed models of the oligosaccharide-antibody complexes.     

Chemo-enzymatic synthesis of 4-methylumbelliferyl beta-(1-->4)-D-xylooligosides: new substrates for beta-D-xylanase assays

Transglycosylation catalyzed by a beta-D-xylosidase from Aspergillus sp. was used to synthesize a set of 4-methylumbelliferyl (MU) beta-1-->4-D-xylooligosides having the common structure [beta-D-Xyl-(1-->4)]2-5-beta-D-Xyl-MU. MU xylobioside synthesized chemically by the condensation of protected MU beta-D-xylopyranoside with ethyl 2,3,4-tri-O-acetyl-1-thio-beta-D-xylopyranoside was used as a substrate for transglycosylation with the beta-D-xylosidase from Aspergillus sp. to produce higher MU xylooligosides. The structures of oligosaccharides obtained were established by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. MU beta-D-xylooligosides synthesized were tested as fluorogenic substrates for the GH-10 family beta-D-xylanase from Aspergillus orizae and the GH-11 family beta-D-xylanase I from Trichoderma reesei. Both xylanases released the aglycone from MU xylobioside and the corresponding trioside. With substrates having d.p. 4 and 5, the enzymes manifested endolytic activities, splitting off MU, MUX, and MUX2 primarily.     

A new phenoxyacetate-based linker system for the solid-phase synthesis of oligosaccharides

[structure: see text]. A novel linker system has been designed, and its first application to solid-phase oligosaccharide synthesis is described. The use of the highly reactive o-nitro-phenoxyacetate linker allows a fast and quantitative cleavage using mild basic conditions. This method combined with the trichloroacetimidate glycosylation exhibits highly promising results as demonstrated for the synthesis of tetrasaccharide 1 (n = 3) containing glucose beta(1 --> 4) and beta(1 --> 6) linkages.     

Convergent synthesis of a beta-(1-->3)-mannohexaose

An as yet unknown beta-(1-->3)-mannohexaose has been synthesized by a block route involving the coupling of two trisaccharides. Comparison of three closely related attempted mannohexaose syntheses reinforces the influence of subtle matching and/or mismatching interactions on the outcome of convergent oligosaccharide synthesis.     

Chemical synthesis of cyclic galactooligofuranosides isolated from enzymatic degradation products of cell wall arabinogalactan of Mycobacterium tuberculosis

Synthesis of cyclic tetra-, hexa- and octasaccharides containing alternating (1-->5)-beta- and (1-->6)-beta-galactofuranosyl linkages has been achieved by intramolecular cycloglycosylation of corresponding linear sugars and by cyclooligomerization of 1,6-linked and 1,5-linked disaccharides. In particular, cyclooligomerization of the (1-->6)-beta-galactofuranosyl disaccharide provides an efficient way to secure all three cyclic sugars in one operation.     

Use of insoluble yeast beta-glucan as a support for immobilization of Candida rugosa lipase

In the present study, insoluble yeast beta-glucan (IYG) has been explored as a support matrix for enzyme immobilization. IYG contains mainly beta-(1-3) linkages along with some intra- or inter-molecular branches of beta-(1-6) linkages with large number of free hydroxyl groups. Epichlorohydrin was used to convert these free hydroxyl groups into activated epoxy groups that are capable of forming covalent linkages with various groups of enzyme molecule. The epoxy-activated IYG was evaluated for immobilization of Candida rugosa lipase (CRL). Post-immobilization treatment of 5% glutaraldehyde was given in order to achieve stable and irreversible binding of enzyme on the support. The resultant biocatalytic IYG support expressed lipase activity of 8136.7 U/g and 59.6% activity yield. There was 51.05% retention of synthetic activity after six repeated esterification cycles, indicating its stability and reusability in non-aqueous medium. Moreover, the immobilized lipase gave the storage half-life of about 285 days (at 4 degrees C).     

Rapid synthesis of oligosaccharide moieties of globotriaosylceramide using fluorous protective group

The use of the Bfp (bisfluorous chain type propanoyl) group as a fluorous protective group made it possible to rapidly synthesize galabiose and the Gb3 oligosaccharide derivatives by a simple fluorous-organic extraction purification. The fluorous oligosaccharide synthesis using the Bfp group is an excellent strategic alternative to solid phase oligosaccharide synthesis, and removes some of the disadvantages of the solid phase method.     

Direct chemical synthesis of the beta-mannans: linear and block syntheses of the alternating beta-(1-->3)-beta-(1-->4)-mannan common to Rhodotorula glutinis, Rhodotorula mucilaginosa, and Leptospira biflexa

Two stereocontrolled syntheses of a methyl glycoside of an alternating beta-(1-->4)-beta-(1-->3)-mannohexaose, representative of the mannan from Rhodotorula glutinis, Rhodotorula mucilaginosa, and Leptospira biflexa, are described. Both syntheses employ a combination of 4,6-O-benzylidene- and 4,6-O-p-methoxybenzylidene acetal-protected donors to achieve stereocontrolled formation of the beta-mannoside linkage. The first synthesis is a linear one and proceeds with a high degree of stereocontrol throughout and an overall yield of 1.9%. The second synthesis, a block synthesis, makes use of the coupling of two trisaccharides, resulting in a shorter sequence and an overall yield of 4.4%, despite the poor selectivity in the key step.     

Solution conformations of mycothiol bimane, 1-D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins and 1-D-GlcNAc-alpha-(1 --> 1)-L-myo-Ins

Mycothiol is an abundant small molecular weight thiol found only in actinomycetes, which include mycobacteria. Mycothiol biosynthetic and detoxification enzymes are novel and unique to actinomycetes, thereby representing potential antimycobacterial targets. To better guide inhibitor design, we have determined by NMR the solution conformations of mycothiol bimane (MSmB) and the pseudodisaccharide 1-D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins (D-GI), molecules that represent the natural substrates for the mycothiol-dependent detoxification enzyme mycothiol-S-conjugate amidase (MCA) and the mycothiol biosynthetic enzyme D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins deacetylase (AcGI deacetylase), respectively. Comparison of the mean structure of MSmB and the energy-minimized structures of two competitive spiroisoxazoline-containing MCA inhibitors shows striking similarities between these molecules in the region of the scissile amide bond of MSmB and provides structural evidence that those inhibitors are substrate mimics. Owing to our earlier finding that AcGI deacetylase will not deacetylate the unnatural isomer 1-d-GlcNAc-alpha-(1 --> 1)-L-myo-Ins (L-GI), the solution conformation of L-GI was also determined. The interglycosidic bond angles for all three compounds are comparable. When considered together with the observation that a simplified cyclohexyl thioglycoside mycothiol analogue is a good substrate for MCA, it appears that the stereochemistry of the inositol ring is critical for deacetylase function, superceding the importance of the full complement of hydroxyl groups on the "nonreducing" ring.     

Detection of beta-glucanase activity on various beta-1,3 and beta-1,4-glucans after native and denaturing polyacrylamide gel electrophoresis.

beta-Glucanases were detected after polyacrylamide gel electrophoresis under native and denaturing conditions using various beta-1,3- and beta-1,4-glucans, including mixed glucans (laminarin, pachyman, carboxymethyl cellulose, lichenan and barley beta-glucan). After electrophoresis and incubation of gels, substrates incorporated into polyacrylamide gels were stained with specific fluorochromes, Sirofluor for beta-1,3 linkages and Calcofluor White M2R for beta-1,4 linkages. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. Enzymes of bacterial, fungal and plant sources could be revealed sequentially in gles containing mixed beta-(1,3)(1,4)-glucans by staining first with sirofluor followed by staining with Calcofluor White M2R. Active profiles were more diverse when substrates were stained with sirofluor. The use of purified sirofluor at pH 11.5 compared with Aniline Blue at pH 8.6 allowed better detection of beta-1,3-glucanase activities. In gels containing laminarin stained with sirofluor, bands exhibiting a more intense fluorescence than the background fluorescence were observed in addition to dark nonfluorescent bands. It is postulated that these two types of beta-1,3-glucanase activities differ by their enzymatic action (partial versus extensive hydrolysis). Analysis of fungal extracts using denaturing gels embedded with various beta-glucans displayed lysis bands migrating between 32 and 35 kDa.     

Enantioselective synthesis of ethyl (S)-2-hydroxy-4-phenylbutyrate by recombinant diketoreductase

Recombinant diketoreductase showed excellent stereoselectivity in the double reduction of β,δ-diketo esters. To investigate the substrate specificity and to broaden the applications of this new biocatalyst, a number of ketone substrates were used to evaluate the substrate spectrum and enantioselectivity of this enzyme in the present study. Among the ketone substrates tested, only this enzyme displayed high efficiency and excellent enantioselectivity in the reduction of ethyl 2-oxo-4-phenylbutyrate to ethyl (S)-2-hydroxy-4-phenylbutyrate. After optimizing the reaction conditions, the bio-reduction of ethyl 2-oxo-4-phenylbutyrate at a substrate concentration of 0.8 M (164.8 g/L) was achieved by the recombinant diketoreductase in an aqueous-toluene biphasic system coupled with formate dehydrogenase for the regeneration of cofactor, resulting in an overall hydroxyl product yield of 88.7% (99.5% ee). This new enzymatic transformation may offer a practical method for the preparation of this important chiral building block.     

Ionic-liquid-based MS probes for the chemo-enzymatic synthesis of oligosaccharides

A new N-benzenesulfonyl-based ionic-liquid mass spectroscopy label (I-Tag2) for covalent attachment to substrates has been prepared. I-Tag2 was used to monitor oligosaccharide elongation and serve as a purification handle. Starting from chemically synthesized I-Tag2-labelled N-acetyl glucosamine (GlcNAc) 1, I-Tag2-LacNAc (Galβ(1-4)GlcNAc) 2 and I-Tag2-Lewis(X) (Galβ(1-4)[Fucα(1-3)]GlcNAc) 3, which are oligosaccharides of biological relevance, were enzymatically prepared. The apparent kinetic parameters for the enzyme catalysed transformations with β-1,4-galactosyltransferase (β-1,4-GalT) and fucosyltransferase VI (FucT VI) were measured by LC-MS demonstrating the applicability and versatility of the new I-Tags in enzymatic transformations with glycosyltransferases.     

Effective synthesis of (S)-3,5-bistrifluoromethylphenyl ethanol by asymmetric enzymatic reduction

The synthesis of (S)-3,5-bistrifluoromethylphenyl ethanol, a pharmaceutically important alcohol intermediate for the synthesis of NK-1 receptor antagonists, was demonstrated from a ketone via asymmetric enzymatic reduction. The isolated enzyme alcohol dehydrogenase from Rhodococcus erythropolis reduced the poorly water soluble substrate with excellent ee (>99.9%) and good conversion (>98%). The optimized process was demonstrated up to pilot scale using high substrate concentration (390 mM) using a straightforward isolation process achieving excellent isolation yields (>90%) and effective space time yield (100–110 g/L d). Process improvements, demonstrated at preparative scale, increased the substrate concentration to 580 mM achieving a space time yield of 260 g/L d.     

Oligosaccharide synthesis in microreactors

Described is the combination of microreactors and fluorous phase chemistry to assemble oligosaccharides. The synthesis of a beta-(1-->6) linked D-glucopyranoside homotetramer serves to illustrate this approach. Glycosylations employing a Fmoc-protected glucosyl phosphate building block were performed in a silicon-based micro-structured device to optimize reaction conditions and for reaction scale-up. A perfluorinated linker at the reducing end of the oligosaccharides allowed for purification by fluorous solid-phase extraction (FSPE) and further functionalization.     

Use of 18O water and ESI-MS detection in subsite characterisation and investigation of the hydrolytic action of an endoglucanase

We present a novel method for investigating subsite-substrate interactions of glycoside hydrolases and the determination of the oligosaccharide cleavage point based on the analysis of the hydrolysis products produced in the presence of ¹⁸O-labelled water. Conventional techniques for such determination of the hydrolysis pattern call for the chemical modification of the substrate, whereas the method presented makes it possible to use natural substrates, utilising the selectivity and sensitivity of mass spectrometry. This method is very useful for the detection and analysis of enzyme-catalysed hydrolysis, provided that the conditions are chosen where ¹⁸O incorporation without the presence of the enzyme is absent or undetectable. Such conditions were found and used in incubations of cellopentaose with the well-characterised endoglucanase Cel5A from Bacillus agaradhaerens. We were able to confirm that the preferred glycoside bond to be hydrolysed is the third one counting from the non-reducing end of the cellopentaose. Thus, cellopentaose prefers to bind from the –3 to the +2 subsites, which is in accordance with published crystallographic data. The main advantage of the method presented is that there is no need for a priori chemical modification/labelling of oligosaccharide substrates, which are processes that can disturb the enzyme–substrate interaction. From ¹⁸O incorporation we could demonstrate that the enzyme also has an oxygen-exchange activity on cellotriose and cellobiose. This is in agreement with the mechanism for transglycosylation and indicates that it is possible for the enzyme to perform such reactions.     

Identification of peptidase substrates in human plasma by FTMS based differential mass spectrometry

Approximately 2% of the human genome encodes for proteases. Unfortunately, however, the biological roles of most of these enzymes remain poorly defined, since the physiological substrates are typically unknown and are difficult to identify using traditional methods. We have developed a proteomics experiment based on FTMS profiling and differential mass spectrometry (dMS) to identify candidate endogenous substrates of proteases using fractionated human plasma as the candidate substrate pool. Here we report proof-of-concept experiments for identifying in vitro substrates of aminopeptidase P2, (APP2) and dipeptidyl peptidase 4 (DPP-4), a peptidase of therapeutic interest for the treatment of type 2 diabetes. For both proteases, previously validated peptide substrates spiked into the human plasma pool were identified. Of note, the differential mass spectrometry experiments also identified novel substrates for each peptidase in the subfraction of human plasma. Targeted MS/MS analysis of these peptides in the complex human plasma pool and manual confirmation of the amino acid sequences led to the identification of these substrates. The novel DPP-4 substrate EPLGRQLTSGP was chemically synthesized and cleavage kinetics were determined in an in vitro DPP-4 enzyme assay. The apparent second order rate constant (k_cat/K_M) for DPP-4-mediated cleavage was determined to be 2.3 × 10⁵ M⁻¹ s⁻¹ confirming that this peptide is efficiently processed by the peptidase in vitro. Collectively, these results demonstrate that differential mass spectrometry has the potential to identify candidate endogenous substrates of target proteases from a human plasma pool. Importantly, knowledge of the endogenous substrates can provide useful insight into the biology of these enzymes and provides useful biomarkers for monitoring their activity in vivo.     

Fluorescence modification of Gb3 oligosaccharide and rapid synthesis of oligosaccharide moieties using fluorous protective group

The use of the bisfluorous chain-type propanoyl (Bfp) group as a fluorous protective group made it possible to rapidly synthesize the Gb2 and Gb3 oligosaccharide derivatives by a simple fluorous-organic extraction purification. Furthermore, the fluorescence-labeled Gb2 and Gb3 oligosaccharides were prepared as a potential Vero Toxins detecting reagent.     

Production, purification, and biochemical characterization of two beta-glucosidases from Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum produces beta-glucosidases in liquid culture with a variety of carbon sources, including cellulose (filter paper), xylan, barley straw, oat meal, and xylose. Analysis by native polyacrylamide gel electrophoresis (PAGE) followed by an activity staining with the specific chromogenic substrate, 5-bromo 4-chloro 3-indolyl beta-1,4 glucoside (X-glu) showed that two extracellular beta-glucosidases, designated as beta-glu1 and beta-glu2, were in the filter paper culture filtrate. Only one enzyme designated as beta-glu x was revealed by the same method in the xylose culture filtrate. Beta-glu1 and beta-glu2 were purified to homogeneity. The purification procedure consist of a common step of anion-exchange chromatography on DEAE-Sepharose CL6B, both high-performance liquid chromatography (HPLC) anion-exchange and gel filtration columns for beta-glu1 and only HPLC gel filtration for beta-glu2. Beta-glu1 has a molecular mass of 196 kDa and 96.5 kDa, as estimated by gel filtration and sodium dodecyl sulfate (SDS)-PAGE, respectively, suggesting that the native enzyme may consist of two identical subunits. The same analysis showed that beta-glu2 is a monomeric protein with an apparent molecular mass of about 76.5 kDa. Beta-glu1 and beta-glu2 hydrolyses PNPGlc and cellobiose, with apparent Km values respectively for PNPGlc and cellobiose of 0.1 and 1.9 mM for beta-glu1 and 2.8 and 8 mM for beta-glu2. Both enzymes exhibit the same temperature and pH optima for PNPGlc hydrolysis (60 degrees C and pH 5.0). beta-glu1 was stable over a pH range of 3-8 and kept 50% of its activity after 30 min of heating at 60 degrees C without substrate. It was further characterized by studying the effect of some cations and various reagents on its activity.     

Efficient dynamic kinetic resolution of secondary alcohols with a novel tetrafluorosuccinato ruthenium complex

Dynamic kinetic resolution (DKR) of a series of secondary alcohols has been conducted with a novel dinuclear ruthenium complex, bearing tetrafluorosuccinate and (rac)-BINAP ligands as the racemization catalyst. Novozym 435 has been used as the enzyme, and isopropyl butyrate as the acyl donor. Five substrates underwent DKR successfully: an aliphatic and an aromatic secondary alcohol, an aromatic alcohol with an electron-withdrawing substituent on the phenyl ring, an aromatic alcohol bearing an electron-donating substituent on the ring and a heteroaromatic secondary alcohol. The catalyst performed optimally at 70 °C. Typically the reaction reached complete conversion within 1 day with 0.1 mol % of racemization catalyst relative to the substrate. The addition of the ketone corresponding to the substrate stabilizes the active Ru complex and, therefore, increases the rate of the reaction.     

Triterpene glycosides from the stems of Akebia quinata

The stems of Akebia quinata have been analyzed for their triterpene glycoside constituents, resulting in the isolation of six new triterpene glycosides, along with 19 known ones. On the basis of extensive spectroscopic analysis, including 2D NMR data, and chemical evidence, the structures of the new compounds were deter-mined to be 3beta-[(O-beta-D-glucuronopyranosyl-(1-->3)-alpha-L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3beta-[(O-beta-D-glucuronopyranosyl-(1-->3)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]olean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3beta-[(O-beta-D-glucuronopyranosyl-(1-->3)-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3beta-[(O-beta-D-glucuronopyranosyl-(1-->3)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3beta-[(O-beta-D-glucopyranosyl-(1-->3)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-29-hydroxyolean-12-en-28-oic acid, and 3beta-[(O-beta-D-glucopyranosyl-(1-->3)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl)oxy]-23,29-dihydroxyolean-12-en-28-oic acid, respectively. The main triterpene glycosides contained in the stems of A. quinata were found to have two sugar units at C-3 and C-28 of the aglycone in this study, whereas those of Akebia trifoliate were reported to possess one sugar unit at C-28 of the aglycone. It may be possible to distinguish between A. quinata and A. trifoliate chemically by comparing their triterpene glycoside constituents.