1,6-anhydro-<Emphasis Type="Italic">N</Emphasis>-acetyl-β-<Emphasis Type="Italic">D</Emphasis>-glucosamine in oligosaccharide synthesis: II. The synthesis of the spacered Le<Superscript>y</Superscript> tetrasaccharide

3-Aminopropyl glycoside of 3,2′-di-O-α-L-fucosyl-N-acetyllactosamine (Le^y tetrasaccharide) was synthesized. The glycosyl donor, 2-O-acetyl-2,4,6-tri-O-benzoyl-α-D-galactopyranosyl bromide, was coupled with glycosyl acceptor, 1,6-anhydro-2-acetamido-2-deoxy-β-D-glucopyranose or its 3-O-acetyl derivative, to give the corresponding N-acetyllactosamine derivatives in 20 and 71% yields, respectively. The glycosyl donor was synthesized from 1,2-di-O-acetyl-3,4,6-triO-benzoyl-D-galactopyranose, which was obtained by the treatment of benzobromogalactose with sodium borohydride to yield 1,2-O-benzylidene derivative and subsequent removal of benzylidene group and acetylation. Acidic methanolysis of the disaccharide derivatives resulted in the selective removal of one or both acetyl groups to give the disaccharide acceptor bearing hydroxy groups at C3 of the glucosamine residue and C2 of the galactose residue. The introduction of fucose residues in these positions by the treatment with tetrabenzylfucopyranosyl bromide resulted in a tetrasaccharide derivative, which was converted into 3,2′-di-O-α-L-fucopuranosyl-1,6-anhydro-N-acetyllactosamine peracetate after substitution of acetyl groups for benzoyl and benzyl groups. Opening of the anhydro ring by acetolysis resulted in peracetate, which was then converted into the corresponding oxazoline derivative by two steps. Glycosydation of the oxazoline derivative with 3-trifluoroacetamidopropan-1-ol and removal of O-acetyl and N-trifluoroacetyl protective groups resulted in a free spacered Le^y tetrasaccharide.     

Synthesis of 3′-deoxy-3′-carboxymethylnucleosides,precursors of oligonucleotides with an amide internucleoside bond

An improved method for the synthesis of 3-deoxy-3-carboxymethyl nucleosides was suggested. Oxidation of 5-O-benzoyl-1,2-O-isopropylidene-α-D-xylofuranose resulted in the 3-keto derivative, which was treated with triethylphosphonoacetate in the presence of sodium hydride to obtain the 3-deoxy-3-ethoxycarbonylmethylene derivative. Hydrogenation of the unsaturated compound proceeded strictly stereospecifically and gave the product with the ribo-configuration. Acetolysis of the resulting compound with AcOH-Ac₂O-CH₃SO₃H led to 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-ethoxycarbonylmethyl-D-ribofuranose, whose interaction with persilylated nucleic bases gave 3-deoxy-3-ethoxycarbonylmethylnucleosides in a total yield of 42–49% from the starting compound.     

The preparative method for 2-fluoroadenosine synthesis

The preparative method for the synthesis of 2-fluoroadenosine starting from commercially available guanosine was developed. It included the intermediate formation of 2-amino-6-azido-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine, which was isolated exclusively in the tetrazolo[5,1-i]-form {5-amino-7-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-7H -tetrazolo[5,1-i]purine}. The latter compound was converted by the Schiemann reaction to 6-azido-2-fluoro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine, which was isolated at an 80% yield after careful optimization of the process. The IR and ¹H NMR spectroscopy data indicated the 6-azido-2-fluoropurine structure of the aglycone. The catalytic reduction of the azido group in 6-azido-2-fluoro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine to the amino moiety and the subsequent deacetylation by the routine procedure resulted in 2-fluoroadenosine at a total yield of 74%.     

Anionic polymers of the cell wall of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. VKM Ac-2534

The cell wall of Streptomyces sp. VKM Ac-2534, the causative agent of common scab in potato tubers, which does not synthesize thaxtomin and is phylogenetically close to phytopathogen Streptomyces setonii sp. ATCC 25497, contains two anionic carbohydrate-containing polymers. The major polymer is teichuronic acid, whose repeating unit is disaccharide → 4)-β-D-ManpNAc3NAcyA-(1 → 3)-α-D-GalpNAc-(1→, where Acy is a residue of acetic or L-glutamic acid. The polymer of such structure has been found in Gram-positive bacteria for the first time. The minor polymer is teichoic acid [1,5-poly(ribitol phosphate)], in which a part of the ribitol residues are glycosylated at C4 with β-D-Glcp and, probably, with β-D-GlcpNAc and some residues are O-acylated with Lys residues. The structures were proved by chemical and NMR spectroscopic methods. It is likely that the presence of acidic polysaccharides on the surface of the phytopathogenic streptomycete is necessary for its attachment to the host plant.     

Isolation and characterization of lectin from the surface of <Emphasis Type="Italic">Grifola frondosa</Emphasis> (Fr.) S.F.Gray mycelium

From the surface of the dikaryotic mycelium of the xylotrophic basidiomycete Grifola frondosa 0917 a lectin has been isolated with a molecular mass of 68 ± 1 kDa, consisting of two subunits of 33–34 kDa each. The lectin is a hydrophilic glycoprotein with the protein: glycan ratio of 3: 1. It exhibits high affinity to native rabbit erythrocytes and to human erythrocytes of the 0 blood group, but not to trypsin-treated ones. The hemagglutination (HA) caused by lectin was not blocked by any of the 25 tested mono-, di-, and amino sugars; it was also not blocked by some of glyco derivatives. Only 13.9 μg/ml of the homogeneous preparation of a polysaccharide, a linear D-rhamnan with the structure of the repeated component →2)-β-D-Rhap-(1→3)-α-D-Rhap-(1→3)-α-D-Rhap-(1→2)-α-D-Rhap-(1→2)-α-sD-Rhap-1(→ blocked hemagglutination completely. The analysis of the amino acid composition of the lectin showed the greatest percentage of amino acids with positively charged R groups, arginine, lysine, and histidine, as well as the complete absence of sulfurcontaining amino acids, cysteine, and methionine. D-glucose and D-glucosamine were detected in the carbohydrate part. Original Russian Text ? L.V. Stepanova, V.E. Nikitina, A.S. Boiko, 2007, published in Mikrobiologiya, 2007, Vol. 76, No. 4, pp. 488–493.     

Two new steroid glycosides from the Far East starfish <Emphasis Type="Italic">Hippasteria kurilensis</Emphasis>

Two new steroid glycosides were isolated from the Far East starfish Hippasteria kurilensis collected in the Sea of Okhotsk. They were characterized as (22E,24R)-3-O-(2-O-methyl-β-D-xylopyranosyl)-24-O-[2-O-methyl-β-D-xylopyranosyl-(1→5)-α-L-arabinofuranosyl]-5α-cholest-22-ene-3β,4β,6α,7α,8,15β,24-heptaol (kurilensoside I) and (24S)-3-O-(2-O-methyl-β-D-xylopyranosyl)-24-O-(α-L-arabinofuranosyl)-5α-cholestane-3β,4β,6β,15α,24-pentaol (kurilensoside J). In addition, the earlier known glycosides linkosides F and L1, leviusculoside G, forbeside L, desulfated echinasteroside, and granulatoside A were isolated and identified. The structures of the new compounds were established with the help of two-dimentional NMR spectroscopy and mass- spectrometry.     

Polyhydroxylated steroid compounds from the Far Eastern starfish <Emphasis Type="Italic">Distolasterias nipon</Emphasis>

Two new steroid glycosides: distolasteroside D₆, (24S)-24-O-(β-D-xylopyranosyl)-5α-cholestane-3β,6α,8,15β,16β,24-hexaol, and distolasteroside D₇, (22E,24R)-24-O-(β-D-xylopyranosyl)-5α-cholest-22-ene-3β,6α,8,15β,24-pentaol were isolated along with the previously known distolasterosides D₁, D₂, and D₃, echinasteroside C, and (25S)-5α-cholestane-3β4β,6α,7α,8,15α,16β,26-octaol from the Far Eastern starfish Distolasterias nipon. The structures of new compounds were elucidated by NMR spectroscopy and MALDI TOF mass spectrometry. Like neurotrophins, distolasterosides D₁, D₂, and D₃ were shown to induce neuroblast differentiation in a mouse neuroblastoma C1300 cell culture.     

Neuro-and psychotropic activity of <Emphasis Type="Italic">N</Emphasis>-uronoylamino acids and <Emphasis Type="Italic">N</Emphasis>-uronoylpeptides

Peculiarities of the rat behavior were studied in a series of experimental stress models after a systemic administration of new N-uronoyl derivatives of amino acids. The psychotropic effect was shown to be determined by the nature of the amino acid fragment. N-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyraneuronoyl)-glycylglycine exhibited an anxiolytic effect more pronounced than that of pyracetam, whereas N-(1,2:3,4-di-O-isopropilidene-α-D-galactopyranuronoyl)-glycylglutamic acid has antidepressant action stronger than that of amitriptyline. Mechanisms for the psychotropic effects of the examined derivatives are discussed.     

A completed structure of the O-polysaccharide from <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> type 10

The structure of the O-specific polysaccharide from Shigella dysenteriae type 10, which has been reported previously in Bioorganic chemistry (1977, vol.3, pp. 1219–1225), is refined: →2)-β-D-Manp-(1→3)-α-D-ManpNAc-(1→3)-β-L-Rhap-(1→4)-α-D-GlcpNAc-(1→.     

Glycosidase-catalyzed Deoxy Oligosaccharide Synthesis. Practical Synthesis of Monodeoxy Analogs of Ethyl β-Thioisomaltoside Using Aspergillus niger α-Glucosidase

Enzymatic transglycosylation using four possible monodeoxy analogs of p-nitrophenyl α-D-glucopyranoside (Glcα-O-pNP), modified at the C-2, C-3, C-4, and C-6 positions (2D-, 3D-, 4D-, and 6D-Glcα-O-pNP, respectively), as glycosyl donors and six equivalents of ethyl β-D-thioglucopyranoside (Glcβ-S-Et) as a glycosyl acceptor, to yield the monodeoxy derivatives of glucooligosaccharides were done. The reaction was catalyzed using purified Aspergillus niger α-glucosidase in a mixture of 50 mM sodium acetate buffer (pH 4.0)/CH₃CN (1: 1 v/v) at 37°C. High activity of the enzyme was observed in the reaction between 2D-Glcα-O-pNP and Glcβ-S-Et to afford the monodeoxy analogs of ethyl β-thiomaltoside and ethyl β-thioisomaltoside that contain a 2-deoxy α-D-glucopyranose moiety at their glycon portions, namely ethyl 2-deoxy-α-D-arabino-hexopyranosyl-(1,4)-β-D-thioglucopyranoside and ethyl 2-deoxy-α-D-arabino-hexopyranosyl-(1,6)-β-D-thioglucopyranoside, in 6.72% and 46.6% isolated yields (based on 2D-Glcα-O-pNP), respectively. Moreover, from 3D-Glcα-O-pNP and Glcβ-S-Et, the enzyme also catalyzed the synthesis of the 3-deoxy analog of ethyl β-thioisomaltoside that was modified at the glycon α-D-glucopyranose moiety, namely ethyl 3-deoxy-α-D-ribo-hexopyranosyl-(1,6)-β-D-thioglucopyranoside, in 23.0% isolated yield (based on 3D-Glcα-O-pNP). Products were not obtained from the enzymatic reactions between 4D- or 6D-Glcα-O-pNP and Glcβ-S-Et.     

Oligonucleotides containing substituted 4-nitroindoles: Synthesis and study of their DNA duplexes

The synthesis of oligonucleotides containing 1-(2-deoxy-β-D-ribofuranosyl)-2-methyl-4-nitroindole and 1-(2-deoxy-β-D-ribofuranosyl)-2-phenyl-4-nitroindole is described. The synthesized modified oligonucleotides were used for studying the stability of intermolecular DNA duplexes with one unnatural strand and for evaluation of discriminating potential of 2-methyl-and 2-phenyl-4-nitroindoles toward nucleic bases. For comparison, an unmodified oligonucleotide and oligonucleotides bearing 5-nitroindole were used. It was shown that 2-methyl-4-nitroindole was only insignificantly inferior in stability to 5-nitroindole and characterized by a similar discriminating potential. 2-Phenyl-4-nitroindole provided a more pronounced duplex destabilization, the discrimination toward natural bases being decreased.     

Steroid compounds from two pacific starfish of the genus <Emphasis Type="Italic">Evasterias</Emphasis>

Three new steroid glycosides (evasteriosides C, D, and E) along with six known compounds were isolated from two Pacific starfish of the genus Evasterias. Evasterioside C from E. retiferacollected from the Sea of Japan was identified as (20R, 22E)-3-O-(β-D-xylopyranosyl)-24-nor-5α-cholest-22-ene-3β,6β,15α,26-pentaol 26-sulfate sodium salt. The structures of evasteriosides D and E from E. echinosoma (collected from the Gulf of Shelichov, the Sea of Okhotsk) were established as (20R, 24S)-24-O-(β-D-glucopyranosyl)-5α-cholestane-3β,6α,8,15β,24-pentaol and (20R,24S)-3,24-di-O-(β-D-xylopyranosyl)-cholest-4-ene-3β,6β,8,15α,24-pentaol, respectively. In addition, the known compounds pycnopodiosides A and C, luridoside A, 5α-cholestane-3β,6α,8,15β,16β,26-hexaol. 5α-Cholestane-3β,6α,8,15β,24-pentaol 24-sulfate sodium saltand marthasterone sulfate sodium salt were identified in E. echinosoma. The structures of the isolated compounds were established on the basis of spectroscopic analyses, using 1D and 2D NMR techniques, mass spectrometry, and some chemical transformations.     

Steroid Compounds from Far Eastern Starfishes <Emphasis Type="Italic">Henricia aspera</Emphasis> and <Emphasis Type="Italic">H. tumida</Emphasis>

Six new natural compounds were isolated from two Far Eastern starfish species, Henricia aspera and H. tumida, collected in the Sea of Okhotsk. Two new glycosylated steroid polyols were obtained from H. aspera: asperoside A and asperoside B, which were shown to be (20R,24R, 25S)-3-O-(2,3-di-O-methyl-β -D-xylopyranosyl)-24-methyl-5α-cholest-4-ene-3β, 6β,8,15α,16β,26-hexaol and (20R, 24R,25S,22E)-3-O-(2,4-di-O-methyl-β-D-xylopyranosyl)-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,26-hexaol, respectively. Two other glycosylated polyols, tumidoside A, with the structure elucidated as (20R, 22E)-3-O-(2,4-di-O-methyl-β -D-xylopyranosyl)-26,27-dinor-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,25-hexaol, and tumidoside B, whose structure was elucidated as (20R,24S)-3-O-(2,3-di-O-methyl-β-D-xylopyranosyl)-5α-cholestan-3β,4β,6β,8,15α,24-hexaol, were isolated from the two starfish species. (20R, 24S)-5α-Cholestan-3β,6β,15α,24-tetraol and (20R, 24S)-5α-cholestan-3β,6β,8,15α,24-pentaol were identified only in H. tumida. The known monoglycosides henricioside H1 and laeviuscolosides H and G were also identified in both species.     

Synthesis of oligosaccharide fragments of mannan from <Emphasis Type="Italic">Candida albicans</Emphasis> cell wall and their BSA conjugates

3-Aminopropyl glycosides of α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, α-D-mannopyranosyl-(1→3)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, and α-D-mannopyranosyl-(1→2)-[α-D-mannopyranosyl-(1→3)]-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose were efficiently synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-1-thio-α-D-mannopyranoside, and 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl bromide. The oligosaccharide chains synthesized correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the third pentasaccharide with bovine serum albumin was prepared using the squarate method.     

Synthesis of 11-phenoxyundecyl phosphate and its use as a substrate-acceptor in the reaction with UDP-GlcNAc: polyprenyl phosphate GlcNAc-phosphotransferase from <Emphasis Type="Italic">Salmonella arizona</Emphasis> O:59

A new scheme of synthesis of 11-phenoxyundecyl phosphate from 11-bromoundecanoic acid was suggested; its ability to serve as an acceptor of 2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate in a reaction catalyzed by UDP-N-acetylglucosamine: polyprenyl phosphate N-acetylglucosamine phosphotransferase from Salmonella arizona O:59 was demonstrated.     

General assay for enzymes in the heptose biosynthesis pathways using electrospray ionization mass spectrometry

The ADP-l-glycero-β-d-manno-heptose and the GDP-6-deoxy-α-d-manno-heptose biosynthesis pathways play important roles in constructing lipopolysaccharide of Gram-negative bacteria. Blocking the pathways is lethal or increases antibiotic susceptibility to pathogens. Therefore, the enzymes involved in the pathways are novel antibiotic drug targets. Here, we designed an efficient method to assay the whole enzymes in the pathways using mass spectrometry and screened 148 compounds. One promising lead is (?)-nyasol targeting d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) included in the GDP-6-deoxy-α-d-manno-heptose biosynthesis pathway from Burkholderia pseudomallei. The inhibitory activity of the lead compound against HddC has been confirmed by blocking the system transferring the guanosine monophosphate (GMP) moiety to α-d-glucose-1-phosphate. (?)-Nyasol exhibits the half maximal inhibitory concentration (IC50) value of 17.6 μM. A further study is going on using (?)-nyasol derivatives to find better leads with high affinity.     

Characterization of two novel bacterial type A <Emphasis Type="Italic">exo</Emphasis>-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-d-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N′-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-d-glucosaminide (1 → 4)-β-linkages and are thus “exo-chitobiose hydrolases.” In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.     

Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives

Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG₁, SG_?1, SG_?2, SG_?3, SG_?4, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG₁ and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1–4Glcα1–4Glcα1–4Glcα1–4Glcα1–4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc _m -AltNAc-Glc _n -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG₁ –SG_?4 subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V _derivative) with that toward PA-0 (V _PA-0), we found that the value of V _derivative/V _PA-0 decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.     

Synthesis of 2-Acetamido-2-deoxy-1,6-dithio-d-glucose and its Derivatives

2-Acetamido-3,4-di-Oacetyl-2,6-dideoxy-6-S-acetyl-6-thio-d-glucopyranosyl chloride (III) was condensed with potassium thiolacetate, potassium ethylxanthate or thiourea to give three crystalline derivatives of 2-acetamido-2-deoxy-1,6-dithio-d-glucose. An attempt to prepare 2-acetamido-1,2,6-trideoxy-1,6-dimercapto-D-glucose (VII) from 2-acetamido-3,4-di-O-acetyl-1,2,6-trideoxy-1,6-di-S-acetyl-1,6-dithio-β-d-glucopyranose was described. 2-Acetamido-3,4-di-O-acetyl-1,2,6-trideoxy-1-mercapto-6-S-acetyl-6-thio-β-d-glucopyranose (VIII) was synthesized from the condensation product of III with thiourea.     

Synthesis of heteroaromatic <Emphasis Type="Italic">N</Emphasis>-β-glycosides of <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine under phase transfer conditions: II. Indolin-2-one glycosaminides

Regioselective N-β-glucosamination of various unsubstituted or C4-, C5-, or C6-monosubstituted indolin-2-ones under phase transfer conditions was studied. The regioselectivity was unambiguously proved by ¹H NMR spectroscopy and X-ray analysis. The presence of substituent at C7 of the aromatic ring leads to the formation of either a mixture of isomeric N-β-and O-β-D-glucosaminides, or only oxazoline and/or 2-acetamidoglycal irrespective of the reaction conditions.