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The scarcity and expense of access to L-sugars and other rare sugars have prevented the exploitation of their biological potential; for example D-psicose, only recently available, has been recognized as an important new food. Here we give the definitive and cheap synthesis of 99.4% pure L-glucose from D-glucose which requires purification of neither intermediates nor final product other than extraction into and removal of solvents; a simple crystallization will raise the purity to >99.8%.

Microbial oxidation of 2-C-methyl-d-ribitol and 2-C-methyl-d-arabinitol by Gluconobacter thailandicus NBRC 3254 produces 4-C-methyl-l-ribulose and 4-C-methyl-d-ribulose, respectively. Further, 4-C-methyl-l-ribulose and 4-C-methyl-d-ribulose were equilibrated by d-tagatose-3-epimerase (DTE) with 4-C-methyl-l-xylulose and 4-C-methyl-d-xylulose, respectively. These transformations demonstrate that polyol dehydrogenase and DTE act on branched synthetic sugars. The green preparation of all of the stereoisomers of 4-C-methyl pentuloses illustrates the ability of biotechnology to generate novel branched monosaccharides. © 2008 Elsevier Ltd. All rights reserved.

We biosynthesized 6-deoxy- l -talose, 6-deoxy- l -sorbose, 6-deoxy- l -gulose, and 6-deoxy- l -idose, which rarely exist in nature, from l -fucose by coupling and sequential enzymatic reactions. The first product, 6-deoxy- l -talose, was directly produced from l -fucose by the coupling reactions of immobilized d -arabinose isomerase and immobilized l -rhamnose isomerase. In one-pot reactions, the equilibrium ratio of l -fucose, l -fuculose, and 6-deoxy- l -talose was 80:9:11. In contrast, 6-deoxy- l -sorbose, 6-deoxy- l -gulose, and 6-deoxy- l -idose were produced from l -fucose by sequential enzymatic reactions. d -Arabinose isomerase converted l -fucose into l -fuculose with a ratio of 88:12. Purified l -fuculose was further epimerized into 6-deoxy- l -sorbose by d -allulose 3-epimerase with a ratio of 40:60. Finally, purified 6-deoxy- l -sorbose was isomerized into both 6-deoxy- l -gulose with an equilibrium ratio of 40:60 by l -ribose isomerase, and 6-deoxy- l -idose with an equilibrium ratio of 73:27 by d -glucose isomerase. Based on the amount of l -fucose used, the production yields of 6-deoxy- l -talose, 6-deoxy- l -sorbose, 6-deoxy- l -gulose, and 6-deoxy- l -idose were 7.1%, 14%, 2%, and 2.4%, respectively.

Addition of human milk oligosaccharides (HMO) to baby foods may protect infants from disease. As many simple HMOs are fucosylated this is likely to increase the demand for L-fucose as a synthetic building block. Any chemical synthesis must be cheap to compete with a biotechnological process. Acetonide is the only protecting group we have used in this new synthesis of L-fucose from vitamin C in 27% overall yield (purification by recrystallization; no chromatography required in the entire sequence).

Sucrose phosphorylase (SPase) from Leuconostoc mesenteroides exhibited activity towards eight ketohexoses, which behaved as D-glucosyl acceptors, and α-D-glucose-1-phosphate (G1P), which behaved as a donor. All eight of these ketohexoses were subsequently transformed into the corresponding d-glucosyl-ketohexoses. Of the eight ketohexoses evaluated in the current study, d-allulose behaved as the best substrate for SPase, and the resulting d-glucosyl-d-alluloside product was found to be a non-reducing sugar with a specific optical rotation of [α]D(20) + 74.36°. D-Glucosyl-D-alluloside was identified as α-D-glucopyranosyl-(1→2)-β-D-allulofuranoside by NMR analysis. D-Glucosyl-D-alluloside exhibited an inhibitory activity towards an invertase from yeast with a Km value of 50 mM, where it behaved as a competitive inhibitor with a Ki value of 9.2 mM. D-Glucosyl-D-alluloside was also successfully produced from sucrose using SPase and D-tagatose 3-epimerase. This process also allowed for the production of G1P from sucrose and d-allulose from D-fructose, which suggested that this method could be used to prepare d-glucosyl-d-alluloside without the need for expensive reagents such as G1P and d-allulose.

6-Deoxy-l-glucose, 6-deoxy-l-altrose, and 6-deoxy-l-allose were produced from l-rhamnose with an immobilized enzyme that was partially purified (IE) and an immobilized Escherichia coli recombinant treated with toluene (TT). 6-Deoxy-l-psicose was produced from l-rhamnose by a combination of l-rhamnose isomerase (TT-PsLRhI) and d-tagatose 3-epimerase (TT-PcDTE). The purified 6-deoxy-l-psicose was isomerized to 6-deoxy-l-altrose and 6-deoxy-l-allose with l-arabinose isomerase (TT-EaLAI) and l-ribose isomerase (TT-AcLRI), respectively, and then was epimerized to l-rhamnulose with immobilized d-tagatose 3-epimerase (IE-PcDTE). Following purification, l-rhamnulose was converted to 6-deoxy-l-glucose with d-arabinose isomerase (TT-BpDAI). The equilibrium ratios of 6-deoxy-l-psicose:6-deoxy-l-altrose, 6-deoxy-l-psicose:6-deoxy-l-allose, and l-rhamnulose:6-deoxy-l-glucose were 60:40, 40:60, and 27:73, respectively. The production yields of 6-deoxy-l-glucose, 6-deoxy-l-altrose, and 6-deoxy-l-allose from l-rhamnose were 5.4, 14.6, and 25.1%, respectively. These results indicate that the aldose isomerases used in this study acted on 6-deoxy aldohexoses.

l-Ribose, a pentose, is not known to exist in nature. Although organisms typically do not have a metabolic pathway that uses l-ribose as a carbon source, prokaryotes use various sugars as carbon sources for survival. Acinetobacter sp. DL-28 has been shown to express the novel enzyme, l-ribose isomerase (AcL-RbI), which catalyzes reversible isomerization between l-ribose and l-ribulose. AcL-RbI showed the highest activity to l-ribose, followed by d-lyxose with 47% activity, and had no significant amino acid sequence similarity to structure-known proteins, except for weak homology with the d-lyxose isomerases from Escherichia coli O157 : H7 (18%) and Bacillus subtilis strain (19%). Thus, AcL-RbI is expected to have the unique three-dimensional structure to recognize l-ribose as its ideal substrate. The X-ray structures of AcL-RbI in complexes with substrates were determined. AcL-RbI had a cupin-type β-barrel structure, and the catalytic site was found between two large β-sheets with a bound metal ion. The catalytic site structures clearly showed that AcL-RbI adopted a cis-enediol intermediate mechanism for the isomerization reaction using two glutamate residues (Glu113 and Glu204) as acid/base catalysts. In its crystal form, AcL-RbI formed a unique homotetramer with many substrate sub-binding sites, which likely facilitated capture of the substrate. Database The atomic coordinates and structure factors of AcL-RbI/l-ribose, AcL-RbI/l-ribulose, AcL-RbI/ribitol, E204Q/l-ribose and E204Q/l-ribulose have been deposited in the Protein Data Bank under accession codes, 4Q0P, 4Q0Q, 4Q0S, 4Q0U and 4Q0V. Structured digital abstract • AcL-RbI and AcL-RbI bind by x-ray crystallography (View interaction).

Only D-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to D-allose. D-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-D-allose, a structural derivative of D-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding D-allose kinase to increase D-allose 6-phosphate synthesis were more sensitive to D-allose, but E. coli AlsI encoding D-allose 6-phosphate isomerase expression to decrease D-allose 6-phosphate reduced sensitivity. A D-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, D-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of D-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of D-allose to D-allose 6-phosphate, and treatment with D-allose might prove to be useful for reducing disease development in rice.

L-Ribulose 3-epimerase (L-RE) fromMesorhizobium lotihas been identified as the first ketose 3-epimerase that shows the highest observed activity towards ketopentoses. In the present study, the crystal structure of the enzyme was determined to 2.7 Å resolution. The asymmetric unit contained two homotetramers with the monomer folded into an (α/β)8-barrel carrying four additional short α-helices. The overall structure ofM. lotiL-RE showed significant similarity to the structures of ketose 3-epimerases fromPseudomonas cichorii,Agrobacterium tumefaciensandClostridium cellulolyticum, which use ketohexoses as preferred substrates. However, the size of the C-terminal helix (α8) was much larger inM. lotiL-RE than the corresponding helices in the other enzymes. InM. lotiL-RE theα8 helix and the following C-terminal tail possessed a unique subunit–subunit interface which promoted the formation of additional intermolecular interactions and strengthened the enzyme stability. Structural comparisons revealed that the relatively small hydrophobic pocket of the enzyme around the substrate was likely to be the main factor responsible for the marked specificity for ketopentoses shown byM. lotiL-RE.

A general and efficient method has been developed for the synthesis of sugar-derived azepane nitrones starting from aldohexoses, with an intramolecular condensation of aldehyde and hydroxylamine as the key step. Through this strategy, each aldohexose produced a pair of azepane nitrones, which are precursors of various azepane iminosugars.

We previously reported that a rare sugar D-allose, which is the D-glucose epimer at C3, inhibits the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half seeds in rice (Fukumoto et al. 2011). D-Allose suppresses expressions of gibberellin-responsive genes downstream of SLR1 protein in the gibberellin-signaling through hexokinase (HXK)-dependent pathway. In this study, we discovered that D-allose induced expression of ABA-related genes including OsNCED1-3 and OsABA8ox1-3 in rice. Interestingly, D-allose also up-regulated expression of OsABF1, encoding a conserved bZIP transcription factor in ABA signaling, in rice. The D-allose-induced expression of OsABF1 was diminished by a hexokinase inhibitor, D-mannoheptulose (MNH). Consistently, D-allose also inhibited Arabidopsis growth, but failed to trigger growth retardation in the glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1. D-Allose activated AtABI5 expression in transgenic gin2 over-expressing wild-type AtHXK1 but not in gin2 over-expressing the catalytic mutant AtHXK1(S177A), indicating that the D-allose phosphorylation by HXK to D-allose 6-phosphate (A6P) is the first step for the up-regulation of AtABI5 gene expression as well as D-allose-induced growth inhibition. Moreover, overexpression of OsABF1 showed increased sensitivity to D-allose in rice. These findings indicated that the phosphorylation of D-allose at C6 by hexokinase is essential and OsABF1 is involved in the signal transduction for D-allose-induced growth inhibition.

l-Rhamnose isomerase (l-RhI) catalyzes the reversible isomerization of l-rhamnose to l-rhamnulose. Previously determined X-ray structures of l-RhI showed a hydride-shift mechanism for the isomerization of substrates in a linear form, but the mechanism for opening of the sugar-ring is still unclear. To elucidate this mechanism, we determined X-ray structures of a mutant l-RhI in complex with l-rhamnopyranose and d-allopyranose. Results suggest that a catalytic water molecule, which acts as an acid/base catalyst in the isomerization reaction, is likely to be involved in pyranose-ring opening, and that a newly found substrate sub-binding site in the vicinity of the catalytic site may recognize different anomers of substrates., Highlights: ▸ l-Rhamnose isomerase catalyzes the reversible isomerization of l-rhamnose to l-rhamnulose. ▸ The catalytic reaction includes opening of the sugar-ring, but the mechanism is unclear. ▸ The structure of the enzyme with substrates in a pyranose ring form was determined. ▸ A catalytic water molecule is probably involved in opening the pyranose ring. ▸ A newly found substrate sub-binding site recognizes the anomers of substrates.

A newly isolated bacterium, Cellulomonas parahominis MB426, produced l -ribose isomerase (CeLRI) on a medium containing l -ribose as a sole carbon source. A 32 kDa protein isomerizing l -ribose to l -ribulose was purified to homogeneity from this bacterium. A set of degenerated primers were synthesized based on amino acid sequences of the purified CeLRI, and a 747 bp gene encoding CeLRI was cloned, sequenced and overexpressed in Escherichia coli . This gene encoded a 249 amino acid protein with a calculated molecular mass of 27,435. The deduced amino acid sequence of this gene showed the highest identity with l -ribose isomerase from Acinetobacter calcoaceticus DL-28 (71%). The recombinant l -ribose isomerase (rCeLRI) was optimally active at pH 9.0 and 40°C, and was stable up to 40°C for 1 h and not dependent for metallic ions for its activity. The rCeLRI showed widely substrate specificity for the rare sugar which involved l -erythro form such as l -ribose, d -lyxose, d -talose, d -mannose, l -gulose, and l -allose.

The enantiomers of tagatose are converted to L-DGJ [a noncompetitive inhibitor of human lysosome α-galactosidase A (α-Gal A), K(i) 38.5 μM] and DGJ [a competitive inhibitor of α-Gal A, K(i) 15.1 nM] in 66% yield. L-DGJ and DGJ provide the first examples of pharmacological chaperones that (a) are enantiomeric iminosugars and (b) have synergistic activity with implications for the treatment of lysosomal storage disorders and other protein deficiencies.

l -Tagatose was produced directly from l -psicose by subjecting the same biomass suspension to microbial reduction followed by oxidation using a newly isolated bacteria Enterobacter aerogenes 230S. After various optimizations, it was observed that cells grown on xylitol have the best conversion potential. Moreover, E. aerogenes 230S converted l -psicose to l -tagatose at a faster rate in the presence of polyols such as glycerol, d -sorbitol, ribitol, l -arabitol, d -mannitol and xylitol. At 5% substrate concentration, the conversion ratio of l -psicose to l -tagatose was above 60% in the presence of glycerol. Identity of crystalline l -tagatose was confirmed by HPLC analysis, 13 C-NMR spectra, and optical rotation.

1-De-oxy-d-galactitol, C(6)H(14)O(5), exists in the crystalline form as hydrogen-bonded layers of mol-ecules running parallel to the ac plane, with each mol-ecule acting as a donor and acceptor of five hydrogen bonds.

X-ray crystallography showed that the title compound, C(6)H(12)O(5), crystallizes in the α-pyran-ose form with the six-membered ring in a chair conformation. The crystal structure exists as a three-dimensional hydrogen-bonded network of mol-ecules with each mol-ecule acting as a donor and aceptor for four hydrogen bonds. The absolute configuration was determined by the use of l-fucose as starting material.