The synthesis of 6-deoxy-6-fluoro-α,α-trehalose and related analogues

Selective acid-catalysed methanolysis of 2,3,2′,3′-tetra-O-benzyl-4,6:4′,6′-di-O-benzylidene-α,α-trehalose yielded the monobenzylidene derivative, which was converted into the 4,6-dimesylate. Selective nucleophilic displacement of the primary sulphonyloxy group then gave 2,3-di-O-benzyl-6-deoxy-6-fluoro-4-O-mesyl-α-d-glucopyranosyl 2,3-di-O-benzyl-4,6-O-benzylidene-α-d-glucopyranoside. Removal of the protecting groups then yielded 6-deoxy-6-fluoro-α,α-trehalose. In addition, 6-deoxy-6-fluoro-4-O-mesyl-α,α-trehalose and a derivative of 4-chloro-4,6-dideoxy-6-fluoro-α-d-galactopyranosyl α-d-glucopyranoside were also prepared from the same substrate. Iodide displacement of 2,3-di-O-benzyl-4,6-di-O-mesyl-α-d-glucopyranosyl 2,3-di-O-benzyl-4,6-di-O-mesyl-α-d-glucopyranoside afforded the 6-iodide and 6,6′-di-iodide in yields of 31 and 36%, respectively. Similarly, the 6-azide and 6,6′-diazide were isolated in yields of 17 and 21%, respectively.     

Synthesis and moisture absorption and retention activities of a carboxymethyl and a quaternary ammonium derivative of α,α-trehalose

Carboxymethyl α,α-trehalose (CMT) and a quaternary ammonium derivative of α,α-trehalose (QT) were successfully prepared, and their moisture absorption and retention activities were assessed. Results showed that both CMT and QT had better moisture absorption abilities at 43% and 81% relative humidity (RH) than α,α-trehalose. In addition, the two α,α-trehalose derivatives had better moisture retention abilities than α,α-trehalose under three humidity conditions: 81% RH, 43% RH, and under dry conditions. Therefore, carboxymethylation and quaternarization could improve the moisture absorption and retention abilities of α,α-trehalose. CMT and QT showed better moisture absorption ability and moisture retention ability than that of hyaluronan (HA), and could potentially find a use as moisture retention ingredient, for example, in cosmetics.     

Synthesis of phenyl 2-acetamido-2-deoxy-3-O-α-l-fucopyranosyl-β-d-glucopyranoside and related compounds

The reaction of phenyl 2-acetamido-2-deoxy-4,6- O-(p-methoxybenzylidene)-β-d-glucopyranoside with 2,3,4-tri-O-benzyl-α-l-fucopyranosyl bromide under halide ion-catalyzed conditions proceeded readily, to give phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-3-O-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)-β-d-glucopyranoside (8). Mild treatment of 8 with acid, followed by hydrogenolysis, provided the disaccharide phenyl 2-acetamido-2-deoxy-3-O-α-l-fucopyranosyl-β-d-glucopyranoside. Starting from 6-(trifluoroacetamido)hexyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranoside, the synthesis of 6-(trifluoroacetamido)hexyl 2-acetamido-2-deoxy-3-O-β-l-fucopyranosyl-β-d-glucopyranoside has been accomplished by a similar reaction-sequence. On acetolysis, methyl 2-acetamido-2-deoxy-3-O-α-l-fucopyranosyl-α-d-glucopyranoside gave 2-methyl-[4,6-di-O-acetyl-1,2-dideoxy-3-O-(2,3,4-tri-O-acetyl-α-l-fucopyranosyl)-α-d-glucopyrano]-[2, 1-d]-2-oxazoline as the major product.     

Experimental and theoretical electron density distribution of α,α-trehalose dihydrate

α,α-Trehalose is of interest because of its cryoprotective and antidessicant properties, and because it possesses various technical anomalies such as ¹³C NMR spectra that give misleading indications of intramolecular structural symmetry. It is a non-reducing disaccharide, with the glycosidic oxygen atom shared by the anomeric carbon atoms of the two glucose rings, and is therefore subject to a proposed ‘overlapping’ exo-anomeric effect. We report here a study of the electron density of trehalose with X-ray diffraction and quantum mechanics calculations, similar to a recent study of sucrose, also a non-reducing molecule. In particular we studied the electron density around the glycosidic linkage and the hydrogen bonding with both deformation density and Atoms in Molecules (AIM) analyses. A total of 129,952 single crystal X-ray intensity measurements were collected on α,α-trehalose dihydrate to a resolution of sin θ/λ = 1.18 Å⁻¹ at 100 K and refined with an aspherical multipole model to a final agreement factor of R₁ = 0.0160. Wavefunctions were calculated at three levels of theory. Redistribution of electron density due to anomeric effects was reduced in trehalose, compared to sucrose. Five new C-H?O hydrogen bonds were confirmed with bond critical points and bond paths from AIM analyses, as were the previously proposed O-H?O hydrogen bonds.     

N-Aminoacyl and N-hydroxyacyl derivatives of diosgenyl 2-amino-2-deoxy-β-d-glucopyranoside: Synthesis,antimicrobial and hemolytic activities

Diosgenyl 2-amino-2-deoxy-β-d-glucopyranoside is a semisynthetic saponin with antimicrobial and antitumor activities. To search for more effective analogues, N-aminoacyl and N-hydroxyacyl derivatives of this saponin were synthesized conventionally and with microwave assistance, and tested against the human pathogenic fungi and Gram-positive and Gram-negative bacteria. None of the tested compounds exhibit activity against Gram-negative bacteria. Almost all of the synthesized N-aminoacyl saponins exhibit antifungal activity and act effectively against Gram-positive bacteria, some better than the parent compound. The best acting saponins are the same size and possess sarcosine or l- or d-alanine attached to the parent glucosaminoside. Shorter and longer aminoacyl residues are less advantageous. d-Alanine derivative is the most effective against Gram positive bacteria. Structure-activity relationship (SAR) analysis indicates that the free α-amino group in aminoacyl residue is necessary for antimicrobial activities of the tested saponins. (N-Acetyl)aminoacyl and N-hydroxyacyl analogs are inactive. Measurements of the hemolytic activities demonstrate that the best acting saponins are not toxic towards human red blood cells.     

Catalytic versatility of trehalase: Synthesis of α-d-glucopyranosyl α-d-xylopyranoside from β-d-glucosyl fluoride and α-d-xylose

Trehalase was previously shown (see ref. 5) to hydrolyze α-d-glucosyl fluoride, forming β-d-glucose, and to synthesize α,α-trehalose from β-d-glucosyl fluoride plus α-d-glucose. Present observations further define the enzyme's separate cosubstrate requirements in utilizing these nonglycosidic substrates. α-d-Glucopyranose and α-d-xylopyranose were found to be uniquely effective in enabling Trichoderma reesei trehalase to catalyze reactions with β-d-glucosyl fluoride. As little as 0.2mm added α-d-glucose (0.4mm α-d-xylose) substantially increased the rate of enzymically catalyzed release of fluoride from 25mm β-d-glucosyl fluoride at 0°. Digest of β-d-glucosyl fluoride plus α-d-xylose yielded the α,α-trehalose analog, α-d-glucopyranosyl α-d-xylopyranoside, as a transient (i.e., subsequently hydrolyzed) transfer-product. The need for an aldopyranose acceptor having an axial 1-OH group when β-d-glucosyl fluoride is the donor, and for water when α-d-glucosyl fluoride is the substrate, indicates that the catalytic groups of trehalose have the flexibility to catalyze different stereochemical reactions.     

Stereocontrolled preparation of C-2-deoxy-α- and -β-d-glucopyranosyl compounds from tributyl-(2-deoxy-α- and -β-d-glucopyranosyl)stannanes

tributylstannyllithium treatment of 3,4,6-tri-O-benzyl-2-deoxy-α-d-arabino-hexopyranosyl chloride (2) provided selectively tributyl (3,4,6-tri-O-benzyl-2-deoxy-β-d-arabino-hexopyranosyl)stannane (3) in 85% yield. Isomeric tributyl (3,4,6-tri-O-benzyl-2-deoxy-α-d-arabino-hexopyranosyl)stannane (6) could be prepared in 70% yield by reductive lithiation of 2 and reaction with tributyltin chloride. Tin—lithium exchange reaction, performed on 3 and 6 with butyllithium in oxolane at −78°, generated the corresponding, configurationally stable 2-deoxy-β- and -α-d-hexopyranosyllithium compounds which reacted with electrophilic compounds with retention of configuration. Addition to these glycosyllithium reagents to prochiral carbonyl compounds gave variable degrees of facial selectivity. A significant diastereofacial discrimination (10:1) was observed by condensation of 3,4,6-tri-O-benzyl-2-deoxy-α-d-arabino-hexopyranosyllithium reagent with hexanal and isobutyraldehyde. The structure of all C-glycopyranosyl compounds obtained was established by ¹H-n.m.r. spectroscopy.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-β-d-galactopyranoside and methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-O-β-d-galactopyranosyl-(1→6)-β-d-galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α-d-galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β-d-galactopyranoside (4) gave a fully acetylated (1→6)-β-d-galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α-d-galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β-d-galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β-d-galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Use of 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-acetyl-α-d-galactopyranosyl bromide as a glycosyl donor: Synthesis of p-nitrophenyl 3-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-β-d-galactopyranoside

Acetolysis of methyl 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-acetyl-α-d-galactopyranoside afforded 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-1,2,4,6-tetra-O-acetyl-d-galactopyranose (2). Treatment of 2 in dichloromethane with hydrogen bromide in glacial acetic acid gave 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)- 2,4,6-tri-O-acetyl-α-d-galactopyranosyl bromide (3). The α configuration of 3 was indicated by its high, positive, specific rotation, and supported by its ¹H-n.m.r. spectrum. Reaction of 3 with Amberlyst A-26-p-nitrophenoxide resin in 1:4 dichloromethane-2-propanol furnished p-nitrophenyl 3-O-(2-acetamido-3,4,6- tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-acetyl-β-d-galactopyranoside (7). Compound 7 was also obtained by the condensation (catalyzed by silver trifluoromethanesulfonate-2,4,6-trimethylpyridine) of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl bromide with p-nitrophenyl 2,4,6-tri-O-acetyl-β-d-galactopyranoside, followed by the usual deacylation-peracetylation procedure. O-Deacetylation of 7 in methanolic sodium methoxide furnished the title disaccharide (8). The structure of 8 was established by ¹³C-n.m.r. spectroscopy.     

Synthesis of 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-β-d-glucopyranosyl chloride

A lactosaminyl donor, 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-β-d- glucopyranosyl chloride, was synthesized in 10 steps, starting from 1,3,4,6-tetra-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranose. Benzyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside was prepared by regioselective benzylation at the primary hydroxyl group by the stannyl method, and was used as a key intermediate.

     

The preparation of 4,6-dichloro-4,6-dideoxy-α-d-galactopyranosyl 6-chloro-6-deoxy-,β-d-fructofuranoside and the conversion of chlorinated derivatives into anhydrides

Under carefully controlled conditions, sucrose is converted by selective reaction with sulphuryl chloride into either 6-chloro-6-deoxy-α-d-glucopyranosyl 6-chloro-6-deoxy-β-d-fructofuranoside or 4,6-dichloro-4,6-dideoxy-α-d-galactopyranosyl 6-chloro-6-deoxy-β-d-fructofuranoside, which could be isolated without recourse to chromatography. Treatment of the dichloride with sodium methoxide gave 3,6-anhydro-β-d-glucopyranosyl, 3,6-anhydro-β-d-fructofuranoside in high yield. In contrast, 4,6-dichloro-4,6-dideoxy-α-d-galactopyranosyl 6-chloro-6-deoxy-β-d-fructofuranoside gave, in two distinct stages, 3,6-anhydro-4-chloro-4-deoxy-α-d-galactopyranosyl 6-chloro-6-deoxy-β-d-fructofuranoside and 3,6-anhydro-4-chloro-4-deoxy-α-d-galactopyranosyl 3,6-anhydro-β-d-fructofuranoside. The structures of these products were ascertained by ¹H-n.m.r. and mass spectrometry.     

Enzymes of sucrose,maltose, and α,α-trehalose catabolism in soybean root nodules

Crude, Sephadex-filtered extracts of soybean (Glycine max (L.) Merr.) root nodules contained invertase (E.C. 3.2.1.26) activity with pH optima at 5.4 and 7.8, ,-trehalase (E.C. 3.2.1.28) activity with pH optima at 3.8 and 6.6, and maltase (E.C. 3.2.1.20) activity with a broad pH optimum between 4.5 and 5.0. Bacteroids and cytosol were separated using Percoll density gradients. Cellulase and pectinase were employed to separate protoplasts from the infected region from the nodule cortex, which remained intract. Assays of disaccharidases from these nodule fractions indicated the following localization of enzymes: (1) Bacteroids lack invertase activity (pH 5.4 and 7.8). (2) Much, if not most, of the invertase activity may be localized in the nodule cortex; this is especially likely for acid invertase. However, there was substantial invertase activity in cytosol from the infected region. (3) Most of the maltase activity (pH 5.0) and trehalase activity (pH 3.8 and 6.6) were localized in the cytosol. It is likely that most of these disaccharidase activities are in the cytosol of the infected region, in contrast to invertase. (4) Bacteroids contain maltase (pH 5.0) and trehalase (pH 3.8 and 6.6), but the amount of these enzyme activities was less than 15% of total activity in nodules. Bacteroids and nodule cortex were capable of in-vivo hydrolysis of [¹⁴C]trehalose and [¹⁴C]maltose. These disaccharides were also hydrolyzed by soybean roots and hypocotyls. Therefore, while ,-trehalose in soybean nodules is probably synthesized by the bacteroids, the capability for utilization of trehalose was not restricted to the bacteroids.Approved for publication as Journal Article 74–81 of the Ohio Agricultural Research and Development Center     

Continuous production of α,α-trehalose by immobilised fungal trehalose phosphorylase

Immobilisation of trehalose phosphorylase from Schizophyllum commune by adsorption onto anion-exchange materials stabilised the enzyme activity at 30°C by approx. 35-fold. Immobilised and free enzymes showed similar pH-dependence of activity but different inactivation behavior above 30°C. A fixed-bed enzyme reactor produced ,-trehalose at a stable substrate conversion of 80% with a productivity of 2.6 g l–1 h–1 for 72 h. Inhibition of trehalose phosphorylase by phosphate limited the productivity of a direct conversion of starch into ,-trehalose.     

Synthesis of 2-amino-2-deoxy- and 3-amino-3-deoxy-α-d-mannopyranosyl α-d-mannopyranoside by sequential osmylations of a dienic disaccharide

Partial oxyamination of 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranosyl 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranoside with chloramine-T and osmium tetraoxide gave 4,6-di-O-acetyl-2-deoxy-2-(p-toluene-sulfonamido)-α-d-mannopyranosyl 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranoside and its 3-deoxy-3-(p-toluenesulfonamido) regioisomer, each in 18–19% isolated yield. Osmium tetraoxide-catalyzed cis-hydroxylation of the remaining alkenic residue in these products led in high yields to the corresponding triols having the α-d-manno, α-d-manno configuration. These were N-desulfonylated (and simultaneously O-deacetylated) by the action of sodium in liquid ammonia to furnish 2-amino-2-deoxy-α-d-mannopyranosyl α-d-mannopyranoside and 3-amino-3-deoxy-α-d-mannopyranosyl α-d-mannopyranoside as new, trehalose-type amino sugars.     

Synthesis of benzyl 2-acetamido-2-deoxy-3-O-β-d-fucopyranosyl-α-d-galactopyranoside and benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2-deoxy-3-O-β-d-fucopyranosyl-α-d-galactopyranoside

Condensation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside with 2,3,4-tri-O-acetyl-α-d-fucopyranosyl bromide in 1:1 nitromethane-benzene, in the presence of powdered mercuric cyanide, afforded benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-(2,3,4-tri-O-acetyl-β-d-fucopyranosyl)-α-d-galactopyranoside (3). Cleavage of the benzylidene group of 3 with hot, 60% aqueous acetic acid afforded diol 4, which, on deacetylation, furnished the disaccharide 5. Condensation of diol 4 with 2-methyl-(3,4,6-tri-O-acetyl-1,2-di-deoxy-α-d-glucopyrano)-[2,1-d]-2-oxazoline in 1,2-dichloroethane afforded the trisaccharide derivative (7). Deacetylation of 7 with Amberlyst A-26 (OH^?) anion-exchange resin in methanol gave the title trisaccharide (8). The structures of 5 and 8 were confirmed by ¹³C-n.m.r. spectroscopy.     

Protective mechanisms of α,α-trehalose revealed by molecular dynamics simulations

Molecular dynamics simulations were carried out to model aqueous solution with different concentration of α,α-trehalose, one kind of non-reducing sugars possessing outstanding freeze-drying protective effect on biological system. The dihedral angles of the intraglycosidic linkage in trehalose were measured to estimate its structure rigidity. The dynamics and hydrogen bonding properties were studied by calculating the self-diffusion coefficient of trehalose and the distributions and lifetimes of various types of H-bonds in the solution. Through analysing the results as well as comparison with another common sugar sucrose, the freeze-drying protective mechanism of trehalose was explained at molecule level. First, trehalose is able to maintain the local structure around it as a frame due to its relatively rigid conformation. Second, the addition of trehalose restrains the water molecules from rearrangement as a result of low mobility, thus reduces the probability of freezing; trehalose has lower diffusion coefficient than water and bigger thermal diffusivity, which are favourable for vitrification. Third, the formation of H-bonds between trehalose and water and between trehalose molecules is the essence of the protective effect. Trehalose does not work via strengthening the H-bonds formed between water molecules (W–W H-bonds), instead of which it breaks the potential tetrahedral pattern of W–W H-bonds, thus suppresses the tendency of ice formation. It was also found that trehalose realises protective action better at higher concentration as far as this study is concerned.