Comparative specificities of trehalases from various species.

1. Using derivatives or non-symmetrical analogs of alpha,alpha-trehalose, we studied the catalytic specificities of trehalases from various species: Pseudomonas fluorescens, Melolontha vulgaris, porcine and human kidneys. 2. alpha,Beta-trehalose, beta,beta-trehalose, 6,6'dideoxy alpha,alpha-trehalose, alpha-D-xylopyranosyl alpha-D-xylopyranoside were shown to be neither substrates nor inhibitors. 3. 6'deoxy alpha,alpha-trehalose, alpha-D-glucopyranosyl alpha-D-xylopyranoside, alpha-D-allopyranosyl alpha-D-glucopyranoside and alpha-D-galactosyl alpha-D-glucopyranoside, which all possess an intact alpha-D-glucopyranosyl residue, were split by all these trehalases. 4. alpha-D-glucopyranosyl alpha-D-mannopyranoside, alpha,alpha-trehalosamine are competitive inhibitors. 5. These results show the importance of the primary alcohol group at C-6, of the equatorial configuration of the OH groups at C-2, C-3 and C-4 and of the modification of the structure at C-2 of the substrate for the catalytic activity.     

Sulfatides of Mycobacterium tuberculosis. Synthesis of the core alpha,alpha-trehalose 2-sulfate

alpha,alpha-Trehalose 2-sulfate, the core carbohydrate of sulfatides of Mycobacterium tuberculosis, and the 3-sulfate isomer were synthesized by sulfation of 4,6:4',6'-di-O-benzylidene-alpha,alpha-trehalose with pyridine-sulfur trioxide complex to give the 2- and 3-sulfates, which were separated by column chromatography. The ammonium 2-sulfate salt wa was identical with the natural product obtained from the principal sulfatide (SL-I) of M. tuberculosis.     

Essential role of trehalose in the synthesis and subsequent metabolism of corynomycolic acid in Corynebacterium matruchotii

A previous paper indicated that corynomycolates synthesized by the fluffy layer fraction prepared from Corynebacterium matruchotii cells appeared exclusively as alpha-trehalose 6-monocorynomycolate (TMM) (T. Shimakata, K. Tsubokura, T. Kusaka, and K. Shizukuishi, 1985, Arch. Biochem. Biophys. 238, 497-508). In the present communication, the role of trehalose in the synthesis and subsequent metabolism of corynomycolic acids was reexamined. Consequently the following facts were clarified: (i) trehalose 6-phosphate (T-6-P), but not trehalose, stimulated corynomycolate synthesis from palmitate in the presence of ATP; the immediate product was TMM, which showed a rapid turnover. Since the turnover was blocked by addition of alpha-trehalose, only TMM accumulated among corynomycolate-containing substances. These results strongly suggested that T-6-P is an essential component as the acceptor in corynomycolate-synthetic system; (ii) TMM was the precursor not only to alpha-trehalose 6,6'-dicorynomycolate (TDM) and free corynomycolic acids but also to cell wall corynomycolate; (iii) addition of alpha-trehalose blocked the transfer of the corynomycolate moiety from TMM to cell wall corynomycolate, TDM, and free corynomycolic acids to a similar extent. These results clearly indicate that trehalose plays an essential role in the metabolism of corynomycolate after Claisen condensation and subsequent reduction in C. matruchotii.     

Glycosides of 2-C-methyl-D-erythritol from the fruits of anise,coriander and cumin

Eight glycosides of 2-C-methyl-D-erythritol (1) were isolated from the fruit of anise, and their structures were clarified as 1-O-beta-D-glucopyranoside, 3-O-beta-D-glucopyranoside, 4-O-beta-D-glucopyranoside, 1-O-beta-D-fructofuranoside, 3-O-beta-D-fructofuranoside, 4-O-beta-D-fructofuranoside, 1-O-beta-D-(6-O-4-hydroxybenzoyl)-glucopyranoside and 1-O-beta-D-(6-O-4-methoxybenzoyl)-glucopyranoside of 2-C-methyl-D-erythritol (2-9), respectively. Furthermore, 2 and 4 were isolated from the fruit of coriander, and 2, 3 and 4 were isolated from the fruit of cumin. Though the phosphate of 1 was known to be one of the first precursors of isoprenoids in the non-mevalonate pathway, and 1 is considered to be a common constituent in Umbelliferous plants, the glycosides of 1 are found for the first time.     

Flavonol and drimane-type sesquiterpene glycosides of Warburgia stuhlmannii leaves

An investigation of methanolic extract of Warburgia stuhlmannii leaves has led to the isolation of two new drimane-type sesquiterpene glycosides characterized as mukaadial 6-O-beta-D-glucopyranoside, mukaadial 6-O-alpha-L-rhamnopyranoside together with two other novel flavonol glycosides identified as 3',5'-O-dimethylmyricetin 3-O-beta-D-2",3"-diacetylglucopyranoside and 3'-O-methylquercetin 3-O-beta-D-2",3",4"-triacetylglucopyranoside. The known compounds; mukaadial, deacetylugandensolide, quercetin, kaempferol, kaempferol 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-beta-D-glucopyranoside, kaempferol 7-O-beta-D-glucopyranoside, myricetin 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-sophoroside and isorhamnetin 3-O-beta-D-glucopyranoside were also isolated from the same extract.     

A novel acetylated 3-deoxyanthocyanidin laminaribioside from the fern Blechnum novae-zelandiae

The major 3-deoxyanthocyanin isolated from the fern Blechnum novae-zelandiae was determined to be luteolinidin 5-O-beta-D-[3-O-beta-D-glucopyranosyl-2-O-acetylglucopyranoside] and was found to co-occur with quercetin 3-O-beta-D-[6-O-caffeoylglucopyranoside], quercetin 3-O-beta-D-[6-O-caffeoylgalactopyranoside] and kaempferol 3-O-beta-D-glucuronopyranoside. These pigments were identified by using HPLC chromatography, NMR (1D, 2D) and electrospray mass spectroscopy.     

Lipid peroxidation inhibitory compounds from daylily (Hemerocallis fulva) leaves

Daylilies (Hemerocallis spp.) have been used as food and in traditional medicine for thousands of years in eastern Asia. The leaves of the plant are used in the treatment of inflammation and jaundice. In studies of the aqueous methanol extracts of fresh Hemerocallis fulva leaves, 1',2',3',4'-tetrahydro-5'-deoxy-pinnatanine (1), pinnatanine (2), roseoside (3), phlomuroside (4), lariciresinol (5), adenosine (6), quercetin 3-O-beta-D-glucoside (7), quercetin 3,7-O-beta-D-diglucopyranoside (8), quercetin 3-O-alpha-L-rhamnopyransol-(1-->6)-beta-D-glucopyranosol-7-O-beta-D-glucopyranoside (9), isorhamnetin-3-O-beta-D-6'-acetylglucopyranoside (10) and isorhamnetin-3-O-beta-D-6'-acetylgalactopyranoside (11) were isolated. All of these compounds were tested for their in vitro lipid peroxidation inhibitory activities. Compounds 3-5 and 7-11 were found to possess strong antioxidant properties, inhibiting lipid oxidation by 86.4, 72.7, 90.1, 79.7, 82.4, 89.3, 82.2, and 93.2%, respectively at 50 microg/mL. Compound 1 is novel and compounds 3-6 and 8-11 described here in are isolated for the first time from daylily leaves.     

Steroidal polyols from Far Eastern starfish Henricia sanguinolenta and H. leviuscula leviuscula

Two new asterosaponins, (20R)-3-O-beta-D-(2-O-methylxylopyranosyl)-24-propylcholest-4-ene-3 beta,6 beta, 8, 15 alpha, 16 beta, 29-hexaol (sanguinoside A) and (20R,24S)-3-O-beta-D-(2,3,4-tri-O-methylxylopyranosyl)-5 alpha-cholestane-3 beta, 4 beta, 6 beta, 8, 15 alpha, 24-hexaol (sanguinoside B), were isolated from two species of Pacific Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula, collected in the Sea of Okhotsk. Both glycosides contain aglycones with pentahy-droxysteroid nuclei of similar structures, which are substituted at the 3-hydroxy group with differently methylated beta-D-xylosyl residues. Sanguinoside A has an unusual structure of its aglycone side chain, whereas sanguinoside B has a unique permethylated carbohydrate chain. In addition, laevisculoside G, a known glycoside, was identified in the H. leviuscula starfish. The structures of the isolated glycosides were established by interpreting their spectral data and by comparing their spectral characteristics with those of known compounds. The English version of the paper. Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Synthesis of the tetrasaccharide core region of antigenic lipo-oligosaccharides characteristic of Mycobacterium kansasii

The oligosaccharide core region, beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-alpha-D-Glcp- 1----1)-alpha-D-Glcp (1), of the lipo-oligosaccharide-type antigens isolated from M. kansasii has been synthesised from 2,3,2',3',4',6'-hexa-O-benzyl-6-O-(1-phenylethyl)-alpha, alpha-trehalose (4). Compound 4 was obtained by LiAlH4-AlCl3-type hydrogenolysis of 2,3,2',3',4',6'-hexa-O-benzyl-4,6-O-(S)-(1-phenylethylidene)-alpha , alpha-trehalose. The beta-laminaribiosyl part of the molecule was built-up by sequential glycosylation steps using 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl bromide in the presence of HgBr2 and methyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranoside promoted by methyl triflate. The complete a priori 13C-n.m.r. spectrum assignment of 1 was achieved by applying 2D methods.     

Antioxidant and cytotoxic flavonols from Calotropis procera

Phytochemical investigations of Calotropis procera leaves have led to the isolation of two new compounds: quercetagetin-6-methyl ether 3-O-beta-D-4C1-galacturonopyranoside (3) and (E)-3-(4-methoxyphenyl-2-O-beta-D-4C1 -glucopyranoside)-methyl propenoate (4), along with eleven known metabolites: nine flavonol and two cinnamic acid derivatives. All metabolites were isolated for the first time from the genus Calotropis, except for 1 isolated previously from Calotropis gigantea. The structures were determined by spectroscopic methods (UV, ESI-MS, 1H, 13C NMR, 1H-1H COSY, HSQC, and HMBC). The radical scavenging activity of the aqueous methanol extract and compounds 8-13 was measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. Cytotoxic screening of the same compounds was carried out on brine shrimps as well.     

Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of alpha,alpha-trehalose

Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through approximately 500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of approximately 55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of alpha,alpha-trehalose, yielding alpha-d-glucose 1-phosphate (alphaGlc 1-P) and alpha-d-glucose as the products. The optimum pH of PoTPase for alpha,alpha-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (K(m)) were determined at pH 6.8 and 30 degrees C: alpha,alpha-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); alphaGlc 1-P (4.1mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with alphaGlc 1-P, and robust activity with d-mannose (3%), 2-deoxy d-glucose (8%), 2-fluoro d-glucose (15%) and 2-keto-d-glucose (50%) was detected. Arsenate replaces, with 30% relative activity, phosphate in the conversion of alpha,alpha-trehalose, and vanadate strongly inhibits the enzyme activity (K(i) approximately 4 microM). PoTPase has a half-life (t(0.5)) of approximately 1 h at 30 degrees C in the absence of stabilizing compounds such as alpha,alpha-trehalose (300 mM; t(0.5)=11.5 h), glycerol (20%, w/v; t(0.5)=6.5h) or polyethylenglycol (PEG) 4000 (26%, w/v; t(0.5)=70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to alpha,alpha-trehalose in approximately 60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.     

Conformations in solution of alpha,alpha-trehalose, alpha-D-glucopyranosyl alpha-D-mannopyranoside, and their 1-thioglycosyl analogs, and a tentative correlation of their behaviour with respect to the enzyme trehalase

The conformation in solution of alpha-D-glucopyranosyl alpha-D-glucopyranoside (alpha,alpha-trehalose, 1), alpha-D-glucopyranosyl alpha-D-mannopyranoside (3) and their corresponding 1-thioglycosyl analogs, alpha-D-glucopyranosyl 1-thio-alpha-D-glucopyranoside (1-thio-alpha,alpha-trehalose, 2) and alpha-D-glucopyranosyl 1-thio-alpha-D-mannopyranoside (4) were established from high-resolution 1H-NMR and 13C-NMR measurements. These experimental results are in good agreement with the conformations as inferred from hard-sphere calculations. The dihedral angles phi H and psi H are not significantly different for the O-glycosyl disaccharides 1 and 3 compared with their 1-thioglycosyl analogs 2 and 4; however, the internuclear H-1--H-1' and H-1--H-5' distances appear to be longer for 1-thiodisaccharides. This may account for the differences in affinities of cockchafer trehalase which have been observed. This enzyme exhibits less affinity for the competitive inhibitor alpha-D-glucopyranosyl 1-thio-alpha-D-mannopyranoside (4) than for its O-glycosyl analog 3 (Ki 0.055 mM versus 0.0057 mM). From the similarity in Ki between 1-thio-alpha, alpha-trehalose and alpha-D-glucopyranosyl 1-thio-alpha-D-mannopyranoside (0.050 mM versus 0.055 mM), it is possible to assume a similar decrease in the enzymic affinity between the natural substrate (1) and the corresponding 1-thioglycosyl inhibitor (2), which can together be ascribed to the aforementioned difference in the conformation of the molecules.     

Alpha,alpha-trehalose derivatives bearing guanidino groups as inhibitors to HIV-1 Tat-TAR RNA interaction in human cells

Replication of HIV-1 requires specific interactions of Tat protein with TAR RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6'-diamino-6,6'-dideoxy-alpha,alpha-trehalose was obtained from selective bromination of, alpha,alpha-trehalose at C-6,6', followed by acetylation, azide displacement, deacetylation, and reduction. Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated the novel alpha,alpha-trehalose derivatives. Their abilities to inhibit Tat-TAR RNA interaction in human cells were determined by a Tat-dependent HIV-1 LTR-driven CAT assays.     

The pregnane glycoside marsdekoiside A from Marsdenia koi.

A new pregnane glycoside, marsdekoiside A was isolated from the stems of Marsdenia koi (Asclepiadaceae) and its structure was elucidated from chemical and spectral data as 12-cinnamoyl-dihydrosarcostin-3-O-methyl-6-deoxy-beta-D-allopyr ano syl-(1----4)-O-beta-D-oleandropyranosyl-(1----4)-O-beta-D-++ +cymar opyranoside.     

An unusual C6-C6" linked flavonoid from Miconia cabucu (Melastomataceae)

Chromatographic fractionation of the methanolic extract from the leaves of Miconia cabucu Hoehne (Melastomataceae) afforded the first example of a C(6)-C(6") linked flavone dimer, 5-hydroxy-4',7-dimethoxyflavone-(6-C-6')-5'-hydroxy-3',4',7'-trimethoxyflavone as well as the known compounds, quercetin-3-O-alpha-l-rhamnopyranosyl-(2-->1)-O-beta-D-xylopyranoside, quercetin-3-O-alpha-l-rhamnopyranoside, myricetin-3-O-alpha-l-rhamnopyranoside, quercetin-3-O-beta-D-glucopyranoside, kaempferol-3-O-beta-D-(6'-coumaroyl)-glucopyranoside and gallic acid. Their chemical identities were established by application of NMR spectroscopic methods including 2D-NMR, as well as UV and ESI-MS analyses.     

Synthesis of new heterocyclic derivatives of alpha,alpha-trehalose

Several novel N-1, N-2, and S-5 tetrazole and 1,3,4-oxadiazole derivatives of alpha,alpha-trehalose disubstituted at C-6,6', with potential synthetic and pharmacological interest were prepared from commercial tetrazoles and 1,3,4-oxadiazoles in reaction with hexa-O-benzyl-6,6'-di-O-triflyl-alpha,alpha-trehalose.     

The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies

Schizophyllum communealpha,alpha-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into alpha-d-glucose 1-phosphate and alpha-d-glucose. Recruitment of phosphate by the free enzyme induces alpha,alpha-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (kcat/Km=21,000 m(-1).s(-1)) was dramatic (>or=10(7)-fold) in purified Arg507-->Ala (R507A) and Lys512-->Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in kcat (Arg507) and Km (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H2PO4-, the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with alpha,alpha-trehalose (Ki=0.4 microm) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (Ki=300 microm). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward alpha,alpha-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively.     

Cord-factor analogs: synthesis of 6,6'-di-O-mycoloyl- and -corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside)

Appropriate solvolysis of 2,3,2',3'-tetra-O-benzyl-4,6,4', 6'-tetra-O-mesyl-alpha,alpha-trehalose gave 2,3,2',3' -tetra-O-benzyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (2). Selective tosylation or mesylation of 2 respectively gave the 6, 6'-ditosylate (3) and 6,6'-dimesylate (4), the structures of which were confirmed by the 1H-n.m.r. spectra of the corresponding 4,4'-di-O-acetyl derivatives. Treatment of 3 with potassium mycolate in toluene, and subsequent hydrogenolysis, gave the 6'-mycolate 6-tosylate derivative. Treatment of 3 with potassium mycolate or potassium corynomycolate in hexamethylphosphoric triamide, followed by catalytic hydrogenolysis, yielded the respective cord-factor analogs 6,6'-di-O-mycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) and 6,6'-di-O-corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside). The same 6,6'-diesters were obtained from the 6,6'-dimesylate 4. Putative 4,6-anhydro-6'-monomycolates are also described.     

Flavonoid triglycosides from the seeds of Syzygium aromaticum

Two new apigenin triglycosides, apigenin 6-C-[beta-D-xylopyranosyl-(1'-->2')-beta-D-galactopyranoside]-7-O-beta-D-glucopyranoside and apigenin 6-C-[beta-D-xylopyranosyl-(1'-->2')-beta-D-galactopyranoside]-7-O-beta-D-(6-O-p-coumarylglucopyranoside) were isolated from the ethanol extract of the seeds of Syzygium aromaticum. Their structures were elucidated by chemical and spectral analysis (UV, FABMS, 1H, 13C NMR, HMQC, HMBC, NOESY and DEPT).     

Derivatives of glutamic acid and trehalose which can be transformed into long-living free radicals by the loss of an electron are identified in some bacteria

A derivative of glutamic acid (ammonigenin) and a trisaccharide named lysodektose which are converted into long-living free radicals by the loss of one electron were isolated from Brevibacterium ammoniagenes and Micrococcus lysodeikticus. Structural formulae suggested for both substances based on ESR-, NMR- and mass spectra, isotopic substitution experiments and other data are: lactone of N-hydroxy-N-(2-carbamoylethyl)-glutamyl-4-amino-2-hydroxybutyric amide and 6-O-[2-deoxy-2-(N-methyl)-hydroxylamino-beta-D-glucopyranosyl]- alpha, alpha-trehalose. Radical forms appear on reversible oxidation of hydroxylamino groups to nitroxyl groups. Participation in the protection of bacterial cells and regulation of their metabolism is suggested for these compounds.