Effects of carboxyl group on the anticoagulant activity of oxidized carrageenans.

In this paper, carrageenans having distinct sulfation patterns (κ-, ι-, ι/ν-, θ- and λ-carrageenans), were fully or partially oxidized at C-6 of the ß-d-Galp units using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and trichloroisocyanuric acid (TCCA) in bicarbonate buffer. The modified carrageenans were characterized by mono- and bidimensional 1H and 13C NMR spectroscopy. The influence of the sulfate and carboxyl groups onto anticoagulant activity was evaluated using Activated Partial Thromboplastin Time (aPTT) in vitro assay. The results showed a synergic effect of the carboxyl groups on the anticoagulant activity, which was dependent on the regiochemistry of the sulfate groups in the polysaccharide backbone. Sulfate groups at C2 of the ß-d-GalAp units appeared to positively influence the anticoagulant effect in comparison to C4-sulfate samples. Also, the partially oxidized κ-carrageenan derivative (κLO) showed better anticoagulant effect than the fully oxidized carrageenan (κHO).

C-3 epimers of sugar amino acids as foldameric building blocks: improved synthesis, useful derivatives, coupling strategies.

To obtain key sugar derivatives for making homooligomeric foldamers or α/ß-chimera peptides, economic and multigram scale synthetic methods were to be developed. Though described in the literature, the cost-effective making of both 3-amino-3-deoxy-ribofuranuronic acid (H-t X-OH) and its C-3 epimeric stereoisomer, the 3-amino-3-deoxy-xylofuranuronic acid (H-c X-OH) from D-glucose is described here. The present synthetic route elaborated is (1) appropriate for large-scale synthesis; (2) reagent costs reduced (e.g. by a factor of 400); (3) yields optimized are ~80% or higher for all six consecutive steps concluding -t X- or -c X- and (4) reaction times shortened. Thus, a new synthetic route step-by-step optimized for yield, cost, time and purification is given both for D-xylo and D-ribo-amino-furanuronic acids using sustainable chemistry (e.g. less chromatography with organic solvents; using continuous-flow reactor). Our study encompasses necessary building blocks (e.g. -X-OMe, -X-OiPr, -X-NHMe, Fmoc-X-OH) and key coupling reactions making -Aaa-t X-Aaa- or -Aaa-t X-t X-Aaa- type "inserts". Completed for both stereoisomers of X, including the newly synthesized Fmoc-c X-OH, producing longer oligomers for drug design and discovery is more of a reality than a wish.

Benzylidene Acetal Protecting Group as Carboxylic Acid Surrogate: Synthesis of Functionalized Uronic Acids and Sugar Amino Acids.

Direct oxidation of the 4,6-O-benzylidene acetal protecting group to C-6 carboxylic acid has been developed that provides an easy access to a wide range of biologically important and synthetically challenging uronic acid and sugar amino acid derivatives in good yields. The RuCl3 -NaIO4 -mediated oxidative cleavage method eliminates protection and deprotection steps and the reaction takes place under mild conditions. The dual role of the benzylidene acetal, as a protecting group and source of carboxylic acid, was exploited in the efficient synthesis of six-carbon sialic acid analogues and disaccharides bearing uronic acids, including glycosaminoglycan analogues.

Antioxidant activities of a polyglucuronic acid sodium salt obtained from TEMPO-mediated oxidation of xanthan.

A xanthouronic acid sodium salt called xanthouronan was produced from xanthan by regioselective oxidation with NaOCl/NaBr using 2,2,6,6-tetramethylpiperidine-1-oxy radical (TEMPO) as catalyst. The efficiency of the one pot TEMPO-mediated oxidation was confirmed by HPAEC-PAD, (13)C NMR, and FT-IR. The oxidation degree was close to 98% and the mass yield of this new polyglucuronic acid was higher than 90% (w/w). The macromolecular characterization of xanthouronan using SEC-MALLS showed a molecular size reduced by a third due to the oxidation treatment and the degree of polymerization (DP) of the xanthouronan form was about 665. The evaluation of the enzymatic degradation of this C-6 carboxylated xanthan by various polysaccharide hydrolases and one polysaccharide lyase showed its high resistant to biodegradation. The antioxidant activity of xanthouronan was also tested by using the 2,2'-diphenyl-1-picrylhydrazyle (DPPH) and hydroxyl radical procedures. At 1 g/L, xanthouronan presented 75% of the ascorbic acid antioxidant activity.

Synthesis of Staphylococcus aureus type 5 capsular polysaccharide repeating unit using novel L-FucNAc and D-FucNAc synthons and immunochemical evaluation.

Staphylococcus aureus is a major cause of nosocomial infections. Glycoconjugates of type 5 and 8 capsular polysaccharides have been investigated for vaccine application. The proposed structure of type 5 polysaccharide is: →4-ß-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-ß-D-FucNAc-(1→. The stereocontrolled insertion of these three glycosydic bonds is a real synthetic challenge. In the present paper we report the preparation of two novel versatile L- and D-fucosamine synthons from commercially available starting materials. In addition we applied the two building blocks to the synthesis of type 5 trisaccharide repeating unit. The immunochemical properties of the synthesized trisaccharide were assessed by competitive ELISA and by immunodot blot analysis using sera of mice immunized with type 5 polysaccharide conjugated to CRM(197). The results suggest that although the type 5 S. aureus trisaccharide is recognized by specific anti polysaccharide antibodies in dot blot, structures longer than the trisaccharide may be needed in order to significantly compete with the native type 5 polymer in the binding with sera from mice immunized with S. aureus type 5 polysaccharide-CRM(197) conjugate.

Benzoylated uronic acid building blocks and synthesis of N-uronate conjugates of lamotrigine.

A chemoenzymatic approach towards benzoylated uronic acid building blocks has been investigated starting with benzoylated hexapyranosides using regioselective C-6 enzymatic hydrolysis as the key step. Two of the building blocks were reacted with the antiepileptic drug lamotrigine. Glucuronidation of lamotrigine using methyl (2,3,4-tri-O-benzoyl-α-D-glycopyranosyl bromide)uronate proceeded to give the N2-conjugate. However, lamotrigine-N2-glucuronide was most efficiently synthesised from methyl (2,3,4-tri-O-acetyl-α-D-glucopyranosyl bromide)uronate. Employing nitromethane as solvent with CdCO(3) as a base lamotrigine-N2 glucuronide was prepared in a high yield (41%). Also methyl (2,3-di-O-benzoyl-4-deoxy-4-fluoro-α-D-glucosyl bromide)uronate underwent N-glucuronidation, but the product was unstable, eliminating hydrogen fluoride to give the corresponding enoate conjugate.

Stereoselective synthesis of 2,3-diamino-2,3-dideoxy-ß-D-mannopyranosyl uronates.

With the aim to find an efficient synthetic procedure for the construction of 2,3-diamino-2,3-dideoxy-ß-D-mannuronic acids, we evaluated three mannosyl donors: (S)-phenyl 4,6-di-O-acetyl-2,3-diazido mannopyranoside, (S)-phenyl 2,3-diazido-4,6-O-benzylidene mannopyranoside, and (S)-phenyl 2,3-diazido mannopyranosyl methyl uronate. The first two mannosylating agents are rather unselective or slightly α-selective in their condensation with three different acceptors. The mannuronic acid donor on the other hand reliably provides the desired ß-mannosidic linkage. A mechanistic rationale is put forward to account for the different behavior of the three donor types. Suitably protected 2,3-diazido mannuronic acids were employed to construct the all-cis-linked tetrasaccharide repeating unit of the capsular polysaccharide of Bacillus stearothermophilus , featuring two 2,3-diacetamido-2,3-dideoxy-ß-D-mannuronic acids.

Mannosazide methyl uronate donors. Glycosylating properties and use in the construction of ß-ManNAcA-containing oligosaccharides.

Mannosazide methyl uronate donors equipped with a variety of anomeric leaving groups (ß- and α-S-phenyl, ß- and α-N-phenyltrifluoroacetimidates, hydroxyl, ß-sulfoxide, and (R(s))- and (S(s))-α-sulfoxides) were subjected to activating conditions, and the results were monitored by (1)H NMR. While the S-phenyl and imidate donors all gave a conformational mixture of anomeric α-triflates, the hemiacetal and ß- and α-sulfoxides produced an oxosulfonium triflate and ß- and α-sulfonium bistriflates, respectively. The ß-S-phenyl mannosazide methyl uronate performed best in both activation experiments and glycosylation studies and provided the 1,2-cis mannosidic linkage with excellent selectivity. Consequently, an α-Glc-(1→4)-ß-ManN(3)A-SPh disaccharide, constructed by the stereoselective glycosylation of a 6-O-Fmoc-protected glucoside and ß-S-phenyl mannosazide methyl uronate, was used as the repetitive donor building block in the synthesis of tri-, penta-, and heptasaccharide fragments corresponding to the Micrococcus luteus teichuronic acid.

Synthesis of both enantiomers of hydroxypipecolic acid derivatives equivalent to 5-azapyranuronic acids and evaluation of their inhibitory activities against glycosidases.

We have synthesized 3-hydroxy- and 3,4,5-trihydroxypipecolic acid derivatives corresponding to 5-aza derivatives of uronic acids and evaluated their inhibitory activities against various glycosidases including beta-glucuronidase. Compounds 4 and 5 were chosen as common intermediates for the synthesis of 3,4,5-trihydroxypipecolic acids and 3-hydroxypipecolic acids as well as for 3-hydroxybaikiain, a unique natural product isolated from a toxic mushroom. Cross aldol reaction of N-Boc-allylglycine derivative with acrolein followed by the ring-closing metathesis gave 4 and 5 as a mixture of diastereomers which could be separated by silica gel column chromatography. By employing lipase-catalyzed kinetic resolution, the synthesis of both L- and D-isomers of 3,4,5-trihydroxy- and 3-hydroxypipecolic acids was achieved. None of the compounds tested showed inhibitory activity against alpha- and beta-glucosidases. On the other hand, L-23 and L-29 were found to have potent inhibitory activity against beta-glucuronidase. In addition, it is interesting that some uronic-type azasugar derivatives showed moderate inhibitory activities against beta-N-acetylglucosaminidase.

De novo synthesis of uronic acid building blocks for assembly of heparin oligosaccharides.

An efficient de novo synthesis of uronic acid building blocks is described. The synthetic strategy relies on the stereoselective elongation of thioacetal protected dialdehydes 12 a and 17. The dialdehydes are prepared from D-xylose, a cheap and commercially available source. A highly stereoselective MgBr(2)OEt(2)-mediated Mukaiyama aldol addition to C4-aldehyde 12 a is performed to obtain D-glucuronic acid building block 16, whereas L-iduronic acid building block 22 is prepared by MgBr(2)OEt(2)-mediated cyanation of C5-aldehyde 17. Synthesis of a heparin disaccharide demonstrates the utility of the de novo strategy for the assembly of glycosaminoglycan oligosaccharides.

Glycosidation-anomerisation reactions of 6,1-anhydroglucopyranuronic acid and anomerisation of beta-D-glucopyranosiduronic acids promoted by SnCl(4).

The reaction of silylated nucleophiles with 6,1-anhydroglucopyranuronic acid (glucuronic acid 6,1-lactones) catalysed by tin(IV) chloride provides 1,2-trans or 1,2-cis (deoxy)glycosides in a manner dependent on the donor structure. The alpha-glycoside was obtained for reactions of the donor with the 2-acyl group and 2-deoxydonors, whereas the 2-deoxy-2-iodo donor gave the beta-glycoside. Experimental evidence shows that when 1,2-cis-glycoside formation occurs, the anomerisation of initially formed 1,2-trans-glycosides catalysed by SnCl(4) is possible. The anomerisation of beta-D-glucopyranosiduronic acids was found to be faster, in some cases, than anomerisation of related beta-D-glucopyranosiduronic acid esters and beta-D-glucopyranoside derivatives and the rates are dependent on the structure of the aglycon. Moreover, the rates of anomerisation of beta-D-glucopyranuronic acid derivatives can be qualitatively correlated with rates of hydrolysis of beta-D-glucopyranosiduronic acids. Mechanistic possibilities for the reactions are considered.

Direct oxidation of sugar nucleotides to the corresponding uronic acids: TEMPO and platinum-based procedures.

The direct oxidation of UDP-alpha-d-glucose and UDP-N-acetyl-alpha-d-glucosamine to the corresponding uronic acids was explored using either TEMPO or platinum-catalysed oxidation with molecular oxygen. Whilst TEMPO-based procedures gave rise to substantial over-oxidation and/or degradation of UDP-glucose, oxidation of UDP-N-acetyl-glucosamine to UDP-N-acetyl-glucosaminuronic acid was achieved with >90% conversion and ca. 65% isolated yield using a platinum-catalysed procedure.

Synthesis and characterization of the hexenuronic acid model methyl 4-deoxy-beta-L-threo-hex-4-enopyranosiduronic acid.

A facile synthetic scheme for the preparation of methyl 4-deoxy-beta-L-threo-hex-4-enopyranosiduronic acid utilizing the commercially available methyl alpha-D-galactopyranoside as starting material has been developed. The synthesis sequence comprises six high yielding reaction steps: TEMPO oxidation, acetylation, methanolysis of the lactone, acetylation, beta-elimination, and final removal of the protecting groups. Only one column chromatographic purification is needed throughout the whole sequence. The overall yield is 60%. The final product has been characterized by NMR, Raman, UVRR, FTIR, and HRMS.

An atypical approach identifies TYR234 as the key base catalyst in chondroitin AC lyase.

Chondroitin AC lyase from Flavobacterium heparinum catalyses the degradation of chondroitin by an anionic E1cb elimination mechanism that involves proton abstraction from C5 of glucuronic acid. The lyase also carries out efficient proton transfer to a sugar nitronate anion, which was designed originally as an inhibitor of the enzyme, with a second-order rate constant of kcat/Km=2.7x10(6) M(-1) s(-); this is very similar to that of the natural chondroitin substrate (kcat/Km=1.3x10(6) M(-1) s(-1)). Studies with this nitronate should therefore provide insight into the proton-transfer step (general base catalysis) within this mechanism. Indeed, the Tyr234Phe mutant of the enzyme was essentially inactive with the natural substrate and correspondingly did not catalyse proton transfer to the nitronate, thereby implicating this residue as the general base catalyst. Parallel studies designed to identify the acid catalyst were carried out by using a substrate with a 2,4-dinitrophenol leaving group that needs no acid assistance for departure. These results are consistent with Tyr234 also playing the role of acid catalyst. Not only do these studies confirm the suspected role of Tyr234, but also they validate a new methodology for identification of acid/base catalysts in lyases and epimerases of this type. In addition a structural and mechanistic rationale is provided for different active-site acid/base configurations in syn and anti lyases.

Structure-activity relationships of 2-chloro-N6-substituted-4'-thioadenosine-5'-uronamides as highly potent and selective agonists at the human A3 adenosine receptor.

We have established structure-activity relationships of novel 4'-thionucleoside analogues as the A(3) adenosine receptor (AR) agonists. Binding affinity, selectivity toward other AR subtypes, and efficacy in inhibition of adenylate cyclase were studied. From this study, 2-chloro-N(6)-methyl-4'-thioadenosine-5'-methyluronamide (36a) emerged as the most potent and selective agonist at the human A(3) AR. We have also revealed that, similar to 4'-oxoadenosine analogues, at least one hydrogen on the 5'-uronamide moiety was necessary for high-affinity binding at the human A(3) AR, presumably to allow this group to donate a H bond within the binding site. Furthermore, bulky substituents on the 5'-uronamide reduced binding affinity, but in some cases large 5'-uronamide substituents, such as substituted benzyl and 2-phenylethyl groups, maintained moderate affinity with reduced efficacy, leading to A(3) AR partial agonists or antagonists. In several cases for which the corresponding 4'-oxonucleosides have been studied, the 4'-thionucleosides showed higher binding affinity to the A(3) AR.

Synthesis of methyl 1-O-(4-hydroxymethamphetaminyl)-alpha-D-glucopyranouronate.

For the purpose of the direct characterization of the intact conjugated form in the urine of a methamphetamine (MA) abuser, 4-hydroxymethamphetamine (4-OHMA) glucuronate, corresponding to one of the metabolites of MA, was synthesized from the commercially available methyl 4-hydroxyphenylacetate.

Preparation of 1-thio uronic acid lactones and their use in oligosaccharide synthesis.

The chemo- and regioselective TEMPO/BAIB-mediated oxidation of 2,6- and 3,6-dihydroxy 1-thio glycopyranosides to the corresponding 1-thio uronic acid lactones is described. These locked 1-thio glycuronides can directly be used as donors in glycosidation reactions using the Ph(2)SO/Tf(2)O reagent system. Alternatively, selective opening of the lactone bridge liberates a hydroxyl function for ensuing glycosylations.

A modular strategy toward the synthesis of heparin-like oligosaccharides using monomeric building blocks in a sequential glycosylation strategy.

A novel flexible assembly strategy is described for the modular synthesis of heparin and heparan sulfates. The reported strategy uses monomeric building blocks to construct the oligosaccharide chain to attain a maximum degree of flexibility. In the assembly, 1-hydroxyl glucosazido- and 1-thio uronic acid donors are combined in a sequential glycosylation protocol using sulfonium triflate activator systems. The key 1-thio uronic acids were obtained in an efficient manner from diacetone glucose employing a chemo- and regioselective oxidation of partially protected glucose and idose thioglycosides.

An efficient approach to N-acetyl-D-glucosaminuronic acid-based sialylmimetics as potential sialidase inhibitors.

A novel approach to the synthesis of beta-glycosides of N-acetyl-D-glucosaminuronic acid, in six steps and good overall yield from N-acetyl-d-glucosamine, has been developed. The key synthetic step was the Lewis acid mediated O-glycosidation of methyl 1,3,4-tri-O-pivaloyl-N-acetyl-D-glucosaminuronate (11). Elaboration of glucosaminuronides 15 and 18 provided novel sialylmimetics 21 and 22, which showed inhibition of Vibrio cholerae sialidase.