Mild and General Protocol for Selective Deacetylation of Acetyl/Benzoyl-Protected Carbohydrates.

Herein, we report a mild and general protocol for chemoselective deacetylation of mixed acetyl- and benzoyl-protected carbohydrates under mild acidic conditions. The protocol allows quick access to partially protected carbohydrates, which serve as versatile synthetic intermediates during the total synthesis of various mono- and oligosaccharide targets. The applicability of the developed protocol was successfully demonstrated on a range of carbohydrate substrates of various configurations and substitution patterns featuring functionalized aliphatic and aromatic aglycones. The protocol has shown excellent compatibility with the widely used O-anomeric protecting groups, prespacer aglycones, and thioglycoside glycosyl donors.

Unusual triflic acid-promoted oligomerization of arabinofuranosides during glycosylation.

We discovered an unusual triflic acid-promoted oligomerization of arabinofuranosides during glycosylation of the primary hydroxy group of α-(1 â†’ 5)-linked tetraarabinofuranoside bearing 4-(2-chloroethoxy)phenyl aglycone with α-(1 â†’ 5), ß-(1 â†’ 2)-linked tetraarabinofuranoside containing N-phenyltrifluoroacetimidoyl leaving group, which led to octa-, dodeca- and hexadecaarabinofuranosides. The possible mechanism of triflic acid-promoted oligomerization was proposed. The choice of promoter was found to be a critical factor for the discovered oligomerization of arabinofuranosides. The obtained octa-, dodeca- and hexadecaarabinofuranosides may serve as useful blocks in the synthesis of oligosaccharide fragments of polysaccharides of Mycobacterium tuberculosis.

Comparison of glycosyl donors: a supramer approach.

The development of new methods for chemical glycosylation commonly includes comparison of various glycosyl donors. An attempted comparison of chemical properties of two sialic acid-based thioglycoside glycosyl donors, differing only in the substituent at O-9 (trifluoroacetyl vs chloroacetyl), at different concentrations (0.05 and 0.15 mol·L-1) led to mutually excluding conclusions concerning their relative reactivity and selectivity, which prevented us from revealing a possible influence of remote protective groups at O-9 on glycosylation outcome. According to the results of the supramer analysis of the reaction solutions, this issue might be related to the formation of supramers of glycosyl donors differing in structure hence chemical properties. These results seem to imply that comparison of chemical properties of different glycosyl donors may not be as simple and straightforward as it is usually considered.

Synthesis and study of the glycosyl-donor properties of 2-(2,2,2-trichloroethoxy)-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-galactopyrano)-[2,1-d]-2-oxazoline.

A synthesis of 2-(2,2,2-trichloroethoxy)-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-galactopyrano)-[2,1-d]-2-oxazoline - a previously unknown 2-alkoxy glyco-[2,1-d]-2-oxazoline derivative with d-galacto configuration was carried out. Glycosylating activity of the obtained galactooxazoline has been studied and it has been shown that in the presence of a weak protic acid, such as sym-collidinium triflate, this substance exhibits properties of a reactive and 1,2-trans-stereoselective glycosyl donor. The homopolymerization reaction of oxazoline derivatives of sugars has been found to proceed under the same conditions, leading to the formation of pseudo-oligosaccharide products. It has been found that this undesirable side reaction could be suppressed by changing the acid catalyst concentration, resulting in the development of efficient methods for the synthesis of glycoside and oligosaccharide derivatives of ß-d-galactosamine using the synthesized 2-(2,2,2-trichloroethoxy)-2-oxazoline glycosyl donor under very mild conditions.

Synthesis of sialyl halides with various acyl protective groups.

Glycosyl halides are historically one of the first glycosyl donors used in glycosylation reactions, and interest in glycosylation reactions involving this class of glycosyl donors is currently increasing. New methods for their activation have been proposed and effective syntheses of oligosaccharides with their participation have been developed. At the same time, the possibilities of using these approaches to the synthesis of sialosides are restricted by the limited diversity of known sialyl halides (previously, mainly sialyl chlorides, less often sialyl bromides and sialyl fluorides, with acetyl (Ac) groups at the oxygen atoms and AcNH, Ac2N and N3 groups at C-5 were used). This work describes the synthesis of six new N-acetyl- and N-trifluoroacetyl-sialyl chlorides and bromides with O-chloroacetyl and O-trifluoroacetyl protective groups. Preparation of N,O-trifluoroacetyl protected derivatives was made possible due to development of the synthesis of sialic acid methyl ester pentaol with N-trifluoroacetyl group.

TIPS group-assisted isomerization of benzyl protected d-manno- and d-glucopyranose to d-fructofuranose derivatives.

Base-promoted (MeONa in MeOH or imidazole in DMF) isomerization of a series of 3,4,6-tri-O-benzyl-d-gluco- and d-mannopyranose derivatives with triisopropylsilyl (TIPS) substituents was studied. The presence of a bulky TIPS group at O-1 or O-2 was shown to be favorable for the isomerization of benzyl protected d-gluco- and d-mannopyranose derivatives to d-fructofuranose derivatives, in which the bulky silyl group occupies less sterically hindered primary position. The highest yield (33%) of the fructofuranose derivative was achieved when 3,4,6-tri-O-benzyl-2-O-triisopropylsilyl-d-mannopyranose was treated with MeONa in MeON at 50 °C.

New methods for the synthesis of 2-(2,2,2-trichloroethoxy)-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline and its use for stereo-, chemo- and regioselective glycosylation.

New methods for the synthesis of the title oxazoline 2 from the corresponding 2-deoxy-2-(2,2,2- trichloroethoxycarbonylamino)glucosyl bromide were developed. The target 2-(2,2,2-trichloroethoxy) gluco-[2,1-d]-2-oxazoline 2 can be synthesized under conditions of halide ion catalysis, using triethylamine as a base. The synthesized 2-(2,2,2-trichloroethoxy)-2-oxazoline glycosyl donor was used for stereo-, regio-, and chemoselective glycosylation reactions under extremely mild conditions. The undesirable side reaction of intermolecular aglycone transfer between an ethyl thioglycoside glycosyl acceptor and the 2-(2,2,2-trichloroethoxy)-2-oxazoline glycosyl donor occurred to a relatively small extent. Regio-, and chemoselectivity of the disaccharide synthesis with the oxazoline glycosyl donor depended on the reaction conditions.

Exhaustive Conformational Search for Sialyl Cation Reveals Possibility of Remote Participation of Acyl Groups.

Finding convenient ways for the stereoselective α-sialylation is important due to the high practical significance of α-sialic acid-containing glycans and neoglycoconjugates. It was proposed that sialylation stereoselectivity is determined by the structure of the sialyl cation (also known in biochemistry as "sialosyl cation"), a supposed intermediate in this reaction. Here we design a new approach for studying the conformational space of highly flexible sialyl cation and find 1625 unique conformers including those stabilized by covalent remote participation (also known as long-range participation) of 4-O-acetyl (4-OAc), 5-N-trifluoroacetyl (5-NTFA), as well as 7,8,9-OAc from both α and ß sides. The most energetically stable sialyl cation conformers are featured by 4-OAc participation, closely followed by 5-NTFA- and 7-OAc-stabilized conformers; unstabilized sialyl cation conformers are ∼10 kcal mol-1 less stable than the 4-OAc-stabilized ones. Analysis of all the obtained conformers by means of substituents positions, side chain conformations and ring puckering led us to a new "eight-conformer hypothesis" which describes interconversions among the most important sialyl cation conformers and predicts that stronger remote participation of acyl groups favors ß-anomers. Thus, selective synthesis of the desired α-sialosides requires minimization of acyl groups participation.

Pediatric Microbial Keratitis With Overnight Orthokeratology in Russia.

OBJECTIVES: To estimate the incidence of microbial keratitis in children wearing overnight orthokeratology lenses in Moscow, Russia. METHODS: To estimate the number of children wearing overnight orthokeratology lenses in 2018, 1,368 patient records from 13 Doctors Lens clinics were selected at random and extrapolated to the entire patient base. To identify cases of microbial keratitis, all episodes of infiltrative keratitis from Morozovskaya City Children's Clinical Hospital in 2018 were recorded and adjudicated by three experienced ophthalmologists. Incidence was calculated as the number of cases divided by the estimated number of wearers. RESULTS: There were 23,049 overnight orthokeratology fits between 2010 and 2018. Among the 1,368 records surveyed, 1,078 (79%) were younger than 18 years at initial fitting. Based on a 7% or 10% annual discontinuation rate, the estimated number of children wearing lenses in 2018 was 10,307 or 9,422, respectively. During 2018, there were 139 cases of corneal infiltrative keratitis in children and 45 were adjudicated as microbial keratitis (32%). Of these, 20 cases were associated with contact lens wear (44%): 15 soft lens wearers (33%) and 5 overnight orthokeratology wearers (11%). Based on the 5 cases and assuming a 7% or 10% discontinuation rate, the annual incidence is 4.9 (95% confidence interval[CI]: 2.1-11.4) or 5.3 (95% CI: 2.3-12.4) per 10,000 patient years. CONCLUSION: The incidence of microbial keratitis in children wearing overnight orthokeratology is lower than a previous United States study and similar to rates associated with use of daily wear soft contact lenses.

Heptabladed ß-propeller lectins PLL2 and PHL from Photorhabdus spp. recognize O-methylated sugars and influence the host immune system.

O-methylation is an unusual sugar modification with a function that is not fully understood. Given its occurrence and recognition by lectins involved in the immune response, methylated sugars were proposed to represent a conserved pathogen-associated molecular pattern. We describe the interaction of O-methylated saccharides with two ß-propeller lectins, the newly described PLL2 from the entomopathogenic bacterium Photorhabdus laumondii, and its homologue PHL from the related human pathogen Photorhabdus asymbiotica. The crystal structures of PLL2 and PHL revealed up to 10 out of 14 potential binding sites per protein subunit to be occupied with O-methylated structures. The avidity effect strengthens the interaction by 4 orders of magnitude. PLL2 and PHL also interfere with the early immune response by modulating the production of reactive oxygen species and phenoloxidase activity. Since bacteria from Photorhabdus spp. have a complex life cycle involving pathogenicity towards different hosts, the involvement of PLL2 and PHL might contribute to the pathogen overcoming insect and human immune system defences in the early stages of infection. DATABASES: Structural data are available in PDB database under the accession numbers 6RG2, 6RGG, 6RFZ, 6RG1, 6RGU, 6RGW, 6RGJ, and 6RGR.

Pectin-glycerol gel beads: Preparation, characterization and swelling behaviour.

Low methyl-esterified pectin (AU701) was found to form gel beads with glycerol. Wet AU701-glycerol gel beads exhibited similar diameter and hardness compared to the AU701-Ca gel beads, prepared by ionotropic gelation with Ca2+ and used for comparison. The morphology of dry pectin gel beads determined by scanning electron microscopy revealed that the beads exhibited rough and grooved surface. The AU701-glycerol gel beads absorbed more grams of water than AU701-Ca gel beads (12.2 g vs 3.9 g per 1 g of the beads). Rheological properties and hardness of the AU701-glycerol gel beads improved with the increase of the pectin/glycerol ratio. Swelling behavior of the AU701-glycerol gel beads was determined after sequential incubation in simulated gastric (SGF) and intestinal (SIF) fluids. The AU701-glycerol gel beads swelled in SGF to a greater extent and revealed higher stability in SIF than the gel beads cross-linked by Ca2+.

Preparation and in vivo evaluation of glyco-gold nanoparticles carrying synthetic mycobacterial hexaarabinofuranoside.

A number of bacterial glycans are specific markers for the detection and the serological identification of microorganisms and are also widely used as antigenic components of vaccines. The use of gold nanoparticles as carriers for glyco-epitopes is becoming an important alternative to the traditional conjugation with proteins and synthetic polymers. In this study, we aimed to prepare and evaluate in vivo glyco-gold nanoparticles (glyco-GNPs) bearing the terminal-branched hexaarabinofuranoside fragment (Ara6) of arabinan domains of lipoarabinomannan and arabinogalactan, which are principal polysaccharides of the cell wall of Mycobacterium tuberculosis, the causative agent of tuberculosis. In particular, we were interested whether the antibodies generated against Ara6-GNPs would recognize the natural saccharides on the cell surface of different mycobacterial strains. Two synthetic Ara6 glycosides with amino-functionalized spacer aglycons differing in length and hydrophilicity were directly conjugated with spherical gold nanoparticles (d = 15 nm) to give two sets of glyco-GNPs, which were used for the immunization of rabbits. Dot assays revealed cross-reactions between the two obtained antisera with the hexaarabinofuranoside and the 2-aminoethyl aglycon used for the preparation of glyco-GNPs. Both antisera contained high titers of antibodies specific for Mycobacteria as shown by enzyme-linked immunosorbent assay using M. bovis and M. smegmatis cells as antigens while there was only a weak response to M. phlei cells and no interaction with E. coli cells. The results obtained suggest that glyco-GNPs are promising agents for the generation of anti-mycobacterial antibodies.

Catalyst-free regioselective acetylation of primary hydroxy groups in partially protected and unprotected thioglycosides with acetic acid.

Highly regioselective acetylation of primary hydroxy groups in thioglycoside derivatives with gluco- and galacto-configurations was achieved by treatment with aqueous or anhydrous acetic acid (60-100% AcOH) at elevated temperatures (80-118 °C), avoiding complex, costly and time-consuming manipulations with protective groups. Acetylation of both 4,6-O-benzylidene acetals and the corresponding diols as well as the unprotected tetraol with AcOH was shown to lead selectively to formation of 6-O-acetyl derivatives. For example, the treatment of phenyl 1-thio-ß-d-glucopyranoside with anhydrous AcOH at 80 °C for 24 h gave the corresponding 6-O-acetylated derivative in 47% yield (71% based on the reacted starting material) and unreacted starting tetraol in 34% yield, which can easily be recovered by silica gel chromatography and reused in further acetylation.

Lectin PLL3, a Novel Monomeric Member of the Seven-Bladed ß-Propeller Lectin Family.

The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed ß-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.

Janus glycosides of next generation: Synthesis of 4-(3-chloropropoxy)phenyl and 4-(3-azidopropoxy)phenyl glycosides.

Efficient procedures for the preparative synthesis of per-O-acyl derivatives of 4-(3-chloropropoxy)phenyl (CPP) glycosides of a series of common mono- and disaccharides (d-glucose, d-galactose, d-mannose, l-rhamnose, d-arabinofuranose, d-glucosamine, lactose) are described. The CPP glycosides obtained were transformed in almost quantitative yields to the corresponding unprotected 4-(3-azidopropoxy)phenyl (APP) glycosides, which could become next-generation Janus glycosides with cleavable spacer aglycon, ready for conjugation or further transformation.

The use of the novel glycosyl acceptor and supramer analysis in the synthesis of sialyl-α(2-3)-galactose building block.

A new glycosyl acceptor to be used in sialylation was designed as a 3-hydroxy derivative of 4-methoxyphenyl ß-d-galactopyranoside with 2-O-acetyl group and O-4 and O-6 protected as benzylidene acetal. Two alternative syntheses of this compound were compared. Sialylation of 3-OH group of the glycosyl acceptor with O-chloroacetylated N-trifluoroacetylneuraminic acid phenyl thioglycoside (NIS, TfOH, MeCN, MS 3 Å, -40 °C) was studied in a wide concentration range (5-150 mmol L-1). The outcome of sialylation generally followed the predictions of supramer analysis of solutions of sialyl donor in MeCN, which was performed by polarimetry and static light scattering and revealed two concentration ranges differing in solution structure and the structures of supramers of glycosyl donor. The optimized conditions of sialylation (C = 50 mmol L-1) were used to synthesize protected Neu-α(2-3)-Gal disaccharide (78%, α:ß = 13:1), which was then converted to sialyl-α(2-3)-galactose imidate building block useful for the synthesis of complex sialo-oligosaccharides.

A Ring Contraction of 2,3-Di- O-Silylated Thiopyranosides To Give Thiofuranosides under Mildly Acidic Conditions.

A pyranose ring contraction of ethyl 1-thio-ß-d-galactopyranosides has been discovered that proceeds with retention of aglycon under mildly acidic conditions (aq TFA in CH2Cl2). Key factors for success of this rearrangement are the presence of bulky silyl (TIPS or TBDPS) substituents at both O-2 and O-3 and a free hydroxy group at C-4 (derivatives with acid-labile protective groups at O-4 will also engage in this reaction). The rearrangement cleanly proceeds for 2,3-di- O-TIPS derivatives with two hydroxy groups at C-4 and C-6, acid-labile TES groups at O-4 and O-6, or one acyl substituent (Bz, ClAc) at O-6. A possibility to switch the direction of the debenzylidenation reaction in 4,6- O-benzylidene-2,3-di- O-TIPS/TBDPS derivatives by the choice of an acid (TFA, which cleanly gives furanose, versus AcOH, which cleaves benzylidene acetal only) may present an advantage in the divergent synthesis of selectively protected glycosyl donors (either in furanose or pyranose form) useful for the synthesis of biologically important oligosaccharides.

Application of a Janus aglycon with dual function in benzyl-free synthesis of spacer-armed oligosaccharide fragments of polysaccharides from rhizobacterium Azospirillum brasilense sp7.

Both protective and pre-spacer features of 4-(2-chloroethoxy)phenyl (CEP) aglycon, which belong to the class of Janus aglycons, were engaged in a benzyl-free synthesis of oligosaccharide fragments of polysaccharides from rhizobacterium Azospirillum brasilense sp7. Introduction of α-1,4-linked L-fucose residue was performed using 3,4-di-O-benzoyl-2-O-triisopropylsilyl-α-L-fucopyranosyl N-phenyltrifluoroacetimidate in excellent stereoselectivity and high yields. The obtained deprotected di-, tri- and tetrasaccharides contain 4-(2-azidoethoxy)phenyl (AEP) spacer aglycon, which allows straightforward preparation of neoglycoconjugates that will be used for the study of the role of lipopolysaccharide of rhizobacterium A. brasilense sp7 in plant-microbe symbiosis. The intermediate protected oligosaccharide building blocks with cleavable CEP/AEP aglycons have a strong potential for further application in the synthesis of more complex oligosaccharides.

Arabinofuranose 1,2,5-orthobenzoate as a single precursor of linear α(1 → 5)-linked oligoarabinofuranosides.

Selectively protected mono-, di- and trisaccharide thioglycoside building blocks with unprotected primary hydroxy group at the non-reducing end, available in only one step from 3-O-benzoyl ß-d-arabinofuranose 1,2,5-orthobenzoate, were used in the synthesis of linear α(1 â†’ 5)-linked oligoarabinofuranosides up to octasaccharide. The obtained oligosaccharides contain 4-(2-chloroethoxy)phenyl (CEP) or 4-(2-azidoethoxy)phenyl (AEP) pre-spacer aglycons that allow preparation of neoglycoconjugates.