Zinc tetrafluoroborate catalyzed α-stereoselective synthesis of pseudoglycals: efficient synthesis of digitoxin α-L-amicetose.

We report here an efficient, fast, and cost-effective strategy for synthesizing pseudoglycals by the reaction of glycals with alcohols or nucleophiles using zinc tetrafluoroborate. This mild, transition metal-free approach allowed the α-selective synthesis of pseudoglycals using a wide range of acceptors containing various protecting groups/functionalities. This method is exemplified by the synthesis of digitoxin α-L-amicetose, a known potential cardiac glycoside anticancer agent. The improved 3-step synthesis from L-rhamnal afforded an overall yield of 54%, thus representing a significant improvement over the previous method.

Zinc Tetrafluoroborate Catalyzed Stereo- and Regioselective O-Glycosylation for the Direct Synthesis of ß-Glycosides from Armed O-Glycosyl Trichloroacetimidates.

Efficient stereo- and regioselective O-glycosylation methods remain essential to capacitate the studies of sugars and sugar derivatives in various disciplines. In this work, we demonstrated an operationally simple and cost-effective strategy for the synthesis of 1,2-trans glycosides by the activation of armed O-glycosyl trichloroacetimidates donor using zinc tetrafluoroborate. This mild, transition metal-free, and scalable approach allowed stereo- and regioselective synthesis of ß-glycosides with a wide range of acceptors containing various protecting groups/functionalities. This method is exemplified by synthesizing a branched trisaccharide fragment related to the cell wall O-polysaccharide of E. Coli O27.

Deoxyfluorinated amidation and esterification of carboxylic acid by pyridinesulfonyl fluoride.

Amide bond synthesis is one of the most used reactions in medicinal chemistry. We report an amide bond formation reaction through deoxyfluorinated carboxylic acids under mild conditions using 2-pyridinesulfonyl fluoride. The reaction procedure has been used in a one-pot synthesis of amides and esters via in situ generation of acyl fluoride. This one-pot synthetic method provides easy access to amides and esters. Using this method, we have sequentially synthesized a tetrapeptide and calceolarioside-B glycoside derivative with good yields.

Molecular Insights into O-Linked Sialoglycans Recognition by the Siglec-Like SLBR-N (SLBRUB10712) of Streptococcus gordonii.

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3'SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections.

Efficient community detection in multilayer networks using boolean compositions.

Networks (or graphs) are used to model the dyadic relations between entities in complex systems. Analyzing the properties of the networks reveal important characteristics of the underlying system. However, in many disciplines, including social sciences, bioinformatics, and technological systems, multiple relations exist between entities. In such cases, a simple graph is not sufficient to model these multiple relations, and a multilayer network is a more appropriate model. In this paper, we explore community detection in multilayer networks. Specifically, we propose a novel network decoupling strategy for efficiently combining the communities in the different layers using the Boolean primitives AND, OR, and NOT. Our proposed method, network decoupling, is based on analyzing the communities in each network layer individually and then aggregating the analysis results. We (i) describe our network decoupling algorithms for finding communities, (ii) present how network decoupling can be used to express different types of communities in multilayer networks, and (iii) demonstrate the effectiveness of using network decoupling for detecting communities in real-world and synthetic data sets. Compared to other algorithms for detecting communities in multilayer networks, our proposed network decoupling method requires significantly lower computation time while producing results of high accuracy. Based on these results, we anticipate that our proposed network decoupling technique will enable a more detailed analysis of multilayer networks in an efficient manner.

Total Synthesis of 6-Deoxy-l-talose Containing a Pentasaccharide Repeating Unit of Acinetobacter baumannii K11 Capsular Polysaccharides.

Herein, we report a concise synthetic approach for the first total synthesis of a pentasaccharide repeating unit of Acinetobacter baumannii K11 capsular polysaccharides containing a rare sugar 6-deoxy-l-talose. The pentasaccharide was synthesized in a convergent manner using a [3 + 2] block glycosylation strategy. During this synthetic strive, we used a 2,2,2-trichloroethoxycarbonyl (Troc)-protected monosaccharide unit to achieve a high yield during the glycosylation to synthesize a trisaccharide, and chemoselective deprotection of the Troc group from the trisaccharide was carried out under a mild, pH-neutral condition, keeping the O-glycosidic bond, azido, and acid/base sensitive group intact. A thiotolylglycoside disaccharide donor containing 6-deoxy-l-talose was synthesized for the first time by the armed-disarmed glycosylation method between two thiotolylglycosides.

Chemoenzymatic Synthesis of Sialosides Containing 7-N- or 7,9-Di-N-acetyl Sialic Acid as Stable O-Acetyl Analogues for Probing Sialic Acid-Binding Proteins.

A novel chemoenzymatic synthon strategy has been developed to construct a comprehensive library of α2-3- and α2-6-linked sialosides containing 7-N- or 7,9-di-N-acetyl sialic acid, the stable analogue of naturally occurring 7-O-acetyl- or 7,9-di-O-acetyl-sialic acid. Diazido and triazido-mannose derivatives that were readily synthesized chemically from inexpensive galactose were shown to be effective chemoenzymatic synthons. Together with bacterial sialoside biosynthetic enzymes with remarkable substrate promiscuity, they were successfully used in one-pot multienzyme (OPME) sialylation systems for highly efficient synthesis of sialosides containing multiple azido groups. Conversion of the azido groups to N-acetyl groups generated the desired sialosides. The hydrophobic and UV-detectable benzyloxycarbonyl (Cbz) group introduced in the synthetic acceptors of sialyltransferases was used as a removable protecting group for the propylamine aglycon of the target sialosides. The resulting N-acetyl sialosides were novel stable probes for sialic acid-binding proteins such as plant lectin MAL II, which bond strongly to sialyl T antigens with or without an N-acetyl at C7 or at both C7 and C9 in the sialic acid.

L. pneumophila CMP-5,7-di-N-acetyllegionaminic acid synthetase (LpCLS)-involved chemoenzymatic synthesis of sialosides and analogues.

5,7-Di-N-acetyllegionaminic acid (Leg5,7Ac2) is a bacterial nonulosonic acid (NulO) analogue of sialic acids, an important class of monosaccharides in mammals and in some bacteria. To develop efficient one-pot multienzyme (OPME) glycosylation systems for synthesizing Leg5,7Ac2-glycosides, Legionella pneumophila cytidine 5'-monophosphate (CMP)-Leg5,7Ac2 synthetase (LpCLS) was cloned and characterized. It was successfully used in producing Leg5,7Ac2-glycosides from chemoenzymatically synthesized Leg5,7Ac2 using a one-pot two-enzyme system or from its chemically synthesized six-carbon monosaccharide precursor 2,4-diacetamido-2,4,6-trideoxymannose (6deoxyMan2,4diNAc) in a one-pot three-enzyme system. In addition, LpCLS was shown to tolerate Neu5Ac7NAc, a C9-hydroxyl analogue of Leg5,7Ac2 and also a stable analogue of 7-O-acetylneuraminic acid (Neu5,7Ac2), to allow OPME synthesis of the corresponding α2-3-linked sialosides, from chemically synthesized six-carbon monosaccharide precursor 4-N-acetyl-4-deoxy-N-acetylmannosamine (ManNAc7NAc).

Regioselective One-Pot Multienzyme (OPME) Chemoenzymatic Strategies for Systematic Synthesis of Sialyl Core 2 Glycans.

O-GalNAc glycans or mucin-type glycans are common protein post-translational modifications in eukaryotes. Core 2 O-GalNAc glycans are branched structures that are broadly distributed in glycoproteins and mucins of all types of cells. To better understand their biological roles, it is important to obtain structurally defined Core 2 O-GalNAc glycans. We present here regioselective one-pot multienzyme (OPME) chemoenzymatic strategies to systematically access a diverse array of sialyl Core 2 glycans. Regioselectivity can be achieved by using OPME systems containing a glycosyltransferase with restricted acceptor specificity or by differentiating the branches using altered glycosylation sequences. This work provides a general regioselective strategy to access diverse Core 2 O-GalNAc glycans which can be extended for the synthesis of other complex branched glycans.

A substrate tagging and two-step enzymatic reaction strategy for large-scale synthesis of 2,7-anhydro-sialic acid.

A sialyltransferase acceptor tagging and two-step enzymatic reaction strategy has been developed for multigram-scale chemoenzymatic synthesis of 2,7-anhydro-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac), a compound that can serve as a sole carbon source for the growth of Ruminococcus gnavus, a common human gut commensal. Different approaches of introducing hydrophobic UV-active tags to lactose as well-suited sialyltransferase acceptors have been explored and a simple two-step high-yield chemical synthetic procedure has been identified. The UV-active hydrophobic tag facilitates monitoring reaction progress and allows facile product purification by C18-cartridges. A two-step enzyme-catalyzed reaction procedure has been established to combine with C18 cartridge-based purification process for high-yield production of the desired product in multigram scales with the recycled use of chromophore-tagged lactoside starting material and sialoside intermediate. This study demonstrated an environmentally friendly highly-efficient synthetic and purification strategy for the production of 2,7-anhydro-Neu5Ac to explore its potential functions.

9-Azido-9-deoxy-2,3-difluorosialic Acid as a Subnanomolar Inhibitor against Bacterial Sialidases.

A library of 2(a),3(a/e)-difluorosialic acids and their C-5 and/or C-9 derivatives were chemoenzymatically synthesized. Pasteurella multocida sialic acid aldolase (PmAldolase), but not its Escherichia coli homologue (EcAldolase), was found to catalyze the formation of C5-azido analogue of 3-fluoro(a)-sialic acid. In comparison, both PmAldolase and EcAldolase could catalyze the synthesis of 3-fluoro(a/e)-sialic acids and their C-9 analogues although PmAldolase was generally more efficient. The chemoenzymatically synthesized 3-fluoro(a/e)-sialic acid analogues were purified and chemically derivatized to form the desired difluorosialic acids and derivatives. Inhibition studies against several bacterial sialidases and a recombinant human cytosolic sialidase hNEU2 indicated that sialidase inhibition was affected by the C-3 fluorine stereochemistry and derivatization at C-5 and/or C-9 of the inhibitor. Opposite to that observed for influenza A virus sialidases and hNEU2, compounds with axial fluorine at C-3 were better inhibitors (up to 100-fold) against bacterial sialidases compared to their 3F-equatorial counterparts. While C-5-modified compounds were less-efficient antibacterial sialidase inhibitors, 9-N3-modified 2,3-difluoro-Neu5Ac showed increased inhibitory activity against bacterial sialidases. 9-Azido-9-deoxy-2-(e)-3-(a)-difluoro- N-acetylneuraminic acid [2(e)3(a)DFNeu5Ac9N3] was identified as an effective inhibitor with a long effective duration selectively against pathogenic bacterial sialidases from Clostridium perfringens (CpNanI) and Vibrio cholerae.

Targeting Base Excision Repair Glycosylases with DNA Containing Transition State Mimics Prepared via Click Chemistry.

DNA glycosylases of the base excision repair (BER) pathway are front-line defenders in removing compromising modifications of the DNA nucleobases. Aberrantly modified nucleobases mediate genomic mutations and inhibit DNA replication leading to adverse health consequences such as cancer, neurological diseases, and aging. In an effort to develop high-affinity transition state (TS) analogues as chemical biology probes for DNA glycosylases, oligonucleotides containing a propargyl-modified pyrrolidine TS mimic nucleotide were synthesized. A small library of TS mimic-containing oligonucleotides was generated using a structurally diverse set of five azides via copper(I)-catalyzed azide-alkyne cycloaddition "click" chemistry. The relative affinity ( Kd) was evaluated for BER glycosylases Escherichia coli MutY, bacterial formamidopyrimidine glycosylase (Fpg), and human OG glycosylase 1 (hOGG1) with the library of TS mimic DNA duplexes. All of the BER glycosylases were found to exhibit extremely high affinities (approximately picomolar Kd values) for the TS mimics. However, binding preferences, distinct for each glycosylase, for the TS mimic library members were observed, suggesting different modes of binding and transition state stabilization among the three glycosylases. Fpg bound all of the TS mimics with exceptionally high affinities, while the MutY binding affinity correlated inversely with the size of the appended moiety. Of note, we identified one member of the small TS mimic library that exhibited a particularly high affinity for hOGG1. These results strongly support the use of the propargyl-TS mimic oligonucleotides and elaboration via click chemistry in screening and identification of high-affinity ligands for BER glycosylases of interest.

Sialidase-catalyzed one-pot multienzyme (OPME) synthesis of sialidase transition-state analogue inhibitors.

Sialidase transition state analog inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en, DANA) has played a leading role in developing clinically used anti-influenza virus drugs. Taking advantage of the Neu5Ac2en-forming catalytic property of Streptococcus pneumoniae sialidase SpNanC, an effective one-pot multienzyme (OPME) strategy has been developed to directly access Neu5Ac2en and its C-5, C-9, and C-7-analogs from N-acetylmannosamine (ManNAc) and analogs. The obtained Neu5Ac2en analogs can be further derivatized at various positions to generate a larger inhibitor library. Inhibition studies demonstrated improved selectivity of several C-5- or C-9-modified Neu5Ac2en derivatives against several bacterial sialidases. The study provides an efficient enzymatic method to access sialidase inhibitors with improved selectivity.

Streamlined chemoenzymatic total synthesis of prioritized ganglioside cancer antigens.

A highly efficient streamlined chemoenzymatic strategy for total synthesis of four prioritized ganglioside cancer antigens GD2, GD3, fucosyl GM1, and GM3 from commercially available lactose and phytosphingosine is demonstrated. Lactosyl sphingosine (LacßSph) was chemically synthesized (on a 13 g scale), subjected to sequential one-pot multienzyme (OPME) glycosylation reactions with facile C18-cartridge purification, followed by improved acylation conditions to form target gangliosides, including fucosyl GM1 which has never been synthesized before.

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di-N-acetyllegionaminic Acid-Containing Glycosides.

A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9-di-N-acetyllegionaminic acid (Leg5,7Ac2 ), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2-3/6-linked Leg5,7Ac2 -glycosides was built by using chemically synthesized 2,4-diazido-2,4,6-trideoxymannose as a chemoenzymatic synthon for highly efficient one-pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d-fucose and had an overall yield of 34-52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac2 monosaccharide was also synthesized by a sialic acid aldolase-catalyzed reaction.

Highly efficient chemoenzymatic synthesis and facile purification of α-Gal pentasaccharyl ceramide Galα3nLc4ßCer.

A highly efficient chemoenzymatic method for synthesizing glycosphingolipids using α-Gal pentasaccharyl ceramide as an example is reported here. Enzymatic extension of the chemically synthesized lactosyl sphingosine using efficient sequential one-pot multienzyme (OPME) reactions allowed glycosylation to be carried out in aqueous solutions. Facile C18 cartridge-based quick (<30 minutes) purification protocols were established using minimal amounts of green solvents (CH3CN and H2O). Simple acylation in the last step led to the formation of the target glycosyl ceramide in 4 steps with an overall yield of 57%.

Synthesis of novel muramic acid derivatives and their interaction with lysozyme: Action of lysozyme revisited.

HYPOTHESIS: The interaction of lysozyme with the N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) unit of peptidoglycan (PGN) polymer of the bacterial cell wall is of immense importance to understand the mechanism of lysozyme on PGN. EXPERIMENTS: The synthesis of three novel NAM derivatives containing fused oxazinone ring to the NAM moiety has been achieved. The synthesized compounds were evaluated for their potential as a glycomimetic acceptor of lysozyme using different biophysical and computational methods such as 1H NMR, STD NMR, DOSY and Molecular docking. FINDINGS: Novel modified muramic acid derivatives have been synthesized in excellent yield containing fused cyclooxazine ring embedded on the muramic acid moiety using a newly developed hydrazinolysis reaction condition. From various biophysical studies, it has been established that the compound containing endo modified muramic acid moiety (compound 1) shows significant binding property for the lysozyme while the other isomer (compound 2) did not bind to the lysozyme. The catalytic residues Glu35 and Asp52 were found to be in the close proximity for the active molecule which justifies the selectivity of this molecule in conjunction to lysozyme enzymatic activity.

Chemoenzymatic synthesis of para-nitrophenol (pNP)-tagged α2-8-sialosides and high-throughput substrate specificity studies of α2-8-sialidases.

para-Nitrophenol (pNP)-tagged α2-8-linked sialosides containing different sialic acid forms were chemoenzymatically synthesized using an efficient one-pot three-enzyme α2-8-sialylation system. The resulting compounds allowed high-throughput substrate specificity studies of the α2-8-sialidase activity of a recombinant human cytosolic sialidase hNEU2 and various bacterial sialidases. The sialoside substrate profiles obtained can be used to guide the selection of suitable sialidases for sialylglycan analysis and for cell and tissue surface glycan modification. They can also be used to guide sialidase inhibitor design.

Mephebrindole, a synthetic indole analog coordinates the crosstalk between p38MAPK and eIF2α/ATF4/CHOP signalling pathways for induction of apoptosis in human breast carcinoma cells.

The efficacy of cancer chemotherapeutics is limited by side effects resulting from narrow therapeutic windows between the anticancer activity of a drug and its cytotoxicity. Thus identification of small molecules that can selectively target cancer cells has gained major interest. Cancer cells under stress utilize the Unfolded protein response (UPR) as an effective cell adaptation mechanism. The purpose of the UPR is to balance the ER folding environment and calcium homeostasis under stress. If ER stress is prolonged, tumor cells undergo apoptosis. In the present study we demonstrated an 3,3'-(Arylmethylene)-bis-1H-indole (AMBI) derivative 3,3'-[(4-Methoxyphenyl) methylene]-bis-(5-bromo-1H-indole), named as Mephebrindole (MPB) as an effective anti-cancer agent in breast cancer cells. MPB disrupted calcium homeostasis in MCF7 cells which triggered ER stress development. Detailed evaluations revealed that mephebrindole by activating p38MAPK also regulated GRP78 and eIF2α/ATF4 downstream to promote apoptosis. Studies extended to in vivo allograft mice models revalidated its anti-carcinogenic property thus highlighting the role of MPB as an improved chemotherapeutic option.

Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC), is a specific subtype of epithelial breast tumors that are immuno-histochemically negative for the protein expression of the estrogen receptor (ER), the progesterone receptor (PR) and lack over expression/gene amplification of HER2. This subtype of breast cancers is highly metastatic, shows poor prognosis and hence represents an important clinical challenge to researchers worldwide. Thus alternative approaches of drug development for TNBC have gained utmost importance in the present times. Dietary indole and its derivatives have gained prominence as anti-cancer agents and new therapeutic approaches are being developed to target them against TNBC. But a major drawback with 3, 3'di Indolyl methane (DIM) is their poor bioavailability and high effective concentration against TNBC. However, the Aryl methyl ring substituted analogs of DIM display interesting anti-cancer activity in breast cancer cells. In the current study we report the synthesis of a novel synthetic aryl methyl ring substituted analog of DIM, named as Phemindole as an effective anti-tumor agent against TNBC cells. Furthermore, we enumerated that Phemindole caused reactive oxygen species mediated mitochondrial-dependent apoptosis in MDAMB-231 cells. Furthermore, Phemindole mediated Store Operated Calcium Entry (SOCE) retardation favored inactivation of STIM1 and henceforth activated ER stress to induce apoptosis in TNBC cells. Simultaneously, Phemindole was also found to restrict the in vitro cell migration through its anti mitotic property and pFAK regulation. Studies extended to ex ovo and in vivo mice models further validated the efficacy of Phemindole. Thus our results cumulatively propose Phemindole as a new chemotherapeutic regime which might be effective to target the deadly aspects of the TNBC.