Synthesis of rare sugar isomers through site-selective epimerization.

Glycans have diverse physiological functions, ranging from energy storage and structural integrity to cell signalling and the regulation of intracellular processes1. Although biomass-derived carbohydrates (such as D-glucose, D-xylose and D-galactose) are extracted on commercial scales, and serve as renewable chemical feedstocks and building blocks2,3, there are hundreds of distinct monosaccharides that typically cannot be isolated from their natural sources and must instead be prepared through multistep chemical or enzymatic syntheses4,5. These 'rare' sugars feature prominently in bioactive natural products and pharmaceuticals, including antiviral, antibacterial, anticancer and cardiac drugs6,7. Here we report the preparation of rare sugar isomers directly from biomass carbohydrates through site-selective epimerization reactions. Mechanistic studies establish that these reactions proceed under kinetic control, through sequential steps of hydrogen-atom abstraction and hydrogen-atom donation mediated by two distinct catalysts. This synthetic strategy provides concise and potentially extensive access to this valuable class of natural compounds.

Group 8 metallocenes as bulky functional groups in glucopyranosides.

The functionalization of methyl D-glucopyranosides at positions 4 and 6 with bulky moieties was carried out by using ferrocenyl and ruthenocenyl substituents. The synthesis succeeded by reaction of the methyl D-glucopyranosides with the corresponding metallocene monocarbaldehyde dimethyl acetal catalysed by iodine in acetonitrile. The resulting compounds methyl 4,6-O-(metallocenylmethylidene)-α-D-glucopyranoside (M=Fe (1) and M=Ru (3)) and methyl 4,6-O-(metallocenylmethylidene)-ß-D-glucopyranoside (M=Fe (2) and M=Ru (4)) were characterized by (1)H and (13)C NMR spectroscopy, by crystal structure determination as well as elemental analysis.

Aspects of glycosidic bond formation in aqueous solution: chemical bonding and the role of water.

A model of the specific acid-catalyzed glycosidic bond formation in liquid water at ambient conditions is studied based on constrained Car-Parrinello ab initio molecular dynamics. Specifically the reaction of alpha-D-glucopyranose and methanol is found to proceed by a D(N)A(N) mechanism. The D(N) step consists of a concerted protonation of the O(1) hydroxyl leaving group; this process results in the breaking of the C(1)-O(1) bond, and oxocarbenium ion formation involving C(1)=O(5). The second step, A(N), is the formation of the C(1)-O(m) glycosidic bond, deprotonation of the methanol hydroxyl group O(m)H(m), and re-formation of the C(1)-O(5) single bond. A focus of this study is the analysis of the electronic structure during this condensed phase reaction relying on both Boys/Wannier localized orbitals and the electron localization function ELF. This analysis allows the clear elucidation of the chemical bonding features of the intermediate bracketed by the D(N) and A(N) steps, which is a non-solvent equilibrated oxocarbenium cation. Most interestingly, it is found that the oxygen in the pyranose ring becomes "desolvated" upon double bond/oxocarbenium formation, whereas it is engaged in the hydrogen-bonded water network before and after this period. This demonstrates that hydrogen bonding and thus the aqueous solvent play an active role in this reaction implying that microsolvation studies in the gas phase, both theoretical and experimental, might lead to qualitatively different reaction mechanisms compared to solution.

Synthesis and biologic evaluation of (11)c-methyl-d-glucoside, a tracer of the sodium-dependent glucose transporters.

UNLABELLED: This study aimed to synthesize and to evaluate the biologic characteristics of (11)C labeled methyl-D-glucoside, a nonmetabolizable tracer that is selectively transported by sodium-dependent glucose transporters (SGLTs). METHODS: (11)C-Methyl-D-glucoside was prepared by methylation of glucose with (11)C-methyl triflate and was obtained as a mixture of anomers that were separated with high-performance liquid chromatography. The biodistribution of both the D- and L-isomers was determined in mice, and the presence of metabolites in the blood was investigated. The intrarenal distribution of (11)C-methyl-D-glucoside in mouse kidneys was visualized using autoradiography. Transport of alpha-methyl-D-glucoside and beta-methyl-D-glucoside by the human sodium-D-glucose cotransporter hSGLT1 was characterized after expression of hSGLT1 in oocytes of Xenopus laevis. RESULTS: The developed preparation procedure provided (11)C-methyl-D-glucoside in a total synthesis time of 20 min and a yield of 30% (decay corrected). The alpha- and beta-anomers of methyl-D-glucoside were reabsorbed from the primary urinary filtrate and showed only a minimal urinary excretion. Because methyl-L-glucoside was not reabsorbed and the reabsorption of methyl-D-glucoside was blocked by phlorizin, sodium-D-glucose cotransporters were critically involved. beta-Methyl-D-glucoside was accumulated in the kidneys to a higher extent than the alpha-anomer, suggesting that the basolateral efflux from the tubular cells is slower for the beta-anomer. Autoradiography showed that methyl-D-glucoside was accumulated throughout the renal cortex, suggesting that both sodium-D-glucose cotransporters expressed in kidney, SGLT1 and SGLT2, are involved in the uptake. The tracer was found to be metabolically stable and did not accumulate in red blood cells, which indicates that methyl-D-glucoside is not transported by the sodium-independent transporter GLUT1. Electrical measurements in Xenopus oocytes revealed that alpha-methyl-D-glucoside and beta-methyl-D-glucoside are transported by the human SGLT1 transporter with similar maximal transport rates and apparent Michaelis-Menten constant values. CONCLUSION: (11)C-Methyl-D-glucoside is a selective tracer of sodium-dependent glucose transport and can be used to visualize the function of this transporter with PET in vivo.

Synthesis of oxidized methyl 4-O-methyl-beta-D-glucopyranoside and methyl beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside derivatives as substrates for fluorescence labeling reactions.

The synthetic cellulose model compounds methyl 4-O-methyl-beta-D-glucopyranoside and methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside and related 6-O-protected intermediates were oxidized in good to fair yields using Swern-conditions or bromine/bis(tributyltin) oxide, respectively, to afford compounds containing 6-aldehyde, 3-keto, and 2,3-diketo groups. Cellobiose and oxidized monosaccharides were then labeled with the carbonyl-selective fluorescence marker 9-(7-amino-1,4,7-trioxaheptyl)-9H-carbazolecarboxamide (CCOA). The labeled derivatives serve as model compounds for the determination of minute amounts of carbonyl groups in cellulosic polysaccharides.

Scale-up of pseudo solid-phase enzymatic synthesis of alpha-methyl glucoside acrylate.

The successful scale-up of the enzymatic synthesis of alpha-methyl glucoside acrylate from laboratory-scale (milliliter) to pilot-scale (liter) was examined. Specifically, Candida antarctica lipase B (Novozym 435) was used as a biocatalyst to produce alpha-methyl glucoside acrylate via the transesterification of alpha-methyl glucoside (MG) with vinyl acrylate (VA) using acetone as a solvent. This is a pseudo-solid-phase synthesis; only a fraction of the alpha-methyl glucoside and the product are soluble in acetone. Molecular sieves were used to remove traces of water in the reaction medium and to increase enzyme stability by removing the acetaldehyde by-product. A general method was also developed to purify and recover the monoacrylate product from unreacted sugar and undesired diester by a simple crystallization and precipitation process.

The reaction between epichlorohydrin and polysaccharides: Part 2, Synthesis of some model substances, with cyclic substituents.

Eight derivatives of methyl alpha-D-glucopyranoside, in which the substituents are part of cyclic structures, have been prepared as model substances for possible structural elements formed on reaction of polysaccharides with epichlorohydrin. The substances were converted into the permethylated alditol-1-d acetates and characterised by CIMS and EIMS.

The reaction between epichlorohydrin and polysaccharides: Part 1. Syntheses of some model substances with non-cyclic substituents.

Five derivatives of methyl alpha-D-glucopyranoside, in which the substituents form noncyclic structures, have been prepared as model substances for possible structural elements formed on reaction of polysaccharides with epichlorohydrin. The substances were converted into the permethylated alditol-1-d derivatives and characterised by CIMS and EIMS.

Binding characteristics of IgA 16.4.12E, a monoclonal antibody with specificity for the nonreducing terminal epitope of alpha-(1----6)-dextrans. Comparisons between IgA hybridoma 16.4.12E and myeloma W3129.

IgA 16.4.12E is a murine monoclonal antibody obtained following immunization with isomaltohexose linked to keyhole limpet hemocyanin. We have studied its interaction with methyl alpha-D-glucopyranoside and its derivatives bearing deoxy or deoxyfluoro groups, and with the methyl alpha-glycosides of a series of isomalto-oligosaccharides, some bearing deoxy or deoxy-fluoro groups at selected positions. From the data it is concluded that the antibody binds optimally to 4 sequential glucopyranosyl residues and that the protein subsite possessing the major affinity binds the terminal, nonreducing glucosyl group of that antigenic epitope. All the hydroxyl groups of that terminal glucosyl group are involved in hydrogen bonding, some in a donating and some in an accepting capacity. In the last part of the paper we report the construction of a possible model of the antibody, derived from its known amino acid sequence and the known crystalline structures of two closely related antibodies. It shows a pronounced cavity in the general immunoglobulin combining area which is flanked by 2 solvent-exposed tryptophanyl residues. A model recently reported for anti-dextran IgA W3129 shows a similar cavity with one such residue. Guided by hydrogen bonds, experimentally deduced from the comparison of the affinities of variously derivatized ligands, we suggest a speculative fitting for the nonreducing terminus of the dextran antigen, in the respective cavities of both IgA 16.4.12E and W3129.

Methyl 4-O-(4-alpha-D-glucopyranosyloxy-4-methoxybutyl)-alpha-D-+ ++glucopyranosi de, a modified oligosaccharide for studying interactions of carbohydrates with multisite proteins.

Methyl 4-O-(4-alpha-D-glucopyranosyloxy-4-methoxybutyl)-alpha-D-glu copyranoside (9) was synthesised by transacetalation from methyl 2,3,6-tri-O-acetyl-4-O-(4,4-dimethoxybutyl)-alpha-D-glucopyranosid e and trimethylsilyl 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranoside followed by removal of the blocking groups. Compound 9, which is methyl alpha-maltotrioside modified by replacing the middle D-glucosyl residue with an acyclic spacer, competitively inhibits the hydrolysis of p-nitrophenyl alpha-maltotrioside by porcine alpha-amylase.

Stereoselective total synthesis of methyl alpha-D and alpha-L-glucopyranosides.

Methyl alpha-L- and alpha-D-glucopyranosides have been synthesized from methyl (R)- and (S)-(2-furyl)glycolates (3), respectively. The key intermediates, methyl 6-O-benzyl-2,3-dideoxy-L(and D)-hex-2-enopyranosid-4-uloses (13), were obtained in seven steps from the ester 3, without change of configuration of the asymmetric center, which became C-5 in the sugar molecule. Reduction of the ketone group at C-4 in the glycoside 13 with sodium borohydride afforded the corresponding methyl 6-O-benzyl-2,3-dideoxy-erythro-hex-2-enopyranosides (14). Epoxidation of the double bond in 14, followed by oxirane ring-opening in the anhydro sugar 16, and subsequent catalytic hydrogenolysis of the benzyl group led to the title compounds.

A facile synthesis of 2-methyl-[3,4-di-O-acetyl-6-O-(chloroacetyl)-1,2-dideoxy-alpha-D-glucopyrano]-[2',1':4,5]-2-oxazoline.

The use of the chloroacetyl group as a protecting group has been studied for a 2-methylglyco-[2',1':4,5]-2-oxazoline. The reaction of chloroacetyl chloride or chloroacetic anhydride with 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-beta-D-glucopyranose provided 2-acetamido-1,3,4-tri-O-acetyl-6-O-(chloroacetyl)-2-deoxy-beta-D-glucopyranose which, on treatment with anhydrous ferric chloride in dichloromethane, produced the desired oxazoline. The glycosylating capability of the oxazoline has been investigated with aglycon hydroxides, to give the corresponding 2-acetamido-2-deoxy-beta-D-glucopyranosides. The chloroacetyl group can be selectively removed by treatment with thiourea, and migration of O-acetyl groups was not observed under these conditions.