Synthesis Of Disaccharides Research Articles

Synthesis of nucleotide-activated disaccharides with recombinant β3-galactosidase C from Bacillus circulans

Nucleotide-activated di- and oligosaccharides represent a novel class of glycoconjugates. They are components of human milk with still unknown biological function. Synthetic access to a wide range of nucleotide di- and oligosaccharides would also facilitate their utilization as donor substrates or inhibitors of Leloir-glycosyltransferases. We here present for the first time the synthesis of β1-3-linked nucleotide activated disaccharides by recombinant β3-galactosidase C from Bacillus circulans. UDP-Glc, UDP-GlcNAc, and UDP-GalNAc reacted as acceptor substrates in the transglycosylation reaction with p-nitrophenyl-β-galactoside as donor substrate. In an attempt to optimise the transglycosylation reaction, focused microwave irradiation was investigated. In comparison to conventional thermal heating product compositions and product yields were affected by microwave irradiation and depended on the used acceptor substrate. Microwave irradiation was advantageous for syntheses with UDP-GlcNAc as preferred acceptor substrate of β3-galactosidase C. The β1,3 linked UDP-disaccharide was the main product with minor fractions of UDP-tri- and UDP-tetrasaccharide. In summary, access to important UDP-disaccharides such as UDP-LacNAc type 1 and UDP-Thomsen-Friedenreich(T)-antigen was accomplished for further studies of their role as donor substrates or inhibitors of glycosyltransferases.

Design, synthesis and activity of thio-linked arabinofuranosyl disaccharides against mycobacterial tuberculosis (MTB) and Mycobacterium avium complex (MAC)

We report the chemical synthesis of a series of disaccharides of arabinofuranose with a glycosidic sulfur linker as mimics of the acceptor for arabinofuranosyltransferases with and without using any activator to avoid any complex reactions. These analogs were tested for in vitro activity against MTB strain H37Ra and 3 MAC clinical isolates. MICs were determined using a colorimetric microdilution broth assay. Bactericidal activity was studied with kill curves over a period of seven days. Intracellular activity against MTB H37Ra was determined in the Mono Mac 6 (MM6) human monocytic cell line.

The Molecular Mechanism of Thermostable α-Galactosidases AgaA and AgaB Explained by X-ray Crystallography and Mutational Studies

The α-galactosidase AgaA from the thermophilic microorganism Geobacillus stearothermophilus has great industrial potential because it is fully active at 338 K against raffinose and can increase the yield of manufactured sucrose. AgaB has lower affinity for its natural substrates but is a powerful tool for the enzymatic synthesis of disaccharides by transglycosylation. These two enzymes have 97% identity and belong to the glycoside hydrolase (GH) family GH36 for which few structures are available. To understand the structural basis underlying the differences between these two enzymes, we determined the crystal structures of AgaA and AgaB by molecular replacement at 3.2- and 1.8 Å-resolution, respectively. We also solved a 2.8-Å structure of the AgaA(A355E) mutant, which has enzymatic properties similar to those of AgaB. We observe that residue 355 is located 20 Å away from the active site and that the A355E substitution causes structural rearrangements resulting in a significant displacement of the invariant Trp(336) at catalytic subsite -1. Hence, the active cleft of AgaA is narrowed in comparison with AgaB, and AgaA is more efficient than AgaB against its natural substrates. The structure of AgaA(A355E) complexed with 1-deoxygalactonojirimycin reveals an induced fit movement; there is a rupture of the electrostatic interaction between Glu(355) and Asn(335) and a return of Trp(336) to an optimal position for ligand stacking. The structures of two catalytic mutants of AgaA(A355E) complexed with raffinose and stachyose show that the binding interactions are stronger at subsite -1 to enable the binding of various α-galactosides.

Synthesis of selenium-linked neoglycoconjugates and pseudodisaccharides

The introduction of organoselenium moieties within the structure of carbohydrates has received attention recently. Herein, we report on the synthesis of selenium-containing neoglycoconjugates and pseudodisaccharides by the reaction of nucleophilic selenium species, generated from sugar diselenides, with chiral N-Boc aziridines and sugar tosylates. The reaction proceeds with moderate to good yields for various substrates. The introduction of organoselenium moieties within the framework of various sugars, with increased levels of complexity, thus allowing the synthesis of disaccharide and glycoconjugate mimetics.

Synthesis of Disaccharides Containing 6-Deoxy-a-L-talose as Potential Heparan Sulfate Mimetics

A 6-deoxy-α-L-talopyranoside acceptor was readily prepared from methyl α-L-rhamnopyranoside and glycosylated with thiogalactoside donors using NIS/TfOH as the promoter to give good yields of the desired α-linked disaccharide (69–90%). Glycosylation with a 2-azido-2-deoxy-D-glucosyl trichloroacetimidate donor was not completely stereoselective (α:β = 6:1), but the desired α-linked disaccharide could be isolated in good overall yield (60%) following conversion into its corresponding tribenzoate derivative. The disaccharides were designed to mimic the heparan sulfate (HS) disaccharide GlcN(2S,6S)-IdoA(2S). However, the intermediates readily derived from these disaccharides were not stable to the sulfonation/deacylation conditions required for their conversion into the target HS mimetics.

Bio-solvents change regioselectivity in the synthesis of disaccharides using Biolacta β-galactosidase

Bio-solvents are good alternative solvents that avoid the use of classical organic solvents when performing enzymatic reactions. A noticeably change in regioselectivity was observed in the synthetic behaviour of Biolacta β-galactosidase using bio-solvents derived from dimethylamide and glycerol as co-solvents. Under these conditions, the enzyme changes its well known tendency to produce β-(1→4) to β-(1→6) disaccharides. An evaluation of the bio-solvent concentration and the effects of the non proteic additives in commercially available Biolacta β-galactosidase was undertaken in order to optimize the reaction conditions to improve the yield of the β-(1→6) product.

Sequential Directed Epoxydation-Acidolysis from Glycals with MCPBA. A Flexible Approach to Protected Glycosyl Donors

4,6-Di-O-protected glucal and allal derivatives react with MCPBA to afford manno- and allo-1-O-m-chlorobenzoate derivatives, respectively, as a result of a syn epoxidation directed by the allylic hydroxyl group, and consecutive ring-opening by m-ClBzOH. When glucal and allal derivatives are fully protected, initial epoxidation proceeds mainly anti to the allylic group to give, after ring-opening, the corresponding pyranosyl chlorobenzoates. Stereoselectivity in the reaction of fully protected galactal derivatives was complete, although only a moderate increase in the syn epoxidation product was observed in 4,6- and 3,4-di-O-protected derivatives. 1-O-m-Chlorobenzoate 18 was selectively protected and activated as donor in the synthesis of disaccharide 21.

Screening of strains and recombinant enzymes from Thermus thermophilus for their use in disaccharide synthesis

Abstract Cell extracts from ten different strains of Thermus thermophilus have been screened for glycosidase activity. Among these, the sequenced strain HB27 hydrolyzed a wide variety of glycosides and increased six fold its β-glycosidase activity when grown with cellobiose in nutrient-limited media. We selected five genes encoding (putative) glycosidases (TTP0042, TTP0072, TTP0220, TTC0107 and TTP0222) from the genome of this strain, and the corresponding recombinant enzymes were overexpressed and purified. Several transglycosylation reactions using cellobiose-induced HB27 cell extracts and the purified recombinant enzymes were assayed. Biochemical properties and biosynthetic capabilities of the HB27 cell extracts and the TTP0042 enzyme were very similar, suggesting that this enzyme was responsible for most of the β-glycosidase activity detected in the HB27 strain. This was confirmed through the isolation and analysis of a null mutant of its encoding gene. With both, HB27 cell extracts and purified TTP0042 recombinant enzyme, we finally achieved high yields conditions for disaccharide production by transglycosylation with low amounts of self-condensed donor when high concentrations of a 1:5 donor:acceptor molar ratio was used.

Charged Hexosaminides as New Substrates for β‐N‐Acetylhexosaminidase‐Catalyzed Synthesis of Immunomodulatory Disaccharides

AbstractThis work is a structure‐activity relationship study that investigates the influence of the nature and amount of negative charge in carbohydrate substrates on the affinity of β‐N‐acetylhexosaminidases, and on the stimulation of natural killer cells. It describes synthetic procedures yielding novel glycosides that are useful in immunoactivation. Specifically, we present a thorough study on the ability of six C‐6 modified β‐N‐acetylhexosaminides (aldehyde, uronate, 6‐O‐sulfate, 6‐O‐phosphate) to serve as substrates for cleavage and glycosylation by a library of β‐N‐acetylhexosaminidases from various sources. Four novel disaccharides with one or two (negatively) charged groups were prepared in synthetic reactions in good yields. Surprisingly, the 6‐O‐phosphorylated substrate, although cleaved by a number of enzymes from the series, worked neither as a donor nor as an acceptor in transglycosylation reactions. The results of wet experiments were supported by molecular modeling of substrates in the active site of two representative enzymes from the screening. All ten prepared compounds were examined in terms of their immunoactivity, namely as ligands of two activation receptors of natural killer (NK) cells, NKR‐P1 and CD69, both with isolated proteins and whole cells. Sulfated disaccharides in particular acted as very efficient protectants of NK cells against activation‐induced apoptosis, and as stimulants of the natural killing of resistant tumor cells, which makes them good candidates for potential clinical use in cancer treatment.

A potential glucuronate glycosyl donor with 2- O-acyl-6,3-lactone structure: efficient synthesis of glycosaminoglycan disaccharides

Development of β-selective glucuronylation reaction using phenyl 2,4-di- O-acetyl-1-thio-β- d-glucopyranosidurono-6,3-lactone was described. Glycosylations of this glycosyl donor with hexosamine derivatives proceeded with excellent yield and β-stereoselectivity to afford glycosaminoglycan-type disaccharides.

Improved synthesis of disaccharides with Escherichia coli β-galactosidase using bio-solvents derived from glycerol

A noticeably increase in activity, keeping total regioselectivity was found in the synthetic behaviour of Escherichia coli β-galactosidase in glycerol-based solvents using a 1:7 molar ratio of donor ( pNP-β-Gal): acceptor (GlcNAc). Yields of up to 97% of β(1→6) with different solvents were found. These reactions take place without noticeable hydrolytic activity and with total regioselectivity, representing a considerable improvement over the use of aqueous buffer or conventional organic solvents. There is a clear dependence of the catalytic results on the solvent structure, which is analysed in terms of polarity and hydrophobicity.

Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter subterraneus

A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in Escherichia coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His tag chromatography and was found to be specifically active toward trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h of incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determining the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the following order: C-3 > C-2 > C-4. This results in a particularly high activity on the monosaccharides d-fucose, d-xylose, l-arabinose, and d-galactose, as well as on l-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor d-glucose, resulting in drastically increased K(m) values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity make it a valuable candidate for industrial disaccharide synthesis.

A new Kdo derivative for the synthesis of an inner-core disaccharide of lipopolysaccharides and lopooligosaccharides

Methyl (7,8-di- O-benzoyl-4,5- O-isopropylidene-3-deoxy- d- manno-2-oct-ulopyranoside)onate was found to be a useful new intermediate in the synthesis of an inner-core oligosaccharide of lipooligosaccharides and lipopolysaccharides produced by gram-negative bacteria. This intermediate could be converted to the corresponding glycosyl fluoride and 4,5-diol acceptor with ease. Syntheses of dimeric Kdo, O-(sodium 3-deoxy-α- d- manno-2-octuropyranosylonate)-(2–4)-sodium (allyl 3-deoxy-α- d- manno-2-octuropyranoside)onate, and O-(sodium 3-deoxy-α- d- manno-2-octuropyranosylonate)-(2–8)-sodium (allyl 3-deoxy-α- d- manno-2-octuropyranoside)onate were successfully demonstrated.

Synthesis of a Mannose-Bearing Disaccharide with a Thiol Spacer at the Reducing End

The synthesis of a mannose-bearing disaccharide containing a thiol spacer at the reducing end was carried out to provide a tethered sugar suitable for attaching to gold nanoparticles. An array of such sugars is designed to mimic carbohydrates involved in cell-surface interactions. The molecule was constructed via Schmidt glycosylation of an appropriately protected glycosyl donor and an acceptor, followed by removal of protective groups and reductive amination to introduce a protected thiol spacer at the reducing end of the glycan. A new finding that the anomeric reductive amination is capable of concomitantly removing a neighboring N-acetyl group was also observed. Subsequent removal of the thiol protective group and purification of the product gave the target disaccharide in a satisfactory yield. Keywords: Carbohydrate, oligosaccharide, glycosylation, mannose, nanoparticle, thiol, biomineralization, cytotoxicity, globular

N,N′-Carbonyldiimidazole-Mediated DBU-Catalyzed One-Pot Synthesis of Urea-Tethered Glycosyl Amino Acids and Glycoconjugates

An efficient, mild, simple, and alternative one-pot protocol for the synthesis of urea-tethered glycosyl amino acids mediated by N,N'- carbonyldiimidazole employing DBU as a catalyst is described. This protocol is also extended for the synthesis of urea-tethered disaccharides and oligosaccharides. © Georg Thieme Verlag Stuttgart - New York.

Gold‐Catalyzed Reactions of 2‐C‐Branched Carbohydrates: Mild Glycosidations and Selective Anomerizations

Abstract2‐C‐branched methyl glycosides react with various alcohols under gold catalysis to transglycosylated products. The method is applicable for the convenient synthesis of disaccharides. Without nucleophile a selective anomerization occurs, giving first access to α‐configured 2‐C‐nitromethyl glycosides. The results are interesting for the mechanism of gold‐catalyzed glycosidations.

Noteworthy observations accompanying synthesis of the apoptolidin disaccharide

A stereoselective synthesis of the apoptolidin disaccharide is reported. The key chemistry features a new transformation utilizing a highly selective tetramethylalkoxyalanate[v]-directed syn-methylation of a vinylogous ester, isolation of a hydrate of a 2-keto sugar, an eco-friendly radical cleavage of a bromomethyl group, and an efficient preparation of a fluorodisaccharide via the use of XtalFluor-E.

Solvents derived from glycerol modify classical regioselectivity in the enzymatic synthesis of disaccharides with Biolacta β-galactosidase

Green solvents made from glycerol change the classical regioselectivity of Biolacta No 5 β-galactosidase, from β(1→4) to β(1→6) linkages when a 2 M concentration was used. In order to explain these results, the non-proteic compounds present in the Biolacta preparation were separated by precipitation with ammonium sulfate and the remaining protein extract was used to set reactions with appropriate organic solvents to find that the regioselectivity towards the β(1→6) isomer is retained. According to proteomic analysis, a 98% homology between Streptococcus pneumoniae and Biolacta β-galactosidase preparation was found. With these data, molecular modelling was done which predicts a tridimensional interaction in the enzyme active site with the donor (GlcNAc) and the water-solvent mixture which explains this phenomenon.

ChemInform Abstract: Synthesis of Nucleoside Sulfonates and Sulfones.

Abstract Sulfonate analogues of adenine, cytidine and guanine monophosphates have been synthesized for the first time. The synthesis of ribose 3-sulfonate, a synthesis of sulfonyl disaccharides and dinucleotides and an improved synthesis of a ribose 3-sulfone is also reported.

ChemInform Abstract: Stereocontrolled Synthesis of Disaccharides via the Temporary Silicon Connection.

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