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The galectins are a family of β-galactoside-specific animal lectins, and have attracted much attention as novel regulators of the immune system. Galectin-10 is well-expressed in eosinophils, and spontaneously forms Charcot-Leyden crystals (CLCs), during prolonged eosinophilic inflammatory reactions, which are frequently observed in eosinophilic diseases. Although biochemical and structural characterizations of galectin-10 have been done, its biological role and molecular mechanism are still unclear, and few X-ray structures of galectin-10 in complex with monosaccharides/oligosaccharides have been reported. Here, X-ray structures of galectin-10 in complexes with seven monosaccharides are presented with biochemical analyses to detect interactions of galectin-10 with monosaccharides/oligosaccharides. Galectin-10 forms a homo-dimer in the face-to-face orientation, and the monosaccharides bind to the carbohydrate recognition site composed of amino acid residues from two galectin-10 molecules of dimers, suggesting that galectin-10 dimer likely captures the monosaccharides in solution and in vivo. d -Glucose, d -allose, d -arabinose, and D-N-acetylgalactosamine bind to the interfaces between galectin-10 dimers in crystals, and they affect the stability of molecular packing in crystals, leading to easy-dissolving of CLCs, and/or inhibiting the formation of CLCs. These monosaccharides may serve as effectors of G10 to form CLCs in vivo.

The nematocidal activities of the fatty acid esters of d-allose were examined using the larvae of C. elegans. Among the fatty acid esters, 6-O-octanoyl-d-allose (3) showed significant activity. 6-O-octanoyl-d-glucose (5) showed no activity, indicating that the D-allose moiety is essential for the nematocidal activity of 3. A nonhydrolyzable alkoxy analog 6-O-octyl-d-allose (6) also showed activity equivalent to that of 3.

In this study, gum of Araucaria heterophylla was collected. The collected gum was subjected for extraction of polysaccharide using solvent extraction system. Thus, extracted polysaccharide was further purified using solvent method and was characterized using UV-Vis spectroscopy, Phenol sulfuric acid assay, FTIR, TGA, TLC and GC-MS. The gum derived polysaccharide was found to have the following sugars Rhamnose, Allose, Glucosinolate, Threose, Idosan, Galactose and Arabinose. The extracted polysaccharide was tested for various in-vitro bioactive studies such as antibacterial activity, antioxidant activity and anticancer activity. The polysaccharide was found to have antioxidant and anticancer activity. Further, the polysaccharide was subjected for carboxymethylation to favor the nanocarrier synthesis, where it was chelated using Sodium Tri Meta Phosphate (STMP) to form nanocarriers. The nanocarriers so formed were loaded with curcumin and were characterized using FTIR, SEM, EDX and AFM. Both the loaded and unloaded nanocarriers were studied for its in-vitro cytotoxic effect against the MCF7 human breast cancer cell lines. The nanocarriers were found to deliver the drug efficiently against the cancer cell line used in this study.

A linear tetramer of β-(1 → 6)-linked 3-azido-3-deoxy- d -allose containing glycosyl donor and glycosyl acceptor functions in the terminal monosaccharide units was prepared starting from 3-azido-3-deoxy-1,2:5,6-di-O-isopropylidene-α- d -allofuranose. Cyclization of the linear tetramer under glycosylation conditions afforded the corresponding cyclic tetrasaccharide in 77% yield; its deprotection and reduction of the azido groups resulted in the formation of the cyclic tetramer of 3-amino-3-deoxy- d -allose with axial amino groups, a potential scaffold for the synthesis of tetravalent functional clusters.

1,2,6-Trideoxy-6-amido-d-allose derivative was synthesized and found to exhibit higher growth-inhibitory activity against plants than the corresponding deoxy-d-allose ester, which indicates that an amide group at C-6 of the deoxy-d-allose amide enhances inhibitory activity. In addition, the mode of action of the deoxy-d-allose amide was significantly different from that of d-allose which inhibits gibberellin signaling. Co-addition of gibberellin GA3 restored the growth of rice seedlings inhibited by the deoxy-d-allose amide, suggesting that it might inhibit biosynthesis of gibberellins in plants to induce growth inhibition.

A rapid method for the quantitation of sixteen neutral and acidic monosaccharides, from both animal and plant sources was developed using ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC/QqQ-MS) in dynamic multiple reaction monitoring (dMRM) mode. Monosaccharides including three pentoses (ribose, xylose, arabinose), two deoxyhexoses (rhamnose, fucose), five hexoses (fructose, mannose, allose, glucose, galactose), two hexuronic acids (glucuronic acid, galacturonic acid), and two N-acetyl-hexosamines (GlcNAc, GalNAc), were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP), while underivatized sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), were simultaneously analyzed with a 10-minute run. With the optimized UHPLC conditions, baseline separations of the isomers were achieved. The sensitivity and calibration ranges of this method were determined. The limits of detection were between femtomoles and attomoles with linear ranges spanning four to six orders of magnitude and CVs ≤ 7.2%. Spiking experiments performed on a pooled fecal sample demonstrated the high accuracy of this method even when applied to samples with complicated matrices. The validated method was applied to fecal samples from an infant transitioning from breast milk to weaning foods. Major milk monosaccharides including galactose, fucose, glucose, GlcNAc, and Neu5Ac were found to be the most abundant components in the feces of milk-fed infants. PMP-derivatives of other monosaccharides including apiose, lyxose, altrose, talose, gulose, glucosamine, galactosamine, mannosamine, and N-acetylmannosamine (ManNAc) were also tested and could be added to the quantitation method depending on the need. The speed and sensitivity of the method makes it readily adaptable to rapid throughput analysis of monosaccharides in biological samples.

d-Allose is the C3 epimer of d-glucose and has been reported to have beneficial health effects. The transporter mediating intestinal transport of d-allose is unknown. We examined whether d-allose is absorbed via sodium-dependent glucose cotransporter 1 (SGLT1) as well as via glucose transporter type 5 (GLUT5) using rats. For examination of absorption via SGLT1, KGA-2727, an SGLT1-specific inhibitor, and d-allose were orally administered. KGA-2727 blocked the increase of plasma d-allose levels and suppressed them throughout the experiment (0–180 min), whereas without KGA-2727, the plasma d-allose levels peaked at around 60–90 min. For examination of absorption via GLUT5, rats were fed a high-fructose diet for 3weeks to increase the abundance and activity of GLUT5 in the small intestine. High-fructose diet-fed rats did not exhibit significant changes in the plasma d-allose levels compared to control rats fed a high-glucose diet. These results indicate that SGLT1 but not GLUT5 mediates the intestinal absorption of d-allose., Highlights ● Plasma d-allose levels were blocked by KGA-2727, an SGLT1-specific inhibitor. ● Fructose-fed rats which highly express intestinal GLUT5 did not exhibit significant changes in the plasma d-allose levels. ● SGLT1 clearly mediates intestinal d-allose transport.

d -Allose was considered as a kind of rare sugars with testified potential medicinal and agricultural benefits. l -Rhamnose isomerase (L-RI, EC 5.3.1.14), an aldose-ketose isomerase, played a significant part in producing rare sugar. In this article, a thermostable d -allose-producing L-RI was characterized from a thermotolerant bacterium, Thermobacillus composti KWC4. The recombinant L-RI was activated obviously in the presence of Mn2+ with an optimal pH 7.5 and temperature 65 °C. The Michaelis-Menten constant (Km), turnover number (kcat) and catalytic efficiency (kcat/Km) for l -rhamnose were 33.8 mM, 1189.8 min−1 and 35.2 min−1 mM−1, respectively. At a higher temperature, Mn2+ played a pivotal role in strengthening the thermostability of T. composti L-RI. The differential scanning calorimetry (DSC) results showed the denaturing temperature (Tm) of T. composti L-RI was increased by 3 °C in presence of Mn2+. Although the T. composti L-RI displayed the optimum substrate as l -rhamnose, it could also effectively catalyze the isomerization between d -allulose and d -allose. When the reaction reached equilibrium, the sole product d -allose was produced from D-alluose by T. composti L-RI.

d-Allose, a C-3 epimer of d-glucose, is a naturally occurring rare monosaccharide that shows anti-proliferative activity against several human cancer cell lines. However, d-allose requires a relatively high concentration for the activity to be observed. Thus, developing more potent derivatives is needed for application. In cells, d-allose is converted to d-allose-6-phosphate (A6P), which is responsible for the anti-proliferative activity of d-allose. In this study, we synthesized A6P derivative 1 with biodegradable protecting groups, which showed higher anti-proliferative activity than A6P against a MOLT-4F human leukemia cell line. Similarly protected derivative of d-glucose-6-phosphate (G6P) (2) and tetraacetyl-A6P (3) showed weaker and less activity compared with 1, respectively, suggesting that both A6P moiety and the protecting group on the phosphate group are responsible for the activity. In addition, significantly weaker induction of thioredoxin-interacting protein (TXNIP) expression by 1 compared with d-allose suggests that 1 exhibited cytotoxicity through the synergetic effect of inducing TXNIP expression and other mechanisms.

Interactions between proteins and carbohydrates are ubiquitous in biology. Therefore, understanding the factors that determine their affinity and selectivity are correspondingly important. Herein, we have determined the relative strengths of intramolecular interactions between a series of monosaccharides and an aromatic ring close to the glycosylation site in an N-glycoprotein host. We employed the enhanced aromatic sequon, a structural motif found in the reverse turns of some N-glycoproteins, to facilitate face-to-face monosaccharide–aromatic interactions. A protein host was used because the dependence of the folding energetics on the identity of the monosaccharide can be accurately measured to assess the strength of the carbohydrate–aromatic interaction. Our data demonstrate that the carbohydrate–aromatic interaction strengths are moderately affected by changes in the stereochemistry and identity of the substituents on the pyranose rings of the sugars. Galactose seems to make the weakest and allose the strongest sugar–aromatic interactions, with glucose, N-acetylglucosamine (GlcNAc) and mannose in between. The NMR solution structures of several of the monosaccharide-containing N-glycoproteins were solved to further understand the origins of the similarities and differences between the monosaccharide–aromatic interaction energies. Peracetylation of the monosaccharides substantially increases the strength of the sugar–aromatic interaction in the context of our N-glycoprotein host. Finally, we discuss our results in light of recent literature regarding the contribution of electrostatics to CH–π interactions and speculate on what our observations imply about the absolute conservation of GlcNAc as the monosaccharide through which N-linked glycans are attached to glycoproteins in eukaryotes.

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Abnormal sialylation of proteins is highly associated with many major diseases, such as cancers and neurodegenerative diseases. However, this study is challenging owing to the difficulty in enriching trace sialylated glycopeptides (SGs) from highly complex biosamples. The key to solving this problem relies strongly on the design of novel SG receptors to capture the sialic acid (SA) moieties in a specific and tunable manner. Inspired by the saccharide-saccharide interactions in life systems, here we introduce saccharide-based SG receptors into this study. Allose (a monosaccharide) displays specific and pH-sensitive binding toward SAs. Integrating allose units into a polyacrylamide chain generates a saccharide-responsive smart copolymer (SRSC). Such design significantly improves the selectivity of SA binding; meanwhile, this binding can be intelligently triggered in a large extent by solution polarity and pH. As a result, SRSC exhibits high-performance enrichment capacity toward SGs, even under 500-fold interference of bovine serum albumins digests, which is notably higher than conventional materials. In real biosamples of HeLa cell lysates, 180 sialylated glycosylation sites (SGSs) have been identified using SRSC. This is apparently superior to those obtained by SA-binding lectins including WGA (18 SGSs) and SNA (22 SGSs). Furthermore, lactose displays good chemoselectivity toward diverse disaccharides, which indicated the good potential of lactose-based material in glycan discrimination. Subsequently, the lactose-based SRSC facilitates the stepwise isolation of O-linked or N-linked SGs with the same peptide sequence but varied glycans by CH3CN/H2O gradients. This study opens a new avenue for next generation of glycopeptide enrichment materials.

We describe the syntheses of three different deoxy-D-allose analogs [2-deoxy-D-allose (2-DOAll), 1,2-dideoxy-D-allose (1,2-DOAll), and 1,2-didehydro-1,2-dideoxy-D-allose (1,2-DHAll)] and their fatty acid esters via regioselective lipase-catalyzed transesterification. Among them, 2-DOAll and its decanoate (2-DOAll-C10) showed higher inhibitory activity on plant growth, which is similar to D-allose (All) and its decanoate (All-C10). Bioassay results of deoxy-All-C10 on four plant species suggest that the hydroxy group at the C-1 position might be important showing growth inhibitory activity. In addition, co-addition of gibberellin (GA3) with 1,2-DHAll-C10 and 2-DOAll-C10 recovered plant growth, suggesting that they might mainly inhibit biosynthesis of gibberellin.

d -allose is one kind of rare sugar and its biological functions and metabolic pathways in several organisms have been reported, but its function in plants’ resistance has not been well understood. In this research, we analyzed the function of d -allose in plant immunity in tomato. The plants which were pretreated with 5 mM d -allose increased the resistance to Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 compared with the plants treated with water, with lighter symptom. The d -allose treatment induced the resistance to B. cinerea and Pst DC3000 maybe through regulating reactive oxygen species (ROS) accumulation and the expression levels of defense-related genes.

The anti-proliferative activities of the 6-O-acyl derivatives of d-allose against the human leukemia MOLT-4F cell line were examined. The activity of the 6-O-dodecanoyl derivative (3) was approximately 30 times stronger than that of d-allose. An evaluation of the derivatives of 3 that occurred in a furanose form revealed the pyranose forms of 3 to be important for the anti-proliferative activity.

Rare sugars are defined as monosaccharides that exist in nature but are only present in limited quantities. d-Allose is a rare sugar that has been reported to have some unique physiological effects. The present study describes suitable synthetic procedures for novel rare sugars of d-allose that are 18F-labeled at the C-3 and C-6 positions and the preparation of the appropriate labeling precursors. The goal is to facilitate in vivo, noninvasive positron emission tomography (PET) investigation of the behavior of rare sugar analogs of d-allose in organs. We found five precursors that were practical for labeling, three for 3-deoxy-3-[18F]fluoro-d-allose ([18F]3FDA) and two for 6-deoxy-6-[18F]fluoro-d-allose ([18F]6FDA). With manual operation synthesis, the highest radiochemical conversion rates were 75% for [18F]3FDA with a precursor of 1,2,4,6-tetra-O-acetyl-3-O-trifluoromethanesulfonyl-β-d-glucopyranose and 69% for [18F]6FDA with a precursor of 1,2,3,4-tetra-O-acetyl-6-O-trifluoromethanesulfonyl-β-d-allopyranose. Furthermore, the practical yields of [18F]3FDA and [18F]6FDA using an automated synthesizer were also investigated. Radiochemical yields of 67% and 49% were obtained for [18F]3FDA and [18F]6FDA, respectively, in an automated synthesizer. As basic assessment of stability for use in PET scanning, high performance liquid chromatography analysis showed no decomposition of [18F]3FDA and [18F]6FDA after up to 6 h in rabbit blood plasma.

Various glycosyl hydrolase enzymes of the clan GH-H (http:// www.cazy.org/Glycoside-Hydrolases.html) catalyse transglycosylation reactions, mostly by using starch (e.g. , CGTase, family GH13) or sucrose (e.g. , glucansucrase, family GH70) as the donor substrate, thus transferring glucose. Some alternative biocatalysts for glycosyl transfer reactions have also been discovered; these include glycosynthases and glycosyltransferases. In recent years we have introduced the concept of sucrose analogues. Sucrose analogues such as a-d-glycopyranosyl bd-fructofuranosides have been reported as promising tools for the synthesis of functional fructo-oligosaccharides. In recent review articles we have suggested that these analogues can be used as glycopyranosyl donors for artificial transglycosylation reactions, which are not performed in nature. 9] Sucrose analogues are readily available from the cheap and abundant starting material sucrose. They are obtained through a transfructosylation reaction catalysed by levansucrase from Bacillus subtilis or Bacillus megaterium, by transferring fructose from sucrose to mannose, galactose, xylose or fucose. a-d-Allopyranosyl b-d-fructofuranoside (1, All-Fru, Scheme 1) is an allose that contains a sucrose analogue derived from sucrose by dehydrogenisation to 3-ketosucrose and subsequent hydrogenation to All-Fru. This synthesis has been established at an industrial scale. Allose is a rare sugar in nature; only a few plant species, for example, Passiflora edulis, Sideritis grandiflora and Protea rubropilosa, are known to contain glycoconjugates with blinked allose residues. Allose and allosylated glycoconjugates show promising properties for pharmaceutical use in cancer therapy, in immune suppression, for inhibition of neutrophil production and as antioxidants. To the best of our knowledge no a-d-allosyltransferase is known, however, research by Thorson and co-workers indicated a possible b-allosyl transfer performed by the glycosyltransferase OleD enzyme (family GT1). In recent work, we sought to identify enzymes able to transfer d-allose from the sucrose analogue All-Fru as the donor substrate. For this, All-Fru was incubated with various enzymes from the glycosyl hydrolase clan GH-H. As this sucrose analogue is a-linked and enzymes of the GH-H clan generally retain the configuration at the anomeric centre, the formed products should exhibit a-linkage. The sucrose isomerase (SI, GH13) from Protaminobacter rubrum showed no activity towards All-Fru. In contrast, incubation of All-Fru (292 mm) with amylosucrase (NpAs, GH13) from Neisseria polysaccharaea at pH 6.6 and 30 8C showed slow hydrolysis : even after 80 h, consumption of All-Fru was incomplete. Glucansucrases from Lactobacillus reuteri strains 121 (GTFA) and 180 (GTF180), and GTFR from Streptococcus oralis (all GH70) were able to hydrolyse 292 mm All-Fru. As hydrolysis activity on All-Fru was highest for GTFA (complete hydrolysis after 80 h), this enzyme was used in further studies. The glucansucrase GTFA from L. reuteri 121 is an extracellular enzyme that produces the polymer reuteran (mainly a(1!4)-linked glucan, with minor a(1!6) and branches) from sucrose. In contrast, with sucrose as donor, no formation of allose oligoor polysaccharides was observed by TLC. The transferase activity of GTFA-DN-CHis (a N-truncated version was used in all experiments) with All-Fru was observed with several acceptor substrates (note: concentration depended on solubility in the solvent system), thus obtaining a variety of functionalities (Table 1) such as sugars (methyl a-d-glucopyranoside (a-d-Me-Glcp, 2) and methyl 6-O-p-toluenesulfonyl-ad-glucopyranoside (a-d-Me-6-Ts-Glcp, 4)), amino acids (e.g. , N-(tert-butoxycarbonyl)-d-serine methyl ester (N-Boc-d-serineOMe, 6)) and ( )-epicatechin (8). Incubating GTFA with All-Fru (292 mm) and a-d-Me-Glcp (772 mm, 2) as the acceptor resulted in methyl a-d-allopyranosyl-(1!4)-a-d-glucopyranoside (3) as the main product (50% yield). The a-configuration of the glycosidic bond was determined by H NMR (H-1’: d= Scheme 1. The sucrose analogue All-Fru.

Ultrasound-assisted extraction was employed to extract polysaccharide from Virginia (flue-cured) tobacco flowers. The orthogonal matrix method (L9(3)(4)) was used to determine the optimal extraction conditions as to ultrasound power, extraction time, ratio of solvent to solid, and extraction temperature at 300 W, 4 min, 35 (mL/g), and 70 °C respectively. The crude extract was successively purified by chromatography, yielding two major polysaccharide fractions, termed Fr-I and Fr-II. Both fractions are heteropolysaccharides, mainly containing glucose, mannose, and allose with an a-configuration. Thermo gravimetric analysis (TGA) indicated that the degradation temperatures (Td) of Fr-I and Fr-II were 185 °C and 190 °C respectively. The preliminary antioxidant activity test in vitro showed both fractions could potentialize the scavenging effect on hydroxyl and DPPH radicals in a dose-dependent manner. In conclusion, the two polysaccharides may be useful as naturally potential antioxidant agents for application in food and medicinal fields.

D-allose has attracted a great deal of attention in recent years due to its many pharmaceutical activities, which include anti-cancer, anti-tumor, anti-inflammatory, anti-oxidative, anti-hypertensive, cryoprotective, and immunosuppressant activities. D-allose has been produced from D-psicose using D-allose-producing enzymes, including L-rhamnose isomerase, ribose-5-phosphate isomerase, and galactose-6-phosphate isomerase. In this article, the properties, applications, and metabolism of D-allose are described, and the biochemical properties of D-allose-producing enzymes and their D-allose production are reviewed and compared. Moreover, several methods for effective D-allose production are suggested herein.

Ribose-5-phosphate isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k cat/K m) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.

Uridine diphosphogalactose-4-epimerase (UDP-galactose-4-epimerase, GalE, EC 5.1.3.2) mediates the 4-epimerization of nucleic acid-activated galactose into UDP-glucose. To date, no enzyme is known to mediate 4-epimerization of free monosaccharide substrates. To determine the potential activity of GalE for free monosaccharide, Escherichia coli GalE was expressed and purified using Ni-affinity chromatography, and its ability to mediate 4-epimerization of a variety of free keto- and aldohexoses was assessed. Purified GalE was found to possess 4-epimerization activity for free galactose, glucose, fructose, tagatose, psicose, and sorbose at 0.47, 0.31, 2.82, 9.67, 15.44, and 2.08 nmol/mg protein per minute, respectively. No 4-epimerization activity was found for allose, gulose, altrose, idose, mannose, and talose. The kinetic parameters of 4-epimerization reactions were K m = 26.4 mM and k cat = 0.0155 min−1 for d-galactose and K m = 237 mM and k cat = 0.327 min−1 for d-tagatose. The 4-epimerization of tagatose, a reaction of commercial interest, was enhanced twofold (19.79 nmol/mg protein per minute) when asparagine was exchanged with serine at position 179. The novel activity of GalE for free monosaccharide may be beneficial for the production of rare sugars using cheap natural resources. Potential strategies for developing enhanced GalE with increased 4-epimerization activity are discussed in the context of the above findings and an analysis of a 3D structural model.

D-allose binding protein (ALBP) belongs to the family of perisplamic receptors of the bacterial ABC transporter system. ALBP experiences a significant conformational rearrangement upon binding to the sugar. Here, we report the sequential backbone assignment for the ALBP from Escherichia coli in the free form (BMRB no. 16982) and in complex with D-allose (BMRB no. 16984).

The alpha-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic alpha-galactosidases and alpha-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g.L(-1) culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with alpha-(1-->6) regioselectivity from 40 mm 4-nitrophenol alpha-d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenol alpha-D-Galp-(1-->6)-D-Galp, alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp and alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp-(alpha1-->beta2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol alpha-D-galactopyranoside (40 mm), alpha-(1-->6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39-58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, L-arabinose, L-fucose and L-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and L-rhamnose. Structural modelling using Thermotoga maritima GH36 alpha-galactosidase as the template and superimposition of melibiose from the complex with human GH27 alpha-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred alpha-galactosyl to 6-OH of the terminal residue in the alpha-linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the beta-linked lactose, lactulose and cellobiose in 28-38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. alpha-D-Galp-(1-->6)-D-Manp; alpha-D-Galp-(1-->6)-beta-D-Glcp-(1-->4)-D-Glcp; alpha-D-Galp-(1-->6)-beta-D-Galp-(1-->4)-D-Fruf; alpha-D-Galp-(1-->6)-D-Glcp-(alpha1-->alpha1)-D-Glcp; and alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->3)-D-Fruf.

The main functions of glycosylation are stabi-lization, detoxification and solubilization of substrates andproducts. To produce glycosylated products, Escherichia coliwas engineered by overexpression of TDP- L -rhamnose andTDP-6-deoxy- D -allose biosynthetic gene clusters, and flavo-noids were glycosylated by the overexpression of the glyco-syltransferase gene from Arabidopsis thaliana. For theglycosylation, these flavonoids (quercetin and kaempferol)were exogenously fed to the host in a biotransformationsystem. The productswere isolated,analyzed and confirmedby HPLC, LC/MS, and ESI-MS/MS analyses. Several con-ditions (arabinose, IPTG concentration, OD 600 , substrateconcentration, incubation time) were optimized to increasetheproductionlevel.Wesuccessfullyisolatedapproximately24mg/L 3-O-rhamnosyl quercetin and 12.9mg/L 3-O-rhamnosyl kaempferol upon feeding of 0.2mM of therespective flavonoids and were also able to isolate 3-O-allosyl quercetin. Thus, this study reveals a method thatmightbeusefulforthebiosynthesisofrhamnosylandallosylflavonoids and for the glycosylation of related compounds.Biotechnol. Bioeng. 2010;107: 154–162. 2010 Wiley Periodicals, Inc.KEYWORDS: engineered E. coli; flavonoids; glycosyltrans-ferase; TDP

The activity of ribose-5-phosphate isomerases (RpiB) from Clostridium difficile for D-ribose isomerization was optimal at pH 7.5 and 40 degrees C, while that from Thermotoga maritima for L-talose isomerization was optimal at pH 8.0 and 70 degrees C. C. difficile RpiB exhibited activity only with aldose substrates possessing hydroxyl groups oriented in the right-handed configuration (Fischer projections) at the C2 and C3 positions, such as D-ribose, D-allose, L-talose, L-lyxose, D-gulose, and L-mannose. In contrast, T. maritima RpiB displayed activity only with aldose substrates possessing hydroxyl groups configured the same direction at the C2, C3, and C4 positions, such as the D- and L-forms of ribose, talose, and allose.

In the course of our current studies on the reactivity of alkylidenecarbenes, prepared with the trimethylsilylazide/Bu2SnO method, on conveniently functionalized α-cyanomesylates derived from d -allose, d -arabinose, and d -threose, we have observed the unexpected, but mechanistically interesting formation of enantiomerically β-substituted polyhydroxylated furans. These compounds are the result of a series of cascade fragmentation reactions on unstable intermediates obtained during 1,5C–H insertion reactions from alkylidenecarbenes. The mechanism of the reaction has been investigated by computational methods using DFT analysis.

This study reports syntheses of d-allose 6-phosphate (All6P), D-allulose (or D-psicose) 6-phosphate (Allu6P), and seven D-ribose 5-phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichiacoli (EcRpiB). 5-Phospho-D-ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenophospho-D-ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step. Kinetic evaluation on Allpi activity of EcRpiB shows that two of these compounds, 5-phospho-D-ribonohydroxamic acid and N-(5-phospho-D-ribonoyl)-methylamine, indeed behave as new efficient inhibitors of EcRpiB; further, 5-phospho-D-ribonohydroxamic acid was demonstrated to have competitive inhibition. Kinetic evaluation on Rpi activity of both EcRpiB and RpiB from Mycobacterium tuberculosis (MtRpiB) shows that several of the designed 6-carbon high-energy intermediate analogues are new competitive inhibitors of both RpiBs. One of them, 5-phospho-D-ribonate, not only appears as the strongest competitive inhibitor of a Rpi ever reported in the literature, with a K(i) value of 9 microM for MtRpiB, but also displays specific inhibition of MtRpiB versus EcRpiB.

An unexpected and interesting intramolecular side reaction occurred during the attempted synthesis of glycosyl cyanides upon treatment of 1-O-acetyl-3-azido-3-deoxyallose derivatives with TMSCN and different Lewis acids. Exo-1,2-O-cyanoalkylidene derivatives formed by neighboring group participation and attack of cyanide underwent, after Lewis-acid mediated isomerization to the endo-isomer, intramolecular azide cyanide cycloaddition leading to the formation of tetrazoles embedded in bridged tetracyclic ring systems. The efficiency of cycloaddition is dependent on the ring structure of the sugar (pyranose or furanose). Of the studied molecules, 3-azido-1,2-O-cyanoethylidene-3-deoxy-allopyranose provides the most suitable scaffold for intramolecular [2+3] cycloaddition under exceptionally mild conditions. Our results highlight the capability of carbohydrates to act as scaffolds for the precise positioning of functional groups productive for a specific chemical reaction.

Glycoside hydrolase family 31 (GH 31) is one of the most intriguing glycoside hydrolase families. This family contains α-glucosidase, α-xylosidase, α-glucan lyase and isomaltosyltransferase. Escherichia coli YicI (α-xylosidase) is a representative enzyme of GH 31 because its biochemical and structural studies have been thoroughly carried out. YicI is a strict α-xylosidase, which rigidly recognizes α-xyloside at the non-reducing terminal end, even though its amino acid sequence apparently displays similarity with α-glucosidases. Phe277, Cys307, Trp345 and Lys414 at the subsite-1 are important for α-xylosidase activity. The mutant YicI enzymes, which possesses Ile307/Asp308 instead of Cys307/Phe308 and which has a shorter β→α loop 1 of (β/α)8 barrel in place of the original longer loop, respectively, possess α-glucosidase activity. In the transxylosylation of YicI, glucose, mannose and allose are able to act as acceptors, but galactose, talose and gulose never do, implying that equatorial OH-4 of the aldopyranose is crucial for acting as an acceptor. YicI transfers α-xylosyl moieties to a specific hydroxy group in the acceptor sugar (except fructopyranose) showing 1,6 regioselectivity, which is in agreement with the structural feature of the aglycone-biding site. Among the transxylosylation products of YicI, α-D-xylopyranosyl-(1→6)-D-mannopyranose, α-D-xylopyranosyl-(1→6)-D-fructofuranose, and α-D-xylopyranosyl-(1→3)-D-fructopyranose are novel sugars. α-D-Xylopyranosyl-(1→6)-D-mannopyranose and α-D-xylopyranosyl-(1→6)-D-fructofuranose have the ability to inhibit rat intestinal α-glucosidases.

The specificity of the aglycone-binding site of Escherichia coliα-xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation-catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and α-xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl–enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone-binding site. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4-OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone-binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, α-d-Xylp-(16)-d-Manp, α-d-Xylp-(16)-d-Fruf, and α-d-Xylp-(13)-d-Frup, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello-oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by α-1,6-xylosidic linkages. Of the transxylosylation products, α-d-Xylp-(16)-d-Manp and α-d-Xylp-(16)-d-Fruf inhibited intestinal α-glucosidases.

We purified recombinant galactose 6-phosphate isomerase (LacAB) from Lactococcus lactis using HiTrap Q HP and Phenyl-Sepharose columns. The purified LacAB had a final specific activity of 1.79units/mg to produce d-allose. The molecular mass of native galactose 6-phosphate isomerase was estimated at 135.5kDa using Sephacryl S-300 gel filtration, and the enzyme exists as a hetero-octamer of LacA and LacB subunits. The activity of galactose 6-phosphate isomerase was maximal at pH 7.0 and 30 degrees C, and enzyme activity was independent of metal ions. When 100g/L of d-psicose was used as the substrate, 25g/L of d-allose and 13g/L of d-altrose were simultaneously produced at pH 7.0 and 30 degrees C after 12h of incubation. The enzyme had broad specificity for various aldoses and ketoses. The interconversion of sugars with the same configuration except at the C2 position was driven by using a large amount of enzyme in extended reactions. The interconversion occurred via two isomerization reactions, i.e., the interconversion of d-allose--d-psicose--d-altrose, and d-allose to d-psicose reaction was faster than d-altrose to d-psicose reaction.

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.

The present study evaluated an extracellular, novel biopolymer produced by Acinetobacter haemolyticus MG606 for its physicochemical properties and phosphate binding mechanism. The exobiopolymer (EBP) was characterized to be majorly polysaccharide in nature consisting of 48.9 kDa heteropolysaccharide composed of galactose, glucose, xylose, lyxose, allose, ribose, arabinose, mannose and fructose. Maximum phosphate binding efficiency of 25mg phosphate/g of EBP was described by Langmuir isotherm and further, the physicochemical and spectroscopic studies revealed that phosphate appeared to bind predominantly with the polysaccharide fraction, and to a relatively lesser extent to protein fraction of EBP. The electrostatic interactions with amino groups and ligand exchange with hydroxyl groups of EBP were found to be primary basis for phosphate binding mechanism. The results of this study implicate the feasibility of the EBP for commercial bioremediation processes.

The analysis of the polar fraction of Melittis melissophyllum L. subsp. melissophyllum led to the identification of several iridoid glycosides: monomelittoside (1), melittoside (2), harpagide (3), acetyl-harpagide (4) and ajugoside (5). Compounds 3 and 4 are considered marker compounds for the genus and, as well as compounds 1, 2 and 5, were already evidenced in a previous study on the nominal species. It was noteworthy of the presence of allobetonicoside (6) which was never reported for this genus. The isolation of 6 is very relevant because of its allose residue on the structure. Allose has been often found in the species of the subfamily Lamioideae even if it mostly regarded flavonoids considered of chemotaxonomical relevance for some correlated genera of Lamiaceae. Same as allosyl-glycosidic flavonoids, the presence of allosyl-glycosidic iridoids may also be an additional chemosystematic evidence of botanical relationships among Lamiaceae species and genera.

The fungal enzyme pyranose dehydrogenase (PDH) (EC 1.1.99.29) purified to apparent homogeneity from culture media of Agaricus meleagris catalyzed the substrate-dependent C-1, C-2, C-3, C-1,3′ or C-2,3(′) (di)oxidation of a number of mono- and disaccharides with 1,4-benzoquinone as an electron acceptor. d -Ribose, d -allose, d -gulose and d -talose were oxidized to the corresponding aldonic acids. l -Arabinose was converted exclusively to 2-dehydro- l -arabinose ( l -erythro-pentos-2-ulose) whereas d -xylose underwent competing C-2 and C-3 oxidation followed by dioxidation to 2,3-didehydro- d -arabinose ( d -glycero-pentos-2,3-diulose). The major final oxidation products of maltose and cellobiose were the novel compounds 2,3′-didehydromaltose and 2,3′-didehydrocellobiose (α- and β- d -ribo-hexos-3-ulopyranosyl-(1 → 4)- d -arabino-hexos-2-ulose), formed via 2- and 3′-monooxidation intermediates. Minor concomitant (di)oxidation at C-1,(3′) to the corresponding bionic acids also took place. Maltotriose was preferentially oxidized at C-3″ of the terminal glucopyranosyl unit and at C-1 of the reducing moiety. The structures of these sugar oxidation products were established by in situ 1D and 2D NMR spectroscopy and ESI–MS. Based on HPLC analysis, conversions of (glycosyl)aldoses in non-buffered systems were nearly quantitative within 3–24 h, depending on the substrate. As the enzyme allows an easy access to highly reactive di- or tricarbonyl sugars, it might become a useful catalyst in carbohydrate chemistry.

l -Rhamnose isomerase ( l -RhI) from Pseudomonas stutzeri LL172 can convert d -psicose to d -allose. Partially purified recombinant l -RhI from Escherichia coli was immobilized on BCW-2510 Chitopearl beads and utilized to produce d -allose. Total 20,000 units of immobilized enzyme converted d -psicose to d -allose without remarkable decrease in the enzyme activity over 17 days. When 50% d -psicose (w/w) was applied to a column with a flow rate of 0.8 ml/min at 42 °C, approximately 30% d -psicose was isomerized to d -allose for 17 days. However, by reducing the flow rate to 0.4 ml/min after 17 days, d -allose was transformed at the same rate for 13 days. The total of 27 l reaction mixture was separated by Simulated-Moving-Bed Chromatograph system. Approximately 2.2 l/d of 50% (w/w) reaction mixture was separated continuously. After separation, d -allose and d -psicose fractions were 3 l of approximately 10% (w/w) with 95% purity and 10 l of approximately 8% (w/w) with 95% purity per day, respectively. The separated d -allose solution was concentrated up to about 50% and crystallized gradually by being kept at room temperature. Crystals of d -allose were separated from the syrup by filtration and 1.65 kg crystals of 100% purity were obtained. The d -allose crystal yield from the d -psicose substrate was approximately 10%.

The Maillard reaction (MR) is an ubiquitous reaction of condensation of a reducing sugar with amino groups of protein, which may improve the functional and/or biological properties of foods. The present study was carried out to determine the degree of α-lactalbumin (α-LA) conjugation with a rare sugar ( d -allose, All) and two alimentary sugars ( d -fructose, Fru; d -glucose, Glc) through MR and the extent to which these reactions could convey antioxidant activity to α-LA. The MR was generated in dry state at 50 °C and 55% relative humidity for up to 48 h. The results showed that the conjugation rate and fluorescence development of α-LA modified with All were faster than that of those modified with Glc and Fru, respectively. Furthermore, α-LA modified with All exhibited the highest tetrazolium salt (XTT) reducibility and radical cation (ABTS + )-scavenging activity. There was a good correlation between the fluorescence temporal patterns and biochemical activity of the different sugar-protein conjugates. Thus, the protein glycated with All could be used in formulated food as a functional ingredient, with a strong antioxidant activity, for scavenging free radicals and delaying deterioration due to oxidation.

The synthesis and biological evaluation of some new glycose esters bearing the 1,2- O -isopropylidene- d -hexofuranose functionality and belonging to the 3- O -acyl- d -allose and 6- O -acyl- d -glucose series are reported. When the results concerning cell growth inhibition are compared, it appears that the 6- O -acyl- d -glucose derivatives are more active than the 3- O -acyl- d -allose compounds. Within both 6- O -acyl- d -glucose and 3- O -acyl- d -allose derivatives, butyric esters displayed the highest inhibitory effects. Inhibition of cell growth is not associated with high induction levels of erythroid differentiation, despite the fact that pivaloates induce erythroid differentiation to an extent similar to that exhibited by previously reported molecules [ Bioorg. Med. Chem. Lett. 1999 , 9 , 3153–3158].

Replacement of specific hydroxyl groups by fluorine in carbohydrates is an ongoing challenge from chemical, biological, and pharmaceutical points of view. A rapid and efficient two-step, regio- and stereoselective synthesis of 2-deoxy-2-(R)-fluoro-beta-d-allose (2-(R)-fluoro-2-deoxy-beta-d-allose; 2-FDbetaA), a fluorinated analogue of the rare sugar, d-allose, is described. TAG (3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol or 3,4,6-tri-O-acetyl-d-glucal), was fluorinated in anhydrous HF with dilute F(2) in a Ne/He mixture or with CH(3)COOF at -60 degrees C. The fluorinated intermediate was hydrolyzed in 1N HCl and the hydrolysis product was purified by liquid chromatography and characterized by 1D (1)H, (13)C, and (19)F NMR spectroscopy as well as 2D NMR spectroscopy and mass spectrometry. In addition, (18)F-labeled 2-deoxy-2-(R)-fluoro-beta-d-allose (2-[(18)F]FDbetaA) was synthesized for the first time, with an overall decay-corrected radiochemical yield of 33+/-3% with respect to [(18)F]F(2), the highest radiochemical yield achieved to date for electrophilic fluorination of TAG. The rapid and high radiochemical yield synthesis of 2-[(18)F]FDbetaA has potential as a probe for the bioactivity of d-allose.

A recombinant thermophilic Thermus caldophilus GK24 hexokinase, one of the ROK-type (repressor protein, open reading frames, and sugar kinase) proteins, exists uniquely as a 120 kDa molecule with four subunits (31 kDa), in contrast to eukaryotic and bacterial sugar kinases which are monomers or dimers. The optimal temperature and pH for the enzyme reaction are 70–80 °C and 7.5, respectively. This enzyme shows broad specificity toward glucose, mannose, glucosamine, allose, 2-deoxyglucose, and fructose. To understand the sugar specificity at a structural level, the enzyme–ATP/Mg 2+ –sugar binding complex models have been constructed. It has been shown that the sugar specificity is probably dependent on the interaction energy occurred by the positional proximity of sugars bound in the active site of the enzyme, which exhibits a tolerance to modification at C2 or C3 of glucose.

Vibrational circular dichroism (VCD) spectra in the entire 2000-900 cm(-1) region have been recorded, for the first time, for films of carbohydrates prepared from aqueous solutions. Eight different carbohydrates, alpha-D-glucopyranosyl-(1--4)-D-glucose, cyclomaltohexaose, alpha-D-glucopyranosyl alpha-D-glucopyranoside, beta-D-glucopyranosyl-(1--6)-D-glucose, beta-D-glucopyranosyl-(1--4)-D-glucose, D-glucose, and both enantiomers of 6-deoxygalactose and of allose, were investigated. The VCD spectra obtained for films are found to be identical to the corresponding spectra obtained for aqueous solutions of carbohydrates. These measurements demonstrate several advantages of significant importance. The strong infrared absorption of water has prevented, in the past, the pursuit for routine applications of VCD in determining the structures of carbohydrates in aqueous solutions. This limitation is not present for film studies because water solvent is removed in the process of preparing the films. Also, strong infrared absorption of water at 1650 cm(-1) requires the use of very short-pathlength (6 microm) cells for measurements on aqueous solutions. This requirement and concomitant inconveniences (such as laborious assembling of a demountable liquid cell or purchasing an expensive variable pathlength liquid cell) have been eliminated for film measurements. The removal of interfering water absorption in film studies resulted in higher light throughput and better signal-to-noise ratios for VCD measurements. Another point of significance is that the amount of carbohydrate sample required for VCD measurements on films is approximately one to two orders of magnitude smaller than that required for corresponding VCD measurements on aqueous solutions. Since carbohydrate samples can now be studied as films, VCD spectroscopy becomes much more broadly applicable for carbohydrates than previously believed. The present work, in combination with other film measurements in our laboratory, indicate that VCD studies on films can be used more generally, providing a convenient and powerful approach for probing structural information for biologically important compounds.

6,6′-Linked ethers derived from D-allose, D-galactose, D-glucose, and D-mannose have been prepared in order to allow comparisons with the reported 6,6′-linked hexopyranose coyolosa, an hypoglycemic compound which has been isolated by extraction of the root of the palm Acrocomia mexicana. Comparison of NMR data from the ethers and their derived peracetates with corresponding reported data from coyolosa and its peracetate indicate that coyolosa is not a 6,6′-linked disaccharide. The synthesised compounds are representatives of a novel class of disaccharide derivatives which are linked tail to tail in contrast to the more usual head to tail (e.g. maltose) or head to head (e.g. trehalose) disaccharides.

The C‐glycoside of methyl α‐d‐altropyranosyl‐(1→4)‐α‐d‐glucopyranoside 2 was prepared in a convergent fashion, from readily available precursors, 4‐O‐tert‐butyldiphenylsilyl‐1,2‐O‐isopropylidene‐d‐erythro‐S‐phenyl monothiohemiacetal 13 (five steps from D‐ribose) and the known acid, methyl 2,3,6‐tri‐O‐benzyl‐4‐C‐(carboxymethyl)‐4‐deoxy‐α‐d‐glucopyranoside 17 (seven steps from methyl α‐d‐glucopyranoside). The key reactions in the synthesis are the oxocarbenium ion cyclization of thioacetal‐enol ether 19 to a C1 substituted glycal 20, and the stereoselective hydroboration of 20 to the α‐C‐altroside 21. †This paper is Dedicated to Professor Gerard Decotes on the occasion of his 70th birthday.

Conformational changes of periplasmic binding proteins are essential for their function in chemotaxis and transport. The allose-binding protein from Escherichia coli is, like other receptors in its family, composed of two α/β domains joined by a three-stranded hinge. In the previously determined structure of the closed, ligand-bound form (Chaudhuri, B. N., Ko, J., Park, C., Jones, T. A., and Mowbray, S. L. (1999) J. Mol. Biol. 286, 1519–1531), the ligand-binding site is buried between the two domains. We report here the structures of three distinct open, ligand-free forms of this receptor, one solved at 3.1-A resolution and two others at 1.7-A resolution. Together, these allow a description of the conformational changes associated with ligand binding. A few large, coupled torsional changes in the hinge strands are sufficient to generate the overall bending motion, with only minor disruption of the individual domains. Integral water molecules appear to act as structural “ball bearings” in this process. The conformational changes of the related ribose-binding protein follow a distinct pattern. The observed differences between the two proteins can be interpreted in the context of changes in sequence and in crystal packing and provide new insights into the nature of hinge bending motion in this class of periplasmic binding proteins.

Pyrimidine 1′-aza-C-nucleosides are synthesised by the fusion of 5-bromouracil, 5-bromocytosine and 5-bromoisocytosine with (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol in 40–41% yield. A homologue of 1′-aza-Ψ-uridine is obtained in a Mannich reaction in 65% yield by treatment of the azasugar, paraformaldehyde and uracil. N-Alkylation of (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol with 6-chloromethyluracil gives the 6-regioisomeric homologue. (3R,4R)-4-(Hydroxymethyl)pyrrolidin-3-ol is synthesised in 25% overall yield from diacetone- d -glucose via 3-C-(azidomethyl)-3-deoxy- d -allose which is subjected to an intramolecular reductive amino alkylation reaction to give (3R,4S)-4-[(1S,2R)-1,2,3-trihydroxypropyl]pyrrolidin-3-ol followed by Fmoc protection, oxidative cleavage of the triol group with further reduction of the obtained aldehyde and subsequent deprotection of the nitrogen atom.

Intramolecular 1,3-dipolar cycloadditions of nitrones formed from 3-O-allyl- d -glucose and d -altrose (both with threo-configuration at C-2,3) afford oxepanes selectively whereas the same reactions of nitrones derived from 3-O-allyl- d -allose and d -mannose (both with erythro-configuration at C-2,3) give tetrahydropyrans selectively.

The synthesis of 3-spirohydantoin derivatives of d -allose and d -ribose is reported. The key step is the stereoselective conversion of glyco-α-aminonitriles from ulose derivatives of d -glucose and d -xylose using titanium(IV) isopropoxide as a mild and efficient catalyst. Cyclisation of the glyco-α-aminonitriles give the target spirohydantoins.