Synthesis of 2,5-anhydro-(beta-d-glucopyranosyluronate)- and (alpha-l-idopyranosyluronate)-d-mannitol hexa-O-sulfonate hepta sodium salt

Glycosidation of 2,5-anhydro-1,6-di-O-benzoyl-D-mannitol with methyl(2,3,4-tri-O-acetyl-alpha-d-glucopyranosyl-1-O-trichloroacetimidate)uronate in the presence of trimethylsilyl triflate afforded the corresponding 3-O-beta-glycoside, which after deprotection was converted into its hexa-O-sulfate with DMF x SO3 to give after treatment with sodium acetate and subsequent saponification of the methyl ester with sodium hydroxide the hepta sodium salt of 2,5-anhydro-3-O-(beta-d-glucopyranosyl uronate)-D-mannitol hexa-O-sulfate. Glycosidation of the same acceptor with the alpha-thiophenylglycoside of methyl 2,4-di-O-acetyl-3-O-benzyl-L-idopyranosyl uronate in the presence of NIS/TfOH afforded the corresponding 3-O-alpha-glycoside in very low yield, therefore the alpha-thiophenylglycoside of 2-O-acetyl-2,4-O-benzylidene-3-O-benzyl-L-idopyranose was used as donor. The terminal hydroxymethyl group of the obtained disaccharide was subsequently oxidised with NaOCl/TEMPO and the obtained iduronic acid derivative was converted into the hepta sodium salt of 2,5-anhydro-3-O-(-alpha-L-idopyranosyluronate)-D-mannitol hexa-O-sulfonate with DMF x SO3 and subsequent treatment with sodium acetate.     

Synthesis of polyhydroxy amino acids based on D- and L-alanine from D-glycero-D-gulo-heptono-1,4-lactone

2-Amino-2,3-dideoxy-D-manno-heptonic acid (7) has been synthesized from 2,5,6,7-tetra-O-acetyl-3-deoxy-D-gluco-heptono-1,4-lactone (1), which was readily prepared from D-glycero-D-gulo-heptono-1,4-lactone. O-Deacetylation of 1 followed by treatment with 13:1 (v/v) 2,2-dimethoxypropane/acetone in the presence of p-toluenesulfonic acid gave methyl 3-deoxy-4,5:6,7-di-O-isopropylidene-D-gluco-heptonate (3) as a crystalline product (80% yield). The free hydroxyl group (OH-2) of 3 was mesylated and substituted by azide to give the corresponding azide derivative 5. Hydrogenolysis and further hydrolysis of the ester function of 5 afforded alpha-amino acid 7 (43% overall yield from 1). Compound 7 is an analog of L-alanine having a polyhydroxy chain attached to C-3. The diastereoisomer of 7 at C-2, 2-amino-2,3-dideoxy-D-gluco-heptonic acid (12) was also prepared from 3, by a route that involved 2,3-dideoxy-2-iodo derivative 8 as a key intermediate.     

Efficient synthesis of 5-thio-D-arabinopyranose and 5-thio-D-xylopyranose from the corresponding D-pentono-1,4-lactones

5-Thio-D-arabinopyranose (5) and 5-thio-D-xylopyranose (10) were synthesized from the corresponding D-pentono-1,4-lactones. After regioselective bromination at C-5, transformation into 5-S-acetyl-5-thio derivatives, reduction into lactols and deprotection afforded the title compounds in 49 and 42% overall yield, respectively.     

An improved synthesis of 5-thio-D-ribose from D-ribono-1,4-lactone

5-Thio-D-ribopyranose was synthesized from D-ribono-1,4-lactone (1) by two approaches: (i) 5-bromo-5-deoxy-D-ribono-1,4-lactone (2) was successively transformed into 5-bromo-5-deoxy, 5-S-acetyl-5-thio or 5-thiocyanato-D-ribofuranose derivatives; appropriate treatment then lead to 5-thio-D-ribopyranose (7) in 46-48% overall yield and; (ii) 2 was transformed into the 5-S-acetyl-5-thio-D-ribono-1,4-lactone derivative (11). Reduction and deprotection of 11 afforded 5-thio-D-ribopyranose (7) in 57% overall yield.     

Synthesis of 4-cyano and 4-nitrophenyl 1,6-dithio-D-manno-, L-ido- and D-glucoseptanosides possessing antithrombotic activity

1,6-Anhydro-3,4-O-isopropylidene-1-thio-D-mannitol was converted into its sulfoxide which after hydrolysis, acetylation and subsequent Pummerer rearrangement gave the penta-O-acetyl-1-thio-D-mannoseptanose anomers in excellent yield. This anomeric mixture was used as donor for the glycosylation of 4-nitro- and 4-cyanobenzenethiol in the presence of boron trifluoride etherate and trimethylsilyl triflate, respectively, to yield the corresponding thioseptanosides in high yield. The same strategy was applied for the synthesis of the corresponding L-idothioseptanosides using 1,6-anhydro-3,4-O-isopropylidene-1-thio-L-iditol as starting material. The penta-O-acetyl-D-glucothioseptanose donors could not be synthesised the same way, as the Pummerer reaction of the corresponding tetra-O-acetyl-1,6-thioanhydro-1-thio-D-glucitol sulfoxides led to an inseparable mixture of the corresponding L-gulo- and D-glucothioseptanose anomers. Therefore, D-glucose diethyl dithioacetal was converted via its 2,3,4,5-tetra-O-acetyl-6-S-acetyl derivative into an anomeric mixture of its 6-thio-septanose and -furanose peracetates which could be separated by column chromatography. Condensation of the 6-thio-glucoseptanose peracetates with 4-cyano- and 4-nitrobenezenethiol in the presence of boron trifluoride etherate afforded anomeric mixtures of the corresponding thioseptanosides. The D-manno-, L-ido- and D-glucothioseptanosides obtained after Zemplén deacetylation of these mixtures were tested for their oral antithrombotic activity.     

Two approaches to the synthesis of 3-beta-D-glucopyranosyl-D-glucitol

Glycosidation of 1,2:5,6-di-O-isopropylidene-D-glucose with tetra-O-acetyl-glucosyl bromide in 1:1 benzene-MeNO2 afforded approximately equal amounts of the 3-O-beta-D-glycoside and the rearranged 6-O-beta-D-glycoside, while in MeCN only the latter was formed. When tetra-O-acetyl-beta-thiophenylglucoside was used as donor in CH2Cl2 in the presence of NIS/TfOH as activator, the 6-O-beta-D-glycoside and a 3-O-orthoester were formed in a 1:2 ratio at -20 degrees C, while at 20 degrees C only the former could be isolated. Glycosidation of 1-O-benzoyl-2,4-O-benzylidene-5,6-O-isopropylidene-d-glucitol with tetra-O-acetyl-glucosyl bromide in MeCN in the presence of Hg(CN)2 afforded the corresponding 3-O-alpha- and 3-O-beta-glycopyranoside in a 1:4 ratio in MeCN and 1:5 in 1:1 benzene-MeNO2, respectively. When Hg(CN)2/HgBr2 was used as promoter, the corresponding orthoester was also formed. When tetra-O-acetyl-beta-thiophenylglucoside was used as donor, the 3-O-beta-anomer and the orthoester were obtained predominantly in a 3:2 ratio together with traces of the 3-O-alpha-glycoside. Both beta-glycosides could be smoothly converted into 3-beta-D-glucopyranosyl-D-glucitol.     

An efficient synthesis of methyl 1,3-O-isopropylidene-alpha-D-fructofuranoside and 2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal derivatives from sucrose

Acetalation of sucrose with 2,2-dimethoxypropane in 1,4-dioxane in the presence of p-toluenesulfonic acid, followed by acetylation, afforded methyl 4,6-di-O-acetyl-1,3-O-isopropylidene-alpha-D-fructofuranoside and 4-O-acetyl-2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal as major products, while tosylation of the intermediate acetals provided methyl 6-O-tosyl-1,3-O-isopropylidene-alpha-D-fructofuranose.     

Direct epoxidation of D-glucal and D-galactal derivatives with in situ generated DMDO

A multi-gram epoxidation of 3,4,6-tri-O-benzyl-D-glucal and D-galactal with dimethyldioxirane (DMDO) generated in situ from Oxone/acetone in a biphasic system (CH(2)Cl(2)-aqueous NaHCO(3)) resulted in the formation of the corresponding 1,2-anhydrosugars in a 99% yield and 100% selectivity. In a similar way, 3,4,6-tri-O-acetyl-D-glucal afforded a 7:1 mixture of the corresponding gluco and manno derivatives in an 87% overall yield.     

A selective Sc(OTf)3-catalyzed trialkylsilyl ether to acetyl ester exchange reaction with beta-l-idopyranoside and 3,4-O-isopropylidene-beta-D-galactopyranoside derivatives

The selective silylation of monosaccharide building blocks is useful for preparing complex oligosaccharides. We now report that the diol, methyl (dimethylthexylsilyl 3-O-pivaloyl-beta-L-idopyranosyl)uronate, can be selectively silylated at the O-2 position by trialkylsilyl triflates. After protection of O-4, the O-2 silyl group can be selectively replaced by acetate by taking advantage of a trialkylsilyl-acetate exchange reaction catalyzed by Sc(OTf)3 in the presence of acetic anhydride. The high O-2 selectivity is shown for triethylsilyl (TES), tert-butyldimethylsilyl (TBS), and triisopropylsilyl (TIPS). The selective cleavage reaction only worked well for TES and TBS derivatives. A selection of silyl triflates and silyl chlorides were used as silylating reagents with ethyl 3,4-O-isopropylidene-1-thio-beta-D-galactopyranoside. In most cases, silylation afforded 2,6-di-O-silylated products in high yields. Studies on the cleavage reaction showed that only the primary silylated protecting groups were replaced by acetyl groups. This reaction worked with a variety of silyl protecting groups but not the tert-butyldiphenylsilyl (TBDPS) protecting group. Unfortunately, the 1-thioethyl group was also sensitive to the Sc(OTf)3, leading in these conditions to alpha/beta mixtures of the 1-acetates, which compromised the synthetic utility of this reaction for these compounds. The sequence presented here is a useful synthetic route to differentially protected L-iduronic acid building blocks.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Convenient conversion of wheat hemicelluloses pentoses (D-xylose and L-arabinose) into a common intermediate

The transformation of D-xylose and L-arabinose, the two major components of wheat straw and bran, into a unique multifunctional, optically pure, five-carbon synthon has been achieved. The synthetic sequence requires three steps: suitable protection of the hydroxyl groups of the pentoses, introduction of an iodide at the C-5 position and zinc-mediated opening of the furanose ring leading to the formation of a common substituted pent-4-enal.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Synthesis of 2-chloro-4-nitrophenyl alpha-L-fucopyranoside: a substrate for alpha-L-fucosidase (AFU)

An effective method to prepare the substrate of alpha-L-fucosidase (AFU) is described. Ethyl 1-thiofucoside with a free 2-OH group was used as the glycosyl donor, and there was found no self-condensed side product. The use of the HF.pyridine reagent to remove the silyl protecting group in the last step afforded a target molecule of high purity.     

Purification,characterization, and gene cloning of a novel aminoacylase from Burkholderia sp. strain LP5_18B that efficiently catalyzes the synthesis of N-lauroyl-l-amino acids

ABSTRACT

An N-lauroyl-l-phenylalanine-producing bacterium, identified as Burkholderia sp. strain LP5_18B, was isolated from a soil sample. The enzyme was purified from the cell-free extract of the strain and shown to catalyze degradation and synthesis activities toward various N-acyl-amino acids. N-lauroyl-l-phenylalanine and N-lauroyl-l-arginine were obtained with especially high yields (51% and 89%, respectively) from lauric acid and l-phenylalanine or l-arginine by the purified enzyme in an aqueous system. The gene encoding the novel aminoacylase was cloned from Burkholderia sp. strain LP5_18B and expressed in Escherichia coli. The gene contains an open reading frame of 1,323 nucleotides. The deduced protein sequence encoded by the gene has approximately 80% amino acid identity to several hydratase of Burkholderia. The addition of zinc sulfate increased the aminoacylase activity of the recombinant E. coli strain.     

Efficient selective preparation of methyl-1,2,4-tri-O-acetyl-3-O-benzyl-beta-L-idopyranuronate from methyl 3-O-benzyl-L-iduronate

Methyl 1,2,4-tri-O-acetyl-3-O-benzyl-L-idopyranuronate 6beta/6alpha, prepared from methyl 3-O-benzyl-L-iduronate (4), is a key synthon in heparin/heparan sulfate synthesis. The 1H and 13C NMR spectra of the furanose-pyranose mixture of 4, after dissolution and equilibration in d(4)-methanol, were fully assigned allowing to expect that 4 could crystallise in the beta-pyranose form. New acetylation conditions able to trap this form were subsequently devised, allowing the isolation of 83% of pure 6beta by simple crystallisation, along with 9% of the 6beta/6alpha mixture. This represents a major advantage over the previously published procedure, especially on multigram scales.     

Evaluation of carbohydrate molecular mechanical force fields by quantum mechanical calculations

A quantitative evaluation of 20 second-generation carbohydrate force fields was carried out using ab initio and density functional methods. Geometry-optimized structures (B3LYP/6-31G(d)) and relative energies using augmented correlation consistent basis sets were calculated in gas phase for monosaccharide carbohydrate benchmark systems. Selected results are: (i). The interaction energy of the alpha-d-glucopyranose.H(2)O heterodimer is estimated to be 4.9 kcal/mol, using a composite method including terms at highly correlated (CCSD(T)) level. Most molecular mechanics force fields are in error in this respect; (ii). The (3)E envelope (south) pseudorotational conformer of methyl 5-deoxy-beta-d-xylofuranoside is 0.66 kcal/mol more stable than the (3)E envelope (north) conformer and the alpha-anomer of methyl d-glucopyranoside is 0.82 kcal/mol more stable than the beta-anomer; (iii). The relative energies of the (gg, gt and tg) rotamers of methyl alpha-d-glucopyranoside and methyl alpha-d-galactopyranoside are (0.13, 0.00, 0.15) and (0.64, 0.00, 0.77) kcal/mol, respectively. The results of the quantum mechanical calculations are compared with the results of calculations using the 20 second-generation carbohydrate force fields. No single force field is consistently better than the others for all the test cases. A statistical assessment of the performance of the force fields indicates that CHEAT(95), CFF, certain versions of Amber and of MM3 have the best overall performance, for these gas phase monosaccharide systems.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

The influence of selected O-alkyl derivatives of cyclodextrins on the enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase

A series of O-alkyl derivatives of cyclodextrin: heksakis[2,3,6-tri-O-(2′-methoxyethyl)]-α-cyclodextrin; heksakis(2,3-di-O-methyl)-α-cyclodextrin; heptakis(2,3-di-O-methyl)-β-cyclodextrin; heksakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-α-cyclodextrin; heptakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-β-cyclodextrin; heksakis[2,3-di-O-(2′-methoxyethyl)]-α-cyclodextrin and heptakis[2,3-di-O-(2′-methoxyethyl)]-β-cyclodextrin have been synthesized. Purity and composition of the obtained substances were examined. The cyclodextrin derivatives listed above as well as (2-hydroxypropyl)-α-cyclodextrin and (2-hydroxypropyl)-β-cyclodextrin, the two commercially available ones, have been investigated as the additives in the course of enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase. It has been found that each of cyclodextrin derivatives causes the inhibition of enzymatic process, both competitive and non-competitive. The competitive inhibition is connected with the formation of inclusion complexes between cyclodextrins and l-tryptophan, related to the geometry of these complexes. The mechanism of the non-competitive inhibition is not so evident; it could be related to the formation of the cyclodextrin complexes on the surface of the enzyme, leading to the change in the flexibility of the enzyme molecule.     

Total synthesis and identification of two diastereoisomers of 1-methyl-2-[N-(p-tolylsulfonyl)-2-pyrrolidinyl]ethyl beta-L-fucopyranoside

The reaction of a racemic mixture of (2R,2'S)- and (2S,2'R)-N-(p-tolylsulfonyl)-2-pyrrolidinyl-2-propanol, prepared from (S)-proline, with 2,3,4-tri-O-acetyl-alpha-L-fucopyranosyl trichloroacetimidate led to both diastereoisomers of the title compound after O-deacetylation.     

Intramolecular interactions in aminoacyl cyclic-3',5'-nucleotides

Polymerization of amino-acid acyl cyclic-3',5'-nucleotides is postulated to be the origin of RNA and associated protein in prebiotic molecular evolution. The enthalpy change in the intramolecular interaction between the nucleotide base and the amino-acid side chain determines the stability of the particular complex, resulting in a preferred association (or coding) of a base for a particular amino acid. The compounds studied were glycine acyl cyclic-3',5'-guanylate where the strong hydrogen bond between protonated glycine and guanine N7 gives an enthalpy change of -0.05 h. Similarly, hydrogen bonds in l-lysine acyl cyclic-3',5'-adenylate give an enthalpy change of -0.06 h. Hydrophobic interactions in l-phenylalanine acyl cyclic-3',5'-uridylate give an enthalpy change of -0.02 h and the corresponding value for l-proline acyl cyclic-3',5'-cytidylate is -0.01 h. These interactions were expected to be modified as the genetic code became a duplet and finally a triplet code. The interactions have been shown to be feasible from the overall enthalpy changes in the ZKE approximation at the MP2/6-31G* level.