Synthesis of docosasaccharide arabinan motif of mycobacterial cell wall

Mycobacterial arabinan is a common constituent of both arabinogalactan (AG) and lipoarabinomannan (LAM). In this study, synthesis of β-Araf containing common arabinan docosasaccharide motif (22 Araf monomer units) of mycobacterial cell wall was achieved. Our synthetic strategy toward arabinan involves (1) the stereoselective β-arabinofuranosylation using both 3,5-O-TIPDS-protected and NAP-protected arabinofuranosyl donors for straightforward intermolecular glycosylation and intramolecular aglycon delivery (IAD), respectively, and (2) the convergent fragment coupling with branched fragments at the linear sequence using thioglycoside donor obtained from the corresponding acetonide at the reducing terminal of each fragment through a three-step procedure. Because the acetonide at the reducing terminal of all fragments would be converted to thioglycoside as the glycosyl donor, and mainly Bn ether protections were used, our strategy will be readily applicable to the synthesis of more complex arabinan, arabinogalactan, and arabinomycolate derived from mycobacterial CWS.     

Rapid and efficient conversion of sialyl thioglycosides to sialyl esters via NIS/BF3OEt2-promoted glycosylation

A rapid and efficient one-step conversion of sialyl thioglycosides to sialyl esters was disclosed. Under the promotion of NIS and BF₃OEt₂, the glycosylation of per-acetylated sialyl thioglycoside with a set of carboxylic acids provided β-sialyl esters as the major products in good to excellent yields within 5 min. Compared with the long-chain alkyl-, aryl- and α,β-unsaturated acids, complete β-selectivities were observed when the short-chain alkyl acids were selected as the coupling partners. The resultant β-selectivity for the glycosylation of the per-acetylated sialyl thioglycoside with acetic acid was compromised when the 5-N,4-O-oxazolidinone protected sialyl thioglycoside was employed as the coupling partner.     

A synthetic glycan array containing Cryptococcus neoformans glucuronoxylomannan capsular polysaccharide fragments allows the mapping of protective epitopes

A convergent synthetic strategy to Cryptococcus neoformans glucuronoxylomannan (GXM) capsular polysaccharide part structures was developed based on di-, tri-, tetra-, penta- and hexasaccharide thioglycoside building blocks. The approach permitted the synthesis of a library of spacer-containing serotype A and D related GXM oligosaccharide structures, ranging from di- to octadecasaccharides. Ten deprotected GXM compounds (mono- to decasaccharide) were printed onto microarray plates and screened with seventeen mouse monoclonal antibodies (mAbs) to GXM. For the first time a GXM oligosaccharide structure (a serotype A decasaccharide), capable of being recognized by neutralizing forms of these GXM-specific mAbs, has been identified, offering insight into the binding epitopes of a range of protective monoclonal antibodies and furthering our efforts to develop semi-synthetic conjugate vaccine candidates against C. neoformans.

A library of Cryptococcus neoformans glucuronoxylomannan (GXM) oligosaccharides was synthesized using a thioglycoside building block strategy. The first GXM microarray was printed, allowing mapping of epitopes of antibodies directed towards GXM.     

Synthesis of a d,d- and l,d-heptose-containing hexasaccharide corresponding to a structure from Haemophilus ducreyi lipopolysaccharides

The synthesis of a linear hexasaccharide, 2-(4-trifluoroacetamidophenyl)ethyl (β-d-galactopyranosyl)-(1→4)-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)-(β-d-galactopyranosyl)-(1→4)-(d-glycero-α-d-manno-heptopyranosyl)-(1→6)-(β-d-glucopyranosyl)-(1→4)-l-glycero-α-d-manno-heptopyranoside, corresponding to a structure found in Haemophilus ducreyi LPS, is described. A Barbier reaction between benzyloxymethyl chloride and a properly protected 6-aldo-1-thio-mannopyranoside yielded both the d,d- and the l,d-heptopyranoside (2 and 3, ratio 2:3), which were separated and both used in the synthesis. p-Methoxybenzyl and chloroacetyl groups were employed as temporary protecting groups, selectively removed in the presence of the persistent benzyl, acetyl, benzoyl and isopropylidene groups by treatment with DDQ/H₂O and hydrazine dithiocarbonate, respectively. Thioglycosides were utilised as donors throughout using either NIS/TfOH or DMTST as promoters. The introduction of the spacer into thioglycoside 5 was high-yielding (95%) but with low stereoselectivity (α:β 5:3). All other glycosylations are completely stereoselective. The target hexasaccharide is obtained via a 3+3 block approach with the yield in the final NIS/TfOH-promoted coupling between an N,N-diacetyl-trisaccharide thioglycosyl donor 20 and a 4′′-OH trisaccharide acceptor 13 being 75%.     

Total Synthesis of a Hyperbranched N‐Linked Hexasaccharide Attached to ATCV‐1 Major Capsid Protein without Precedent

The N‐glycans attached to some chloroviruses comprise a hyperbranched core structure without precedent. We are interested in the chemical synthesis of the hexasaccharide attached to ATCV‐1 (Acanthocystis turfacea Chlorella virus 1) for its distinct structure. After exploring four routes, the target hexasaccharide 2 was successfully synthesized for the first time in overall 10% yield over 8 steps from thioglycoside building blocks. This synthetic protocol is characterized by the three‐component one‐pot glycosylation and the regioselective glycosylation reactions. The disclosed synthetic approach to this new type of N‐glycans will facilitate the in‐depth understanding of their biological functions.     

Synthesis of glycosaminoglycan oligosaccharides. Part 5: Synthesis of a putative heparan sulfate tetrasaccharide antigen involved in prion diseases

The synthesis of the putative prion-associated heparan sulfate tetrasaccharide containing two d-glucuronic acid units is reported. Key to the synthesis were the differentiation of the N-acetylated and N-unsubstituted glucosamine units, the high-yielding glucosylation at O-4 of an N-acetyl-d-glucosamine derivative and the α-selective glycosylation of the 4′-OH group of a β-d-GlcpA-(1→4)-d-GlcpNAc disaccharide building block with a disaccharide thioglycoside donor.     

Chemical trans-glycosylation of bioactive glycolinkage: synthesis of an α-lycotetraosyl cholesterol

The aim of this study was to verify the antitumor role of the β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-d-galactopyranosyl (lycotetraosyl) moiety present in steroidal glycosides from Solanaceous plants. We explored a new chemical trans-glycosylation method using an endoglycosidase called tomatinase that is produced by the tomato pathogen, Fusarium oxysporum f. sp. lycopersici. The lycotetraose, which was prepared by enzymatic hydrolysis of α-tomatine with tomatinase, was converted to glycosyl donors such as trichloroacetimidate, fluoride, and thioglycoside. All obtained glycosyl donors were glycosylated with cholesterol to form α-lycotetraosyl cholesterols in a stereoselective manner. The obtained lycotetraosyl derivatives together with typical natural lycotetraosyl glycosides were examined for their antiproliferative activity.     

Convergent synthesis of the tetrasaccharide repeating unit related to the O-antigenic polysaccharide of Escherichia coli 78

A convergent synthesis of the tetrasaccharide repeating unit of the O-antigenic cell wall polysaccharide of Escherichia coli 78, as the corresponding methyl glycoside (I), is being reported. It involved stereoselective glycosidation of a β-linked mannodisaccharide acceptor with a β-linked glucosamine based disaccharide thioglycoside donor, which were prepared from the corresponding functionalised monosaccharide based glycosyl donors and acceptors. The resulting tetrasaccharide derivative was finally converted to (I) by selective deprotection and also by global protection and deprotection techniques.     

Synthesis of the hexasaccharide repeating unit corresponding to the cell wall lipopolysaccharide of Azospirillum irakense KBC1

A convenient chemical synthesis of the hexasaccharide repeating unit of the cell wall lipopolysaccharide of Azospirillum irakense KBC1 has been successfully achieved. A stereo- and regioselective [4 + 2] block glycosylation strategy has been used to obtain the target hexasaccharide as its octyl glycoside. All synthetic intermediates have been prepared in high yields from commercially available reducing sugars following a series of protection–deprotection reactions. An oxidation–reduction methodology has been applied to convert β-d-glucosidic unit to a β-d-mannosidic moiety.     

Chemical Synthesis of Asparagine‐Linked Archaeal N‐Glycan from Methanothermus fervidus

Several N‐linked glycoproteins have been identified in archaea and there is growing evidence that the N‐glycan is involved in survival and functioning of archaea in extreme conditions. Chemical synthesis of the archaeal N‐glycans represents a crucial step towards understanding the putative function of protein glycosylation in archaea. Herein the first total synthesis of the archaeal L ‐asparagine linked hexasaccharide from Methanothermus fervidus is reported using a highly convergent [3+3] glycosylation approach in high overall yields. The synthesis relies on efficient preparation of regioselectively protected thioglycoside building blocks for orthogonal glycosylations and late stage N‐aspartylation.     

Co- and homocyclotrimerization reactions of protected 1-alkynyl-2-deoxyribofuranose. Synthesis of C-nucleosides, C-di- and C-trisaccharide analogues

Cyclotrimerization of β- or α-ethynyl-3,5-di-O-toluoyl-2-deoxy-d-ribofuranose with α,ω-diynes proceeded smoothly under Rh-catalysis to afford the corresponding β- or α-benzene C-nucleoside derivatives. Analogous co-cyclotrimerization of α- or β-propynyl- and -phenylethynyl-3,5-di-O-toluoyl-2-deoxy-d-ribofuranose with α,ω-diynes gave the corresponding arene derivatives only under microwave irradiation in the presence of a Rh-catalyst in moderate yields. Attempted homocyclotrimerization of β- or α-ethynyl-3,5-di-O-toluoyl-2-deoxy-d-ribofuranose under Rh-catalysis led only to enynes while the use of Ru-catalyst gave the desired 1,2,4- and 1,3,5-tri-(2-deoxyribofuranose-1-yl)benzene.     

Comprehensive synthesis of ER related high-mannose-type sugar chains by convergent strategy

Systematic synthesis of high-mannose-type sugar chains of asparagine-linked glycoproteins is described. To construct the target sugar chains, we employed the convergent route, using three oligosaccharide components, the common hexasaccharide, branched tri-, tetra- and pentasaccharides, and mono-, di-, and triglucosyl fragments. Construction of the β-mannoside linkage was performed using p-methoxybenzyl-assisted intramolecular aglycon delivery. The hexasaccharide fragment was coupled with the branched mannooligosaccharide donors such as M5, M4B, M4C, and M3 to give undecasaccharide (M9), decasaccharide (M8B and M8C), and nonasaccharide (M7), respectively. Incorporation of mono-, di-, and triglucosyl fragments toward them gave tetradecasaccharide (G3M9), tridecasaccharide (G2M9), dodecasaccharide (G1M9), undecasaccharide (G1M8B and G1M8C), and decasaccharide (G1M7), respectively.     

Synthesis of aminoethyl glycoside of the oligosaccharide chain of ganglioside Fuc-GM1

2-Aminoethyl glycoside of the hexasaccharide chain of ganglioside Fuc-GM₁ was synthesized by a [3+3] synthetic scheme. At the key step of the synthetic route, glycosylation of the only hydroxyl group at C(4) of the galactose residue in an α-(N-acetylneuraminyl)-(2→3)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside derivative with a peracetylated thioglycoside of α-L-fucopyranosyl-(1→2)-β-D-galactopyranosyl-(1→3)-2-trichloroacetamido-2-deoxy-β-D-galactopyranose gave a protected hexasaccharide in high yield. Subsequent removal of the protecting groups gave the target 2-aminoethyl glycoside of the oligosaccharide chain of gan-glioside Fuc-GM₁. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 148–153, January, 2006.     

Peculiarities of the Mass Spectrometric Detection of Anthocyanins in High-Performance Liquid Chromatography

The dependence of the degree of fragmentation of anthocyanin “molecular” ions on the cone voltage of a mass spectrometric electrospray ionization detector was studied. It was found that the voltage required for the fragmentation of 50% of original “molecular” ions, E _f (0.5), increased with the number of glycoside residues. The fragmentation of glycosides proceeds with the removal of the entire residue regardless of their structures. In the case of 3,5-diglycosides, two types of fragment ions formed due to the loss of glycosidic residues from different positions; the ratio of their peak intensities is reciprocal to the ratio of the masses of residues eliminated. The values of E _f (0.5) for monoglycosides (190 V), diglycosides (229 V), triglycosides (267 V), and for some acylated cyanidin-3,5-diglycosides are determined. These results were given for the gradient separation of anthocyanins by reversed-phase HPLC in aqueous acetonitrile containing 10 vol % of formic acid using quadrupole mass spectrometric detection.     

Synthesis of penta- and hexasaccharide fragments corresponding to the O-antigen of Escherichia coli O150

The chemical synthesis of a pentasaccharide and a hexasaccharide corresponding to the O-antigen of Escherichia coli O150 has been achieved using sequential glycosylation and [3+3] block glycosylation strategies. Suitably protected monosaccharide synthons have been prepared from the commercially available reducing sugars and then stereoselectively coupled to give the pentasaccharide and a hexasaccharide in excellent yields. 4-Methoxyphenyl and 2-(4-methoxyphenoxy) ethyl groups have been used as the anomeric-protecting groups in the target pentasaccharide and a hexasaccharide, respectively.     

Vilsmeier-Haak formylation of 3,5-dimethylpyrazoles

Formylation of N-alkyl-3,5-dimethyl-1H-pyrazoles according to Vilsmeier-Haak led to the formation of the corresponding 4-formyl derivatives. 3,5-Dimethyl-1H-pyrazole having no substituent on the nitrogen atom failed to undergo formylation at the 4 position under analogous conditions. 3,5-Dimethyl-1H-pyrazole-4-carbaldehyde was synthesized by alkaline hydrolysis of methyl β-(4-formyl-3,5-dimethyl-1H-pyrazol-1-yl)propionate and subsequent heating of the acid thus formed.     

Reactions of 3,5-dibromo-1-(thiiran-2-ylmethyl)-1,2,4-triazole with NH-azoles

Reactions of 3,5-dibromo-1-(thiiran-2-ylmethyl)-1,2,4-triazole with 3,5-dimethylpyrazole, 1,3-dimethyl-3,7-dihydropurine-2,6-dione, 3,5-dibromo-1,2,4-triazole, 2,4,5-tribromoimidazole, and 2-chlorobenzimidazole lead to the formation of 5-azolylmethyl-2-bromo-5,6-dihydrothiazolo[3,2-b]-1,2,4-triazoles. In the case of 8-bromo-1,3-dimethyl-3,7-dihydropurine-2,6-dione the intermediate thiolate anion undergoes cyclization into 7-[(3,5-dibromo-1,2,4-triazol-1-yl)methyl]-1,3-dimethyl-6,7-dihydrothiazolo[2,3-f]purine-2,4(1H,3H)-dione. The structure of reaction products depends on the relative rate of substitution of leaving groups in the reagents.     

Stereoselectivity investigation on glycosylation of oxazolidinone protected 2-amino-2-deoxy-d-glucose donors based on pre-activation protocol

Diverse 2,3-oxazolidinone protected 2-amino-2-deoxy-d-glucose thioglycosides were prepared and studied as glycosyl donors at low temperature by BSM/Tf₂O pre-activation protocol before the addition of glycosyl acceptors. The stereochemistry outcomes of a series of glycosylations were investigated. Different stereoselectivities of the coupling reactions were obtained, arising from the different protecting groups in the oxazolidinone donors. 4,6-Di-O-benzyl-N-benzyl-oxazolidinone protected thioglycoside donor 1c underwent glycosylation with general β-anomeric selectivity and the stereoselectivity could be also affected by glycosylation conditions.     

Synthesis of oligosaccharides as potential novel food components and upscaled enzymatic reaction employing the β-galactosidase from bovine testes

The β-galactosidase from bovine testes (EC 3.2.1.23) promotes the transfer of a galactose unit to glucose or galactose-containing residues in manifold derivatives, establishing β1→3 linkages.The synthesis of several potentially biologically important oligosaccharides β-d-Galp-(1→3)-α-d-Glcp-(1→2)-β-d-Fruf2, β-d-Galp-(1→3)-β-d-Galp-(1→4)-α,β-d-Glcp4, β-d-Galp-(1→3)-α-d-Glcp-(1→4)-d-Glcp-ol/Manp-ol 6, β-d-Galp-(1→3)-α-d-Glcp-(1→6)-β-d-Fruf8, β-d-Galp-(1→3)-α-d-Glcp-(1→6)-[α-d-Glcp-(1↔2)]-β-d-Fruf10, α-d-Galp-(1→6)-[β-d-Galp-(1→3)]-α-d-Glcp-(1↔2)-β-d-Fruf12, β-d-Galp-(1→3)-α-d-Glcp-(1↔2)-β-d-Fruf-(1↔2)-β-d-Fruf14 has been reached in yields between 7 and 44% by implementation of this specific enzyme. In addition, we found that it is feasible to gain high yields without an enzyme-specific buffer and even making upscaled preparation on a gram scale.     

Reaction of acetylated carbohydrates with trimethylaluminum: concise synthesis of 1,2-O-isopropylidene d-ribofuranose

Treatment of β-d-ribose tetraacetate with trimethylaluminum gives α-3,5-O-acetyl-1,2-O-isopropylidene-d-ribofuranoside in excellent yield. This reaction allows for efficient and high-yielding installation of the 1,2-isopropylidene acetal (acetonide), which is difficult to prepare using more traditional acid-catalyzed methods. The reaction of trimethylaluminum with other per-acetylated carbohydrates is also described.