Biocatalyzed Synthesis of Glycostructures with Anti-infective Activity.

Molecules containing carbohydrate moieties play essential roles in fighting a variety of bacterial and viral infections. Consequently, the design of new carbohydrate-containing drugs or vaccines has attracted great attention in recent years as means to target several infectious diseases.Conventional methods to produce these compounds face numerous challenges because their current production technology is based on chemical synthesis, which often requires several steps and uses environmentally unfriendly reactants, contaminant solvents, and inefficient protocols. The search for sustainable processes such as the use of biocatalysts and eco-friendly solvents is of vital importance. Therefore, their use in a variety of reactions leading to the production of pharmaceuticals has increased exponentially in the last years, fueled by recent advances in protein engineering, enzyme directed evolution, combinatorial biosynthesis, immobilization techniques, and flow biocatalysis. In glycochemistry and glycobiology, enzymes belonging to the families of glycosidases, glycosyltransferases (Gtfs), lipases, and, in the case of nucleoside and nucleotide analogues, also nucleoside phosphorylases (NPs) are the preferred choices as catalysts.In this Account, on the basis of our expertise, we will discuss the recent biocatalytic and sustainable approaches that have been employed to synthesize carbohydrate-based drugs, ranging from antiviral nucleosides and nucleotides to antibiotics with antibacterial activity and glycoconjugates such as neoglycoproteins (glycovaccines, GCVs) and glycodendrimers that are considered as very promising tools against viral and bacterial infections.In the first section, we will report the use of NPs and N-deoxyribosyltransferases for the development of transglycosylation processes aimed at the synthesis of nucleoside analogues with antiviral activity. The use of deoxyribonucleoside kinases and hydrolases for the modification of the sugar moiety of nucleosides has been widely investigated.Next, we will describe the results obtained using enzymes for the chemoenzymatic synthesis of glycoconjugates such as GCVs and glycodendrimers with antibacterial and antiviral activity. In this context, the search for efficient enzymatic syntheses represents an excellent strategy to produce structure-defined antigenic or immunogenic oligosaccharide analogues with high purity. Lipases, glycosidases, and Gtfs have been used for their preparation.Interestingly, many authors have proposed the use Gtfs originating from the biosynthesis of natural glycosylated antibiotics such as glycopeptides, macrolides, and aminoglycosides. These have been used in the chemoenzymatic semisynthesis of novel antibiotic derivatives by modification of the sugar moiety linked to their complex scaffold. These contributions will be described in the last section of this review because of their relevance in the fight against the spreading phenomenon of antibiotic resistance. In this context, the pioneering in vivo synthesis of novel derivatives obtained by genetic manipulation of producer strains (combinatorial biosynthesis) will be shortly described as well.All of these strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can contribute to the sustainable development of complex glycan-based therapies and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and selectivity. The use of enzymatic synthesis is becoming a reality in the pharmaceutical industry and in drug discovery to rapidly afford collections of new antibacterial or antiviral molecules with improved specificity and better metabolic stability.

Biocatalysis: A smart and green tool for the preparation of chiral drugs.

Over the last decades, biocatalysis has achieved growing interest thanks to its potential to enable high efficiency, high yield, and eco-friendly processes aimed at the production of pharmacologically relevant compounds. Particularly, biocatalysis proved an effective and potent tool in the preparation of chiral molecules, and the recent innovations of biotechnologies and nanotechnologies open up a new era of further developments in this field. Different strategies are now available for the synthesis of chiral drugs and their intermediates. Enzymes are green tools that offer several advantages, associated both to catalysis and environmentally friendly reactants. Specifically, the use of enzymes isolated from biological sources or of whole-cell represents a valuable approach to obtain pharmaceutical products. The sustainability, the higher efficiency, and cost-effectiveness of biocatalytic reactions result in improved performance and properties that can be translated from academia to industry. In this review, we focus on biocatalytic approaches for synthesizing chiral drugs or their intermediates. Aiming to unveil the potentialities of biocatalysis systems, we discuss different examples of innovative biocatalytic approaches and their applications in the pharmaceutical industry.

Controlled Decoration of [60]Fullerene with Polymannan Analogues and Amino Acid Derivatives through Malondiamide-Based Linkers.

In the last few years, nanomaterials based on fullerene have begun to be considered promising tools in the development of efficient adjuvant/delivery systems for vaccination, thanks to their several advantages such as biocompatibility, size, and easy preparation and modification. In this work we reported the chemoenzymatic synthesis of natural polymannan analogues (di- and tri-mannan oligosaccharides characterized by α1,6man and/or α1,2man motifs) endowed with an anomeric propargyl group. These sugar derivatives were submitted to 1,3 Huisgen dipolar cycloaddition with a malondiamide-based chain equipped with two azido terminal groups. The obtained sugar-modified malondiamide derivatives were used to functionalize the surface of Buckminster fullerene (C60) in a highly controlled fashion, and yields (11-41%) higher than those so far reported by employing analogue linkers. The same strategy has been exploited to obtain C60 endowed with natural and unnatural amino acid derivatives. Finally, the first double functionalization of fullerene with both sugar- and amino acid-modified malondiamide chains was successfully performed, paving the way to the possible derivatization of fullerenes with immunogenic sugars and more complex antigenic peptides.

Developing a Library of Mannose-Based Mono- and Disaccharides: A General Chemoenzymatic Approach to Monohydroxylated Building Blocks.

Regioselective deprotection of acetylated mannose-based mono- and disaccharides differently functionalized in anomeric position was achieved by enzymatic hydrolysis. Candida rugosa lipase (CRL) and Bacillus pumilus acetyl xylan esterase (AXE) were immobilized on octyl-Sepharose and glyoxyl-agarose, respectively. The regioselectivity of the biocatalysts was affected by the sugar structure and functionalization in anomeric position. Generally, CRL was able to catalyze regioselective deprotection of acetylated monosaccharides in C6 position. When acetylated disaccharides were used as substrates, AXE exhibited a marked preference for the C2, or C6 position when C2 was involved in the glycosidic bond. By selecting the best enzyme for each substrate in terms of activity and regioselectivity, we prepared a small library of differently monohydroxylated building blocks that could be used as intermediates for the synthesis of mannosylated glycoconjugate vaccines targeting mannose receptors of antigen presenting cells.

An Enzymatic Flow-Based Preparative Route to Vidarabine.

The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A), catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions. Glyoxyl-agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration) the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor for the preparation of other purine nucleosides.

Epitope and affinity determination of recombinant Mycobacterium tuberculosis Ag85B antigen towards anti-Ag85 antibodies using proteolytic affinity-mass spectrometry and biosensor analysis.

Tuberculosis (TB) is the first cause of death from infectious diseases worldwide. Only a single anti-TB vaccine is currently available for clinical use, but its efficacy is not achieved with certainty. The aim of this work is to provide a basis for the rational design of a neo-glycoconjugate vaccine against TB. Structural characterization of recombinant antigenic proteins from Mycobacterium tuberculosis (MTB) Ag85B (rAg85B, variants, and semi-synthetic glycoconjugates) was initially carried out. Identification of antibody epitope analyses by proteolytic affinity-mass spectrometry and surface plasmon resonance (SPR) biosensor analyses were performed in order to qualitatively identify and quantitatively characterize interaction structures of the antigens with antibodies from different sources. A commercial monoclonal antibody and polyclonal antibodies from different sources (patients with active TB, vaccinated individuals, and a healthy control) were employed to analyze antigen-antibody interactions. These combined approaches provided the identification of different assembled epitope regions on the recombinant MTB antigens, their affinity binding constants in the interactions with specific antibodies, and revealed the importance of protection from excessive glycosylation. The identified epitope peptides should constitute a suitable basis for the design of new specific target vaccines. Graphical abstract ᅟ.

Phytosterol and γ-Oryzanol Conjugates: Synthesis and Evaluation of their Antioxidant, Antiproliferative, and Anticholesterol Activities.

Fifteen new multifunctional conjugates were designed and synthesized by chemically linking the steroidal framework of natural occurring γ-oryzanol and γ-oryzanol-derived phytosterols to a wide range of bioactive natural compounds (fatty acids, phenolic acids, amino acids, lipoic acid, retinoic acid, curcumin, and resveratrol). Starting from γ-oryzanol, which is the main component of rice bran oil, this study was aimed at assessing if the conjugation strategy might enhance some γ-oryzanol bioactivities. The antioxidant activity was evaluated through three different mechanisms, namely, DPPH-scavenging activity, metal-chelating activity, and ß-carotene-bleaching inhibition. Measurement of the in vitro cell growth inhibitory effects on three different human cancer cellular lines was also carried out, and the potential hypocholesterolemic effect was studied. Compounds 10 and 15 displayed an improved antioxidant activity, with respect to that of γ-oryzanol. Compounds 2, 6, and 12 exerted an antiproliferative activity in the low micromolar range against HeLa and DAOY cells (GI50 < 10 µM). As for the claimed hypocholesterolemic effect of γ-oryzanol, none of the synthesized compounds inhibited the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis.

Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates.

Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo-glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.

Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis.

This paper describes the immobilization of the neutral protease from Bacillus subtilis and its application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful for the preparation of anticancer products. Regarding the immobilization study, different results have been obtained depending on the immobilization procedure. Epoxy hydrophobic carriers gave a poorly stable derivative that released almost 50% of the immobilized protein under the required reaction conditions. On the contrary, covalent immobilization on a differently activated hydrophilic carrier (agarose) resulted in very stable enzyme derivatives. In an attempt to explain the obtained enzyme immobilization results, the hypothetical localization of lysines on the enzyme surface was predicted in a 3D structure model of B. subtilis protease N built in silico by using the structure of Staphylococcus aureus metalloproteinase as the template. The immobilized enzyme shown a high regioselectivity in the hydrolysis of different peracetylated nucleosides. A stable enzyme derivative was obtained and successfully used in the development of efficient preparative bioprocesses for the hydrolysis of acetylated nucleosides, giving new intermediates for the synthesis of capecitabine in high yield.

Characterization of intact neo-glycoproteins by hydrophilic interaction liquid chromatography.

In this study, an HPLC HILIC-UV method was developed for the analysis of intact neo-glycoproteins. During method development the experimental conditions evaluated involved different HILIC columns (TSKgel Amide-80 and ZIC-pHILIC), and water-acetonitrile mixtures containing various types of acids and salts. The final selected method was based on a TSKgel Amide-80 column and a mobile phase composed of acetonitrile and water both containing 10 mM HClO4. The influence of temperature and sample preparation on the chromatographic performances of the HILIC method was also investigated. The method was applied to the separation of neo-glycoproteins prepared starting from the model protein RNase A by chemical conjugation of different glycans. Using the method here reported it was possible to monitor by UV detection the glycosylation reaction and assess the distribution of neo-glycoprotein isoforms without laborious sample workup prior to analysis.

Effect of glycosylation on the affinity of the MTB protein Ag85B for specific antibodies: towards the design of a dual-acting vaccine against tuberculosis.

BACKGROUND: To create a dual-acting vaccine that can fight against tuberculosis, we combined antigenic arabino-mannan analogues with the Ag85B protein. To start the process, we studied the impact of modifying different parts of the Ag85B protein on its ability to be recognized by antibodies. RESULTS: Through our research, we discovered that three modified versions of the protein, rAg85B-K30R, rAg85B-K282R, and rAg85B-K30R/K282R, retained their antibody reactivity in healthy individuals and those with tuberculosis. To further test the specificity of the sugar AraMan for AraMan antibodies, we used Human Serum Albumin glycosylated with AraMan-IME and Ara3Man-IME. Our findings showed that this specific sugar was fully and specifically modified. Bio-panning experiments revealed that patients with active tuberculosis exhibited a higher antibody response to Ara3Man, a sugar found in lipoarabinomannan (LAM), which is a major component of the mycobacterial cell wall. Bio-panning with anti-LAM plates could eliminate this increased response, suggesting that the enhanced Ara3Man response was primarily driven by antibodies targeting LAM. These findings highlight the importance of Ara3Man as an immunodominant epitope in LAM and support its role in eliciting protective immunity against tuberculosis. Further studies evaluated the effects of glycosylation on the antibody affinity of recombinant Ag85B and its variants. The results indicated that rAg85B-K30R/K282R, when conjugated with Ara3Man-IME, demonstrated enhanced antibody recognition compared to unconjugated or non-glycosylated versions. CONCLUSIONS: Coupling Ara3Man to rAg85B-K30R/K282R could lead to the development of effective dual-acting vaccines against tuberculosis, stimulating protective antibodies against both AraMan and Ag85B, two key tuberculosis antigens.

Modulation of the microenvironment surrounding the active site of penicillin G acylase immobilized on acrylic carriers improves the enzymatic synthesis of cephalosporins.

The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled synthesis of ß-lactam antibiotics are negatively affected upon immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to improve the synthetic performance of PGA immobilized on epoxy-activated acrylic carriers. First, an aldehyde-based spacer was inserted on the carrier surface by glutaraldehyde activation (immobilization yield = 50%). The resulting 3-fold higher synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit® C and Sepabeads® EC-EP, respectively) with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4) was ascribed to a facilitated diffusion of substrates and products as a result of the increased distance between the enzyme and the carrier surface. A second series of catalysts was prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit® C), followed by quenching of oxiranes not involved in the binding with the protein with different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols). In most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the immobilization yield decreased from 70% (untreated Eupergit® C) to 20%. Glutaraldehyde- and cysteine-treated Eupergit® C catalyzed the synthesis of cefazolin in 88% (±0.9) and 87% (±1.6) conversion, respectively, whereas untreated Eupergit® C afforded this antibiotic in 79% (±1.2) conversion.

l-Theanine Goes Greener: A Highly Efficient Bioprocess Catalyzed by the Immobilized γ-Glutamyl Transferase from Bacillus subtilis.

l-Theanine (l-Th) was synthesized by simply mixing the reactants (l-glutamine and ethylamine in water) at 25 °C and Bacillus subtilis γ-glutamyl transferase (BsGGT) covalently immobilized on glyoxyl-agarose according to a methodology previously reported by our research group; neither buffers, nor other additives were needed. Ratio of l-glutamine (donor) to ethylamine (acceptor), pH, enzymatic units (IU), and reaction time were optimized (molar ratio of donor/acceptor=1 : 8, pH 11.6, 1 IU mL-1 , 6 h), furnishing l-Th in 93 % isolated yield (485 mg, 32.3 g L-1 ) and high purity (99 %), after a simple filtration of the immobilized biocatalyst, distillation of the volatiles (unreacted ethylamine) and direct lyophilization. Immobilized BsGGT was re-used (four reaction cycles) with 100 % activity retention. This enzymatic synthesis represents a straightforward, fast, high-yielding, and easily scalable approach to l-Th preparation, besides having a favorable green chemistry metrics.

From Lactose to Alkyl Galactoside Fatty Acid Esters as Non-Ionic Biosurfactants: A Two-Step Enzymatic Approach to Cheese Whey Valorization.

A library of alkyl galactosides was synthesized to provide the "polar head" of sugar fatty acid esters to be tested as non-ionic surfactants. The enzymatic transglycosylation of lactose resulted in alkyl ß-D-galactopyranosides, whereas the Fischer glycosylation of galactose afforded isomeric mixtures of α- and ß-galactopyranosides and α- and ß-galactofuranosides. n-Butyl galactosides from either routes were enzymatically esterified with palmitic acid, used as the fatty acid "tail" of the surfactant, giving the corresponding n-butyl 6-O-palmitoyl-galactosides. Measurements of interfacial tension and emulsifying properties of n-butyl 6-O-palmitoyl-galactosides revealed that the esters of galactopyranosides are superior to those of galactofuranosides, and that the enantiopure n-butyl 6-O-palmitoyl-ß-D-galactoside, prepared by the fully enzymatic route, leads to the most stable emulsion. These results pave the way to the use of lactose-rich cheese whey as raw material for the obtainment of bio-based surfactants.

Glycovaccine Design: Optimization of Model and Antitubercular Carrier Glycosylation via Disuccinimidyl Homobifunctional Linker.

Conjugation via disuccinimidyl homobifunctional linkers is reported in the literature as a convenient approach for the synthesis of glycoconjugate vaccines. However, the high tendency for hydrolysis of disuccinimidyl linkers hampers their extensive purification, which unavoidably results in side-reactions and non-pure glycoconjugates. In this paper, conjugation of 3-aminopropyl saccharides via disuccinimidyl glutarate (DSG) was exploited for the synthesis of glycoconjugates. A model protein, ribonuclease A (RNase A), was first considered to set up the conjugation strategy with mono- to tri- mannose saccharides. Through a detailed characterization of synthetized glycoconjugates, purification protocols and conjugation conditions have been revised and optimized with a dual aim: ensure high sugar-loading and avoid the presence of side reaction products. An alternative purification approach based on hydrophilic interaction liquid chromatography (HILIC) allowed the formation of glutaric acid conjugates to be avoided, and a design of experiment (DoE) approach led to optimal glycan loading. Once its suitability was proven, the developed conjugation strategy was applied to the chemical glycosylation of two recombinant antigens, native Ag85B and its variant Ag85B-dm, that are candidate carriers for the development of a novel antitubercular vaccine. Pure glycoconjugates (≥99.5%) were obtained. Altogether, the results suggest that, with an adequate protocol, conjugation via disuccinimidyl linkers can be a valuable approach to produce high sugar-loaded and well-defined glycovaccines.

From Batch to Continuous Flow Bioprocessing: Use of an Immobilized γ-Glutamyl Transferase from B. subtilis for the Synthesis of Biologically Active Peptide Derivatives.

γ-Glutamyl-peptides are frequently endowed with biological activities. In this work, "kokumi peptides" such as γ-glutamyl-methionine (1) and γ-glutamyl-(S)-allyl-cysteine (2), as well as the neuroprotective γ-glutamyl-taurine (3) and the antioxidant ophthalmic acid (4), were synthesized through an enzymatic transpeptidation reaction catalyzed by the γ-glutamyl transferase from Bacillus subtilis (BsGGT) using glutamine as the γ-glutamyl donor. BsGGT was covalently immobilized on glyoxyl-agarose resulting in high protein immobilization yield and activity recovery (>95%). Compounds 1-4 were obtained in moderate yields (19-40%, 5-10 g/L) with a variable purity depending on the presence of the main byproduct (γ-glutamyl-glutamine, 0-16%). To achieve process intensification and better control of side reactions, the synthesis of 2 was moved from batch to continuous flow. The specific productivity was 1.5 times higher than that in batch synthesis (13.7 µmol/min*g), but it was not accompanied by a paralleled improvement of the impurity profile.

Design of epidermal growth factor immobilization on 3D biocompatible scaffolds to promote tissue repair and regeneration.

Exogenous application of human epidermal growth factor (hEGF) stimulates epidermal wound healing. The aim of this study was to develop bioconjugates based on hEGF mimicking the protein in its native state and thus suitable for tissue engineering applications, in particular for treating skin-related disorders as burns. Ribonuclease A (RNase A) was used to investigate a number of different activated-agarose carriers: cyanogen bromide (CNBr)-activated-agarose and glyoxyl-agarose showed to preserve the appropriate orientation of the protein for receptor binding. EGF was immobilized on these carriers and immobilization yield was evaluated (100% and 12%, respectively). A peptide mapping of unbound protein regions was carried out by LC-MS to take evidence of the residues involved in the immobilization and, consequently, the flexibility and surface accessibility of immobilized EGF. To assess cell proliferative activities, 10, 25, 50, and 100 ng/mL of each immobilized EGF sample were seeded on fibroblast cells and incubated for 24, 48 and 72 h. The immobilized growth factor showed significantly high cell proliferative activity at 50 and 100 ng/mL compared to control and soluble EGF. Although both of the immobilized samples show dose-dependency when seeded with high number of fibroblast cells, CNBr-agarose-EGF showed a significantly high activity at 100 ng/mL and 72 h incubation, compared to glyoxyl-agarose-EGF.

Characterization and study of the orientation of immobilized enzymes by tryptic digestion and HPLC-MS: design of an efficient catalyst for the synthesis of cephalosporins.

An innovative approach to determine the orientation of penicillin G acylase (PGA) from Escherichia coli covalently immobilized onto solid supports has been developed. This method is based on tryptic digestion of immobilized PGA followed by HPLC-MS analysis of the released peptides which are supposed to be only those exposed toward the reaction medium and not directly bound to the solid support. To this purpose, PGA was immobilized on Eupergit C (acrylic hydrophobic resin) and glyoxyl-agarose (hydrophilic resin) functionalized with epoxy and aldehyde groups, respectively, both involving the Lys residues of the protein. The peptide maps obtained were analyzed to derive the orientation of immobilized PGA, as the position of the detected Lys gave indication concerning the accessibility of the different areas of the protein. The results indicate that PGA immobilization on both supports involves mainly Lys located near the binding pocket (70%). Some differences in the enzyme orientation on the two supports can be deduced by the presence of different unbound Lys residues in the released peptides, specific to each support (Lys 117alpha for PGA-Eupergit C; Lys 163alpha and Lys 165alpha for PGA-glyoxyl-agarose). These results have been correlated with the data obtained in the kinetically controlled synthesis and indicate that the orientation of PGA on both supports is partially unfavorable, driving the active site near the support surface. This type of orientation of the enzyme enhances the effect of the nature of the support and of the binding chemistry on the catalytic properties. The information obtained indicated the most suitable support and activation strategy to design an immobilized acylase with good synthetic properties for preparative processes. The glyoxyl-Eupergit C support with enhanced porosity synergically combines the mechanical stability and synthetic performances of immobilized PGA and was successfully used in the synthesis of several cephalosporins.

Design, synthesis and biological evaluation of new ganglioside GM3 analogues as potential agents for cancer therapy.

Ganglioside GM3 is well known as a tumor-associated carbohydrate antigen on several types of tumors. Many studies have demonstrated that GM3 plays roles in cells proliferation, adhesion, motility and differentiation, which is involved in the process of cancer development. In the present study, we developed methods to synthesize GM3 analogues conveniently. By enzymatic hydrolysis and chemical procedures, two novel analogues and two known analogues were synthesized, containing lactose and glucosamine. Then anti-proliferation and anti-migration activities were evaluated by cytotoxicity assays and wound healing tests, and the data demonstrated that these analogues exhibited anticancer activities. Based on our previous studies, the structure-activity relationships were discussed. This study could provide valuable sight to find new antitumor agents for cancer therapy.

Chemoenzymatic synthesis of arabinomannan (AM) glycoconjugates as potential vaccines for tuberculosis.

Mycobacteria infection resulting in tuberculosis (TB) is one of the top ten leading causes of death worldwide in 2018, and lipoarabinomannan (LAM) has been confirmed to be the most important antigenic polysaccharide on the TB cell surface. In this study, a convenient synthetic method has been developed for synthesizing three branched oligosaccharides derived from LAM, in which a core building block was prepared by enzymatic hydrolysis in flow chemistry with excellent yield. After several steps of glycosylations, the obtained oligosaccharides were conjugated with recombinant human serum albumin (rHSA) and the ex-vivo ELISA tests were performed using serum obtained from several TB-infected patients, in order to evaluate the affinity of the glycoconjugate products for the human LAM-antibodies. The evaluation results are positive, especially compound 21 that exhibited excellent activity which could be considered as a lead compound for the future development of a new glycoconjugated vaccine against TB.