Preparation and conformational analysis of C-glycosyl β- and β/β-peptides

Ten C-glycosyl β²- and β/β²-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble β²-amino acids in which C-glycosylic substituents are attached to the C-2 position of β-amino acids. Conformational analysis of the C-glycosyl β²-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl β/β²-peptides are governed by the configuration at the α-carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl β²-amino acids.     

Indium-mediated alkynylation of sugars: synthesis of C-glycosyl compounds bearing a protected amino alcohol moiety

The coupling of glycals with an alkynyl iodide bearing a protected amino alcohol moiety was achieved in the presence of metallic indium under Barbier conditions. It gave functionalized C-glycosyl compounds, precursors of C-glycosyl amino acids with α configuration.     

N,N-Diacetylsialyl chloride—a novel readily accessible sialyl donor in reactions with neutral and charged nucleophiles in the absence of a promoter

N,N-Diacetylneuraminic acid glycosyl chloride was prepared for the first time and made to react with various nucleophiles to give the corresponding α-glycosyl phosphate, β-glycosyl dibenzyl phosphate, α-glycosyl azide, α-phenyl thioglycoside and α-glycosyl xanthate in 65-82% yields and high stereoselectivity while its reactions with simple alcohols were not stereoselective. The new sialyl donor made possible the first stereoselective synthesis of sialic acid glycosyl phosphate with α-configuration and highly efficient synthesis of β-configured sialic acid glycosyl dibenzyl phosphate.     

Synthesis of C-(d-glycopyranosyl)ethylamines and C-(d-glycofuranosyl)methylamines as potential glycosidase inhibitors

The C-glucosyl aldehyde, 2-C-(2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl)ethanal was prepared from the C-glucopyranosyl propene precursor by ozonolysis. Reductive amination of the C-glucosyl aldehyde and subsequent deprotection gave 1-anilino-2-C-(α-d-glucopyranosyl)ethane. The E and Z isomers of the oxime derivative, 1-C-(α-d-arabinofuranosyl)methanal oxime were prepared by treating their aldehyde precursor with hydroxylamine. Acetylation of the oxime, followed by catalytic hydrogenation and deprotection, gave the corresponding 1-C-(α-d-arabinofuranosyl)methylamine. Reductive amination of ethyl 2,3-O-isopropylidene-α-d-lyxo-pentodialdo-1,4-furanoside using aniline gave ethyl 5-anilino-5-deoxy-d-lyxo-furanoside. Inhibition studies with these compounds on β-d-glucosidase from sweet almond, using o-nitrophenyl d-glucopyranoside as substrate, were carried out.     

C-4′-Branched-chain sugar nucleosides: synthesis of isomers of psicofuranine

Photoamidation of 3-O-acetyl-1,2:5,6-di-O-isopropylidene-α-d-erythro-hex-3-enofuranose (1) afforded 3-O-acetyl-4-C-carbamoyl-1,2:5,6-di-O-isopropylidene-α-d-gulofuranose (2) and 3-O-acetyl-3-C-carbamoyl-1,2:5,6-di-O-isopropylidene-d-α-allofuranose (3) in 65 and 26% yields, respectively (based on consumed1). Treatment of2 with 5% hydrochloric acid in methanol yielded the spiro lactone5, which was deacetylated to yield7. Reduction of5 with sodium borohydride afforded 4-C-(hydroxymethyl)-1,2-O-isopropylidene-α-d-gulofuranose (9) in 79% yield. Oxidation of9 with sodium metaperiodate afforded a dialdose that was reduced with sodium borohydride to give 4-C-(hydroxymethyl)-1,2-O-isopropylidene-α-d-erythro-pentofuranose (11) in 88% yield. Treatment of the acetate12, derived from11, with trifluoroacetic acid, followed by acetylation, afforded the branched-chain sugar acetate14. Condensation of the glycosyl halide derived from14 withN⁶-benzoyl-N⁶, 9-bis-(trimethylsilyl)adenine yielded an equimolar anomeric mixture of protected nucleosides15 and16 in 40% yield. Treatment of the latter compounds with sodium methoxide in methanol afforded 9-[4-C-(hydroxymethyl)-β-d-erythro-pentofuranosyl]-adenine (17) and the α-d anomer18. The structure of3 was determined by correlation with the known 5,3′-hemiacetal of 3-C-(hydroxymethyl)-1,2-O-isopropylidene-α,α′-d-ribo-pentodialdose (25).     

Triterpenoid saponins and C-glycosyl flavones from stem bark of Erythrina abyssinica Lam and their cytotoxic effects.

Six new compounds including two oleanane-type triterpenoid saponins (1, 2) and four C-glycosyl flavones (3–6), along with a known saponin (7), three di-C-glycosyl flavones (8–10) and a glycosyl auronol (11), were isolated from the stem bark of Erythrina abyssinica Lam. The structures of the new compounds, identified as 3-O-[α-l-rhamnopyranosyl-(1 → 2)-β-d-galactopyranosyl-(1 → 2)-β-d-glucuronopyranosyl]-22-O-β-d-glucopyranosyl sophoradiol (1), 3-O-[α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl]-22-O-β-d-glucopyranosyl sophoradiol (2), 6-C-β-glucopyranosyl-8-C-β-quinovopyranosyl apigenin (3), 6-C-β-quinovopyranosyl-8-C-β-glucopyranosyl apigenin (4), 8-C-[6″-O-α-l-rhamnopyranosyl-(1‴ → 6″)]-β-glucopyranosyl 7,4′-dihydroxyflavone (5) and 8-C-[6″-O-β-d-xylopyranosyl-(1‴ → 6″)]-β-glucopyranosyl 7,4′-dihydroxyflavone (6), were determined by comprehensive spectroscopic analysis, including 1D and 2D NMR techniques, mass spectrometry and acid hydrolysis. These new compounds together with the known saponins 7 were evaluated for their cytotoxic activity against MCF-7 (estrogen dependent) and MDA-MB-231 (estrogen independent) cell lines. The new saponin 2 exhibited the highest cytotoxic activity among tested compounds, exerting a selective inhibitory effect against the proliferation of MCF-7 cells, with lower IC₅₀ value (12.90 μM) than that of the positive control, resveratrol (13.91 μM). Structure–activity relationship of these compounds is also discussed.     

Evidence for Deuterium Retention in the Products after Enzymatic C-C and Ether Bond Cleavages of Deuterated Lignin Model Compounds

The mechanism of C-C and ether bond cleavages of C_α-or C_β-deuterated β-O-4 and β-l lignin substructure models and the vicinal diol compounds catalyzed by the enzyme system from Phanerochaete chrysosporium culture was investigated. The enzymatic oxidation of β-O-4 lignin model compounds in the presence of H₂O₂ and O₂ yielded C₆-C_α-derived benzaldehyde, and C_β-C_γ-derived product together with the arylglycerol product. Likewise, the β-l models and the diol compounds also underwent the C-C bond cleavage, yielding C₆-C_β-derived benzaldehyde and the arylglycol product. The results demonstrated that the d-labels at C_α and C_β of the substrates were retained in the products after the C_α-C_β and ether bond cleavages.     

Facile synthesis of 1,2-trans-O-acetyl glycosyl chloride derivatives of cellobiose,lactose, and D-glucose

Solutions of O-acetyl-α-glycosyl bromide derivatives of d-glucose, cellobiose, and lactose in hexamethylphosphoramide were converted into corresponding β-chlorides at room temperature by the action of lithium chloride. At 3:1 mM ratios of chloride ion to glycose, 5–10% w/v solutions of glycosyl bromide formed α- and β-chlorides in ratios of (or greater than) 1:19 within 2–13 min and produced crystalline β-chlorides in 70–80% yields. Anomeric compositions were determined by n.m.r. spectroscopy in hexamethylphosphoramide. Older methods of preparing 1,2-trans-O-acetylgIycosyl chlorides, with aluminum chloride or titanium tetrachloride, gave the α- and β-cellobiosyl and -Iactosyl chlorides in ratios that varied from 2:3 to 1:4 and reached 85–95% levels of β-chloride only with β-d-glucose pentaacetate. When hydrolyzed under conditions that controlled solution acidity, the β-cellobiosyl and -Iactosyl chlorides each gave 2-hydroxy derivatives in yields that could be varied from 16 to 60%. Hepta-O-acetyl-2-O-methyl-α-cellobiose was prepared to demonstrate how these hydrolysis mixtures can be used to synthesize a 2-O-substituted derivative.     

Distribution of flavonoids and their systematic significance in Clematis subsection Viornae

Fifteen flavonoids were identified in the eight taxa of Clematis subsection Viornae. Flavonols, flavones, and C-glycosyl flavones were present. The compounds were based primarily on luteolin, apigenin, quercetin, and kaempferol. The systematic significance of the distribution of these compounds among the taxa is discussed.     

Syntheses stereoselectives de 1-thioglycosides

2,3,4,6-Tetra-O-acetyl-β-d-mannopyranosyl chloride (2) was obtained in 70% yield by the action of lithium chloride on 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide (1) in hexamethylphosphoric triamide. p-Nitrobenzenethiol reacted with 1 and 2 as well as with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide (9) or its β-d-chloro analog (10), giving exclusively and in good yield the corresponding p-nitrophenyl 1-thioglycosides of inverted anomeric configuration. The 1,2-cis-d-manno and -glucop-nitrophenylglycosides were likewise prepared. α-d-Glucopyranosyl 1-thio-α-d-glucopyranoside was similarly obtained by the action of the sodium salt of 1-thio-α-d-glucopyranose on the β-chloride 10 in hexamethylphosphoric triamide, or by treatment of 10 with sodium sulfide, with subsequent deacetylation. Analogous procedures allowed the preparation of β-d-mannopyranosyl 1-thio-β-d-mann opyranoside, the corresponding α,β anomer and α-d-glucopyranosyl 1-thio-α-d-mannopyranoside, starting from bromide 1, 1-thio-α-d-mannopyranose (8),and chloride 10, respectively. When acetone was used as solvent, the reaction between 1 and 8 led instead to the α,α anomer. The thio disaccharides that are interglycosidic 4-thio analogs of methyl 4-O-(β-d-galactopyranosyl)-α-d-galactopyranoside, methyl α-cellobioside, and methyl α-maltoside, respectively, were obtained by way of the peracetates of methyl 4-thio-α-d-galactopyranoside and -glucopyranoside by reaction of the corresponding thiolates with tetra-O-acetyl-α-d-galactopyranosyl bromide, bromide 9, or chloride 10, respectively, in hexamethylphosphoric triamide. These 1-thioglycosides, and (1→1)- and (1→4)-thiodisaccharides, were characterized by ¹H- and ^{1 3}C-n.m.r. spectroscopy. Correlations were established between the polarity of the sulfur atom and certain proton and carbon chemical-shifts in the 1-thioglycosides in comparison with the O-glycosyl analogs; these correlations permitted in particular the unambigous attribution of anomeric configuration.     

Preparation of 2-amino-2-C-glycosyl-acetonitriles from C-glycosyl aldehydes by Strecker reaction

Synthesis of new 2-amino-2-C-d-glycosyl-acetonitriles in a Strecker reaction from various C-glycosyl aldehydes, chiral amines, and HCN was carried out. While aminonitriles from glycal and 2-deoxy-β-d-glycosyl aldehydes were prepared in satisfactory yields, lower yields were obtained with C-glycosyl aldehydes. Strecker reaction with the benzyl-protected 1-C-formyl-d-galactal and S- or R-1-phenylethylamine (S-PEA or R-PEA) yielded predominantly the R-configured C-glycosyl aminoacetonitrile. The direction of the nucleophilic addition appears to be governed by the configuration of the anomeric carbon with β-linked sugars. Since the stereochemistry of the transition state is unknown according to the configuration of the major product a Felkin–Ahn selectivity can be mainly presumed.     

Flavonoids,stilbenoids, and phenolic derivatives from the stems of Gnetum macrostachyum (Gnetaceae)

Gnetum species have been traditionally consumed as food and used as folk medicine to treat various pathological conditions. Ten compounds including three simple phenolic compounds (1–3), five stilbenoids (4, 5, 8–10), and two C-glycosyl flavanones (6 and 7), were isolated from the stems of Gnetum macrostachyum Hook. f. The structures of these compounds were elucidated by the analysis of spectroscopy data and their comparison with the reported values. This is the first report of the isolation of compounds 1–4 and 6–9 from G. macrostachyum. Compounds 1–3, 6, and 7 have not been previously reported from the genus Gnetum. The C-glycosyl flavanones in G. macrostachyum can be used as chemotaxonomic markers.     

Production and characteristics of some new β-agarases from a marine bacterium,Vibrio sp. strain JT0107

Vibrio sp. strain JT0107 is one of the marine bacteria that secrete β-agarases which catalyze the hydrolysis of agarose. The optimum culture conditions for the production of some β-agarases have been determined. To increase agarase activity, aeration and a sufficient concentration of agarose are needed. One of the enzymes that the bacteria secreted into the culture medium was isolated and purified 39-fold using a combination of ultrafiltration and subsequent anion exchange column chromatography. The purified protein migrated as a single band (72 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis and its isoelectric point was 4.7. Amino acid sequence analysis revealed a single N-terminal sequence that had no sequence identity to other marine bacterial agarases. This novel enzyme was found to be an endo-type β-agarase (EC 3.2.1.81) that catalyzes the hydrolysis of the β-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-O-β-d-galactopyranosyl(1→4)-O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d -galactose] and neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d-galactose]. The optimum pH and temperature for obtaining high activity of the enzyme were at around 8 and 30°C, respectively. The enzyme did not degrade sodium alginate, λ-carrageenan, ι-carrageenan or κ-carrageenan.     

Photo-oxygenation of glycosylfurans. Rearrangement of C-glycosyl into O-glycosyl derivatives

Photo-oxygenation of 3-ethoxycarbonyl-5-(2,3-O-isopropylidene-β-d-erythrofuranosyl)-2-methylfuran and 3-hydroxymethyl-5-(2,3-O-isopropylidene-β-d-erythrofuranosyl)-2-methylfuran yields the corresponding endo-peroxides which rearrange at room temperature into the O-glycosyl derivatives ethyl 2,3-O-isopropylidene-β-d-erythrofuranosyl 2-acetylfumarate and 2,3-O-isopropylidene-β-d-erythrofuranosyl 3-acetyl-3-hydroxymethylacrylate, respectively. The endo-peroxides can be reduced without rearrangement, yielding C-glycosyl derivatives. Alcoholysis of the O-glycosyl derivatives yields 2,3-O-isopropylidene-d-erythrose, dialkyl 2-acetyl-3-alkoxysuccinates, 4-ethoxycarbonyl-5-methoxy-5-methyl-2-oxo-2,5-dihydrofuran and 4-hydroxymethyl-5-methoxy-5-methyl-2-oxo-2,5-dihydrofuran.     

Beshornin and beshornoside,steroidal saponins of Beshorneria yuccoides

Two new saponins beshornin and beshornoside have been isolated from the methanolic extract of Beshorneria yuccoides leaves and their structures elucidated. Beshornin is 3-O-[α-l-rhamnopyranosyl-(1 → 4)-β-d-glucopyranosyl- (1 → 2)-[α-l-rhamnopyranosyl-(1 -+ 4)-P-D-glucopyranosyl-(1 → 3)]-β-d-glucopyranosyl-(1 → 4)-β-d- galactopyranosyl-(25R)-5α-spirostan-3β-ol, whereas beshornoside is 3-O-[α-l-rhamnopyranosyl-(1 → 4)- β-d)-glycopyranosyl-(1 → 2)]-[α-l-rhamnopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 3)]-β-d-glucopyranosyl- (1 → 4)-β-d-galactopyranosyl 26-O-[β-d]-glucopyranosyl-(25R)-5α-furostan-3β,22α,26-triol.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a β-glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima β-glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity β-glucosidase and as a member of the β-glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family β-glucosidases, a β-xylosidase, β-1,4-glycanases of the enzyme family F (mostly xylanases), and other families of β-1,4-glycosyl hydrolases. This result indicates that BGA β-glucosidases may comprise one enzyme family within a large ‘enzyme order’ of retaining β-glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

The synthesis of mono- and oligosaccharide 1,2-orthoesters by way of a glycosyl nitrate intermediate

Sugar orthoesters with complex alcohols were obtained in high yield in the reaction of acylated 1,2-cis-glycosyl halides with partially protected sugar derivatives in the presence of silver nitrate and 2,4,6-trimethylpyridine in dry acetonitrile. The reaction has been shown to proceed by way of the acylated 1,2-trans-glycosyl nitrate intermediate.     

C-daunosaminyl derivatives by the wittig reaction

C-Glycosylation of a 2-deoxypyranose has been achieved for the first time by conversion of 4-O-p-nitrobenzoyl-N-trifluoroacetyldaunosamine in a Wittig reaction into the corresponding derivative of ethyl 2-(daunosaminyl)acetate. The product was predominantly (54%) in the desired α-l configuration (separated from the β-l anomer, 15%) required for further elaboration of C-daunosaminyl derivatives. Conversion into the corresponding derivatives of 2-(α-l-daunosaminyl)acetaldehyde and 2-(α-l-daunosaminyl)ethanol was also achieved.     

Synthesis of anomeric sulfonamides and their behaviour under radical-mediated bromination conditions

O-Peracetylated methyl 3-(d-glycopyranosylthio)propanoates of β-d-gluco, and α- and β-d-galacto configurations were oxidized to the corresponding S,S-dioxides (sulfones) by Oxone^® or MCPBA. Oxidation of the β-d-gluco derivative with H₂O₂/Na₂WO₄ gave the corresponding S-oxide (sulfoxide). DBU-induced elimination of methyl acrylate from the β-d-gluco and β-d-galacto configured S,S-dioxides (sulfones) gave O-peracetylated β-d-glycopyranosyl-1-C-sulfinates which, on treatment with H₂NOSO₃H, furnished the corresponding β-d-glycopyranosyl-1-C-sulfonamides. Radical-mediated bromination of the protected methyl 3-(β-d-glycopyranosylthio)propanoate S,S-dioxides gave mixtures of 1-C- and 5-C-bromoglycosyl compounds. Similar brominations of the O-peracetylated β-d-glycopyranosyl-1-C-sulfonamides resulted in the formation of α-d-glycopyranosyl bromides and 1-C- and 5-C-bromoglycosyl sulfonamides. A rationale for these observations was proposed. Methyl 3-(β-d-glucopyranosylthio)propanoate, its S,S-dioxide, and β-d-glucopyranosyl-1-C-sulfonamide proved inefficient when tested as inhibitors of rabbit muscle glycogen phosphorylase b.     

Synthèse,conformation et affinité tréhalasique des α-d-glucopyranosyl-α-d-xylopyranoside, α-d-glucopyranosyl-α-d-mannopyranoside et α-d-allopyranosyl-α-d-glucopyranoside

α-d-Glucopyranosyl α-d-xylopyranoside has been synthesized in 49% yield by treatment of 2,3,4-tri-O-benzyl-α-d-xylopyranosyl bromide with 2,3,4,6-tetra-O-acetyl-d-glucopyranose in nitromethane-benzene with mercuric cyanide and bromide, followed by catalytic hydrogenolysis and O-deacetylation. Condensation with 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide in acetonitrile-dichloromethane with mercuric cyanide, followed by catalytic hydrogenolysis and O-deacetylation, gave α-d-glucopyranosyl α-d-mannopyranoside and β-d-glucopyranosyl β-d-mannopyranoside in 44 and 25% yield, respectively. The mixture was resolved by column chromatography of the fully acetylated derivatives. Selective acetylation of the di-O-benzylidene derivative of trehalose with N-acetylimidazole, followed by oxidation with dimethyl sulfoxide-acetic anhydride at C-3 and stereoselective reduction gave, after removal of the protecting groups, α-d-allopyranosyl α-d-glucopyranoside in 20% overall yield. The structure of the compounds was confirmed by ¹H- and ¹³C-n.m.r., and mass spectrometry. α-d-Glucopyranosyl α-d-xylopyranoside and α-d-allopyranosyl α-d-glucopyranoside are less efficient substrates than trehalose for cockchafer trehalase, but α-d-glucopyranosyl α-d-mannopyranoside is a competitive inhibitor of the enzyme.