Impact of the structures of macrocyclic Michael acceptors on covalent proteasome inhibition.

Molecules that have a reactive functional group within a macrocycle represent a class of covalent inhibitor. The relationship between reactivity and affinity for the target is cooperative and complicated. An understanding and characterization of this class of inhibitor are vital for the development of covalent inhibitors as drug candidates. Herein, we describe a systematic analysis of structure-activity relationships using a series of syringolin analogues, which are irreversible covalent inhibitors of proteasomes. We investigate the detailed mechanistic effects of the macrocycles on affinity and reaction rate.

Isolation and structural analyses of positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) and 6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose.

6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose (beta CD) and three positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) in a mixture of products from beta CD and N-acetylglucosamine by the reversed reaction of beta-N-acetylhexosaminidase from jack bean were isolated and purified by HPLC. The structures of four isomers of di-N-acetylglucosaminyl-beta CDs were determined by FABMS and NMR spectroscopy. The degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying alpha-amylase (BSA) was established by LC-MS methods.

Comparative study of the cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases.

The actions of cyclomaltodextrin glucanotransferases (CGTase; EC 2.4.1.19) from alkalophilic Bacillus sp. strain A2-5a (A2-5a CGTase), Bacillus macerans (Bmac CGTase), and Bacillus stearothermophilus (Bste CGTase) on amylose were investigated. All three enzymes produced large cyclic alpha-1,4-glucans (cycloamyloses) at the early stage of the reaction, but these were subsequently converted into smaller cycloamyloses. However, the rates of this conversion differed among the three enzymes. The product specificity of each CGTase in the cyclization reaction was determined by measuring the amount of each cycloamylose from CD6 to CD31 (CDn, a cycloamylose with a degree of polymerization of n). A2-5a CGTase produced 10 times more CD7, while Bmac CGTase produced 34 times more CD6 than other cycloamyloses. Bste CGTase produced 12 and 3 times more CD6 and CD7 than other cycloamyloses, respectively. The substrate specificities of the linearization reactions of CD6, CD7, CD8, and larger cycloamyloses (a mixture of CD22 to CD50) were investigated, and we found that CD7 and CD8 are extremely poor substrates for both hydrolytic and transglycosidic linearization (coupling) reactions while larger cycloamyloses are linearized at a much higher rate. By repeating these cyclization and linearization reactions, the larger cycloamyloses initially produced are converted into smaller cycloamyloses and finally into mainly CD6, CD7, and CD8. These three enzymes also differ in their hydrolytic activities, which seem to accelerate the conversion of larger cycloamyloses into smaller cycloamyloses.

Hydrolysis of beta-galactosyl ester linkage by beta-galactosidases.

p-Hydroxybenzoyl beta-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of beta-galactosyl ester linkage by beta-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl beta-galactoside (pNP-Gal), a usual substrate with a beta-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that beta-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.

Transfructosylation of thiol group by beta-fructofuranosidases.

Beta-fructofuranosidase fructosylated not only the hydroxyl group but also the thiol group of 2-mercaptoethanol in a transfer reaction using sucrose as a donor substrate. The enzymes from Candida utilis and Saccharomyces cerevisiae (bakers' yeast) were effective catalysts for the thio-fructofuranosylation. The thio-fructosylation product was isolated by activated carbon chromatography and its structure was confirmed by Fab-mass spectrometry and NMR spectroscopy. The thio-fructofuranoside was synthesized effectively at around 3.0 M for the acceptor concentration. The product increased with the sucrose concentration at least up to 1.9 M. O-Fructofuranoside was simultaneously synthesized at an early stage of the reaction, although it was hydrolyzed on further incubation. On the contrary, the thio-fructofuranoside accumulated efficiently after synthesis, indicating it was very stable against the hydrolytic action of the beta-fructofranosidase.

Galactosylation of thiol group by beta-galactosidase.

beta-Galactosidase catalyzed beta-galactosylation not only of a hydroxyl group but also of a thiol group in the condensation reaction of D-galactose and 2-mercaptoethanol. The thio-galactosylation product was confirmed as 2-hydroxyethyl S-beta-D-galactoside on the bases of fast atom bombardment mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectorometry. Aspergillus oryzae beta-galactosidase hydrolyzed p-nitrophenyl S-beta-D-galactoside most rapidly among several beta-galactosidases and produced the thio-galactosylation product most efficiently. The Penicillim multicolor enzyme was as effective as the A. oryzae enzyme. However the enzymes from Escherichia coli, Saccharomyces fragilis, Kluyveromyces lactis, and Bacillus circulans galactosylated hydroxyl groups predominantly to produce O-galactoside. The thio-galactoside was synthesized most effectively at a 2-mercaptoethanol concentration of about 1.25 M. Galactose concentration at 0.8-2.8 M did not affect the synthetic yield of the thiogalactoside so greatly.

Purification and some properties of a beta-glucosidase from Flavobacterium johnsonae.

Flavobacterium johnsonae was isolated as a microorganism that produced a beta-glucosidase with hydrolytic activity of beta-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point of pI 8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature was 45 degrees C, and the enzyme was stable below 35 degrees C. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad beta-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl beta-glucosides such as p-nitrophenyl beta-glucoside, phenyl betaglucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2+, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl beta-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.

Synthesis of novel heterobranched beta-cyclodextrins from alpha-D-mannosylmaltotriose and beta-cyclodextrin by the reverse action of pullulanase, and isolation and characterization of the products.

Alpha-D-mannosyl-maltotriose (Man-G3) were synthesized from methyl alpha-mannoside and maltotriose by the transfer action of alpha-mannosidase. (Man-G3)-betaCD and (Man-G3)2-betaCD were produced in about 20% and 4% yield, respectively when Aerobacter aerogenes pullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 M Man-G3 and 0.16 M betaCD at 50 degrees C for 4 days. The reaction products, (Man-G3)-betaCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)2-betaCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-betaCDs was identified as 6-O-alpha-(6(3)-O-alpha-D-mannosylmaltotriosyl)-betaCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)2-betaCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by alpha-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-betaCDs. The structures of three main components of (Man-G3)2-betaCDs were identified as 6(1),6(2)-, 6(1),6(3)- and 6(1),64-di-O-(63-O-alpha-D-mannosyl-maltotriosyl)-betaCD.

Hydrolysis of beta-glucosyl ester linkage of p-hydroxybenzoyl beta-D-glucose, a chemically synthesized glucoside, by beta-glucosidases.

To investigate the hydrolysis of glucosyl esters by beta-glucosidase, p-hydroxybenzoyl beta-D-glucose (pHBG) was chemically synthesized. The hydrolytic activity of some beta-glucosidases for pHBG was compared to that for p-nitrophenyl beta-glucoside (pNPG). The Clavibacter michiganense and Flavobacterium johnsonae enzymes could hydrolyze pHBG and steviol glycosides which are natural glucosyl esters. The commercial beta-glucosidase originating from Caldocellum saccharolyticum also hydrolyzes pHBG despite having no activity for steviol glycosides. The beta-glucosidase from Aspergillus niger cleaved the glucosyl ester linkage much more weakly than the glucosidic linkage. The pH-activity profile for the hydrolysis of pHBG was similar to that of pNPG by the C. saccharolyticum beta-glucosidase. The similar profiles for these substrates suggested that the active site for the glucosyl ester chemically resembles that for glucoside with respect to catalysis. Kinetic analysis of the C. saccharolyticum beta-glucosidase for mixed substrates of pHBG and pNPG showed that the hydrolysis of pHBG competed with that of pNPG. This result indicated that there is only one active site for both the glucosyl ester and glucoside. Mass spectroscopic analysis of the hydrolysates of pHBG in H218O suggested that beta-glucosidase hydrolyzes glucosyl esters between the anomeric carbon of glucose and the carbonyl oxygen, not between the carbonyl carbon and the carbonyl oxygen.

Enzymatic synthesis of N-acetylglucosaminyl-cyclodextrin by the reverse reaction of N-acetylhexosaminidase from jack bean.

Novel heterobranched cyclodextrins (CDs), N-acetylglucosaminyl-cyclodextrins (GlcNAc-CD), were synthesized from a mixture of GlcNAc and alpha, beta, or gamma CD by the reverse reaction of N-acetylhexosaminidase from jack bean. Optimum pH and temperature for the production of GlcNAc-alpha CD by N-acetylhexosaminidase were pH 4.9 and 50-70 degrees C, respectively. The maximum yield of GlcNAc-alpha CD was 17.5% (mol/mol) at the concentration of 1 M GlcNAc and 0.25 M alpha CD. The reverse reaction product, GlcNAc-alpha CD, was separated into two peaks by HPLC analysis on the ODS column. Their structures were identified as 6-O-beta-D-N-acetylglucosaminyl-alpha CD and 2-O-beta-D-N-acetylglucosaminyl-alpha CD by FAB-MS and NMR spectroscopies. N-Acetylhexosaminidase from jack bean also synthesized N-acetylgalactosaminyl-alpha CD from N-acetylgalactosamine and alpha CD.

Isolation and structural analyses of positional isomers of 6(1),6m-di-O-alpha-D-mannopyranosyl-cyclomaltooctaose (m = 2-5) and 6-O-alpha-(n-O-alpha-D-mannopyranosyl)-alpha-D-mannopyranosyl- cyclomaltooctaose (n = 2, 3, 4, and 6).

Eight positional isomers of 6(1),6m-di-O-alpha-D-mannopyranosyl-cyclomaltooctaose (gamma CD) (m = 2-5) and 6-O-alpha-(n-O-alpha-D-mannopyranosyl)-D-mannopyranosyl-gamma CD (n = 2, 3, 4, and 6) in a mixture of products from gamma CD and D-mannose by condensation reaction of alpha-mannosidase from jack bean were isolated by HPLC. The structures of four isomers of 6-O-alpha-(n-O-alpha-D-mannopyranosyl)-D-mannopyranosyl-gamma CD were elucidated by NMR spectroscopy. On the other hand, four positional isomers of 6(1),6m-di-O-alpha-D-mannopyranosyl-gamma CD were determined by LC-MS analysis of degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying alpha-amylase (BSA), and combination of BSA and glucoamylase. Similarly cyclomaltodextrin glucanotransferase also digested these isomers.

Isolation and characterization of di- and tri-mannosyl-cyclomaltoheptaoses (beta-cyclodextrins) produced by reverse action of alpha-mannosidase from jack bean.

Di- and tri-mannosyl-cyclomaltoheptaoses (beta-cyclodextrins, beta CDs), which were synthesized together with monomannosyl-beta CD in a large-scale production by reverse action of alpha-mannosidase from jack bean, were isolated and purified by HPLC. The structures of seven isomers of di-mannosyl-beta CD and six isomers of tri-mannosyl-beta CD were elucidated by FABMS and NMR spectroscopy, and by enzymatic methods.

Enzymatic synthesis of oligosaccharides containing Gal beta-->4Gal disaccharide at the non-reducing end using beta-galactanase from Penicillium citrinum.

The transglycosylation reaction was done with a beta-galactanase from Penicillium citrinum. The regioselectivity in the transglycosylation reaction was studied using soy bean arabinogalactan as a donor and mono- or disaccharide derivatives containing beta-galactosyl residue as acceptors. We also synthesized oligosaccharides containing Gal beta 1-->4Gal sequence such as Gal beta 1-->4Gal beta1-->4Glc, Gal beta 1-->4Gal beta 1-->3GlcNac, Gal beta 1-->4Gal beta 1-->4GlcNAc, Gal beta 1-->4Gal beta 1-->6GlcNAc, and Gal beta 1-->4Gal beta 1-->3GalNAc for use in the total synthesis of complex sugar chains.

Enzymatic synthesis, isolation, and analysis of novel alpha- and beta-galactosyl-cycloisomalto-octaoses.

Novel branched cycloisomalto-octaoses (CI8s) were enzymatically synthesized by transgalactosylation with alpha-galactosidase from coffee bean and beta-galactosidase preparations from Penicillium multicolor and Bacillus circulans, using melibiose and lactose as donor substrates, and CI8 which is a cyclic homogeneous oligosaccharide composed of eight glucose units bound by alpha-(1-->6)-linkages, as an acceptor. alpha-Galactosyl-CI8s and beta-galactosyl-CI8s obtained were isolated and purified by HPLC. Their structures were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOFMS) and NMR spectroscopy.

Isolation by HPLC of the positional isomers of heterogeneous doubly branched cyclomaltohexaose having one alpha-D-galactosyl and one alpha-D-glucosyl side chain and determination of their structures by enzymatic degradation.

Each of the six and five positional isomers of 2n- and 6n-O-alpha-D-galactopyranosyl-6(1)-O-alpha-D-glucopyranosyl-cyclomalt ohexaose (alpha-cyclodextrin, alpha CD; 2n: n = 1-6, 6n: n = 2-6), which were produced from 6-O-alpha-D-glucopyranosyl-alpha CD and melibiose by transgalactosylation with coffee bean alpha-galactosidase, were isolated by high-performance liquid chromatography (HPLC) on a reversed-phase column and a graphitized carbon column. The structures of those isomers were elucidated by analyses of enzymatic degradation products with cyclomaltodextrin glucanotransferase and glucoamylase.

Isolation and characterization of novel heterogeneous branched cyclomalto-oligosaccharides (cyclodextrins) produced by transgalactosylation with alpha-galactosidase from coffee bean.

Transgalactosylated derivatives of cyclomalto-hexaose (alpha CD), -heptaose (beta CD), and -octaose (gamma CD) were synthesized by alpha-galactosidase from coffee bean using melibiose as a donor and alpha CD, beta CD or gamma CD as an acceptor. Mono- and di-O-alpha-D- galactosylated CDs were isolated and purified by HPLC. Their structures were elucidated by fast-atom bombardment mass spectrometry (FABMS) and 13C NMR spectroscopy. For structural determination of positional isomers of 6(1),6n-di-O-alpha-D-galactosyl-CDs, digestion products with cyclodextrin glucanotransferase were analyzed by HPLC and FABMS.

Synthesis of 2-deoxy-glucooligosaccharides through condensation of 2-deoxy-D-glucose by glucoamylase and alpha-glucosidase.

Glucoamylases from Aspergillus niger and Rhizopus niveus catalyzed condensation of 2-deoxy-D-glucose (dGlc) to yield deoxy-glucooligosaccharides with polymerization degrees of 2-5. The enzymes also gave a small amount of products from 3-deoxy-D-glucose, but no products from 6-deoxy-D-glucose. A. niger alpha-glucosidase also catalyzed condensation of dGlc, while Torula and Saccharomyces alpha-glucosidases had low activity. alpha-1,4-, 1,6-, and 1,3-linked deoxy-glucobioses were isolated and identified as the products of A. niger glucoamylase and A. niger alpha-glucosidase. In the reaction of the glucoamylase, 1,4- and 1,3-linked saccharides decreased with an increase of 1,6-linked one. A. niger alpha-glucosidase produced alpha-1,6-linked disaccharide predominantly during the whole course of the reaction.

Transgalactosylation catalyzed by alpha-galactosidase from Candida guilliermondii H-404.

The thermostable alpha-galactosidase from Candida guilliermondii H-404 synthesized self-transfer products in the absence of a suitable acceptor. The main self-transfer product, using melibiose as a donor substrate, was O-alpha-D-galactosyl-(1,6)-O-alpha-D-galactosyl-(1,6)-D-glucose. This enzyme had a wide acceptor specificity. D-Glucose, D-galactose, maltose, maltitol, and 1,4-butandiol were the most effective acceptors in the transgalactosylation catalyzed by this enzyme. The enzyme could also transfer alpha-galactosyl residues to pentoses (L-arabinose, D-xylose, and D-ribose) and methyl pentoses (D-fucose and L-rhamnose). The main transfer products to lactose, maltose, and sucrose as acceptors were identified as O-alpha-D-galactosyl-(1,6)-O-beta-D-galactosyl-(1,4)-D-glucose, O-alpha-D-galactosyl-(1,6)-O-alpha-D-glucosyl-(1,4)-D-glucose, and O-alpha-D-galactosyl-(1,6)-O-alpha-D-glucosyl-(1,2)-beta-D-fructoside (raffinose), respectively.

Purification and some properties of a trehalase from a green alga, Lobosphaera sp.

An unicellular green alga identified as Lobosphaera sp. by morphological observations was selected as a source of trehalase. The alga grew well heterotrophically and produced intracellular trehalase using Polypepton, yeast extract, and glycerol as nutrients. The enzyme was highly purified by ammonium sulfate fractionation, column chromatography on DEAE-Toyopearl, Sepharose CL-4B, and SP-Toyopearl. The molecular mass was estimated to be 400 kDa by gel filtration. SDS-PAGE indicated that the enzyme consisted of two subunits with a molecular mass range of 180-220 kDa and it contained carbohydrates. The enzyme was most active at pH 5.5 and at 65 degrees C and stable between pH 4-9 and below 65 degrees C. Fe3+ inactivated the enzyme. Sucrose was a competitive inhibitor with a Ki of 7.5 mM. The enzyme specifically hydrolyzed trehalase with a Km of 0.6 mM.