Donor Preactivation-Based Glycan Assembly: from Manual to Automated Synthesis.

Carbohydrates are called the third chain of life. Carbohydrates participate in many important biochemical functions in living species, and the biological information carried by them is several orders of magnitude larger than that of nucleic acids and proteins. However, due to the intrinsic complexity and heterogeneity of carbohydrate structures, furnishing pure and structurally well-defined glycans for functional studies is a formidable task, especially for homogeneous large-size glycans. To address this issue, we have developed a donor preactivation-based one-pot glycosylation strategy enabling multiple sequential glycosylations in a single reaction vessel.The donor preactivation-based one-pot glycosylation refers to the strategy in which the glycosyl donor is activated in the absence of a glycosyl acceptor to generate a reactive intermediate. Subsequently, the glycosyl acceptor with the same anomeric leaving group is added, leading to a glycosyl coupling reaction, which is then iterated to rapidly achieve the desired glycan in the same reactor. The advantages of this strategy include the following: (1) unique chemoselectivity is obtained after preactivation; (2) it is independent of the reactivity of glycosyl donors; (3) multiple-step glycosylations are enabled without the need for intermediate purification; (4) only stoichiometric building blocks are required without complex protecting group manipulations. Using this protocol, a range of glycans including tumor-associated carbohydrate antigens, various glycosaminoglycans, complex N-glycans, and diverse bacterial glycans have been synthesized manually. Gratifyingly, the synthesis of mycobacterial arabinogalactan containing 92 monosaccharide units has been achieved, which created a precedent in the field of polysaccharide synthesis. Recently, the synthesis of a highly branched arabinogalactan from traditional Chinese medicine featuring 140 monosaccharide units has been also accomplished to evaluate its anti-pancreatic-cancer activity. In the spirit of green and sustainable chemistry, this strategy can also be applied to light-driven glycosylation reactions, where either UV or visible light can be used for the activation of glycosyl donors.Automated synthesis is an advanced approach to the construction of complex glycans. Based on the two preactivation modes (general promoter activation mode and light-induced activation mode), a universal and highly efficient automated solution-phase synthesizer was further developed to drive glycan assembly from manual to automated synthesis. Using this synthesizer, a library of oligosaccharides covering various glycoforms and glycosidic linkages was assembled rapidly, either in a general promoter-activation mode or in a light-induced-activation mode. The automated synthesis of a fully protected fondaparinux pentasaccharide was realized on a gram scale. Furthermore, the automated synthesis of large-size polysaccharides was performed, allowing the assembly of arabinans up to an astonishing 1080-mer using the automated multiplicative synthesis strategy, taking glycan synthesis to a new height far beyond the synthesis of nucleic acids (up to 200-mer) and proteins (up to 472-mer).

Sensing of mycobacterial arabinogalactan by galectin-9 exacerbates mycobacterial infection.

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio-synthetical target for anti-tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin-9 and exacerbates mycobacterial infection. Administration of AG-specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb-infected mice or Mycobacterium marinum-infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin-9 with high affinity, and galectin-9 associates with transforming growth factor ß-activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal-regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin-9 or inhibition of MMPs blocks AG-induced pathological impairments in the lung, and the AG-galectin-9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin-9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.

O-GlcNAcylation increases PYGL activity by promoting phosphorylation.

O-GlcNAcylation is a post-translational modification that links metabolism with signal transduction. High O-GlcNAcylation appears to be a general characteristic of cancer cells. It promotes the invasion, metastasis, proliferation and survival of tumor cells, and alters many metabolic pathways. Glycogen metabolism increases in a wide variety of tumors, suggesting that it is an important aspect of cancer pathophysiology. Herein we focused on the O-GlcNAcylation of liver glycogen phosphorylase (PYGL)-an important catabolism enzyme in the glycogen metabolism pathway. PYGL expressed in both HEK 293T and HCT116 was modified by O-GlcNAc. And both PYGL O-GlcNAcylation and phosphorylation of Ser15 (pSer15) were decreased under glucose and insulin, whereas increased under glucagon and Na2S2O4 (hypoxia) conditions. Then, we identified the major O-GlcNAcylation site to be Ser430, and demonstrated that pSer15 and Ser430 O-GlcNAcylation were mutually reinforced. Lastly, we found that Ser430 O-GlcNAcylation was fundamental for PYGL activity. Thus, O-GlcNAcylation of PYGL positively regulated pSer15 and therefore its enzymatic activity. Our results provided another molecular insight into the intricate post-translational regulation network of PYGL.

Synthesis and Immunological Evaluation of Pentamannose-Based HIV-1 Vaccine Candidates.

Dense glycosylation and the trimeric conformation of the human immunodeficiency virus-1 (HIV-1) envelope protein limit the accessibility of some cellular glycan processing enzymes and end up with high-mannose-type N-linked glycans on the envelope spike, among which the Man5GlcNAc2 structure occupies a certain proportion. The Man5GlcNAc2 glycan composes the binding sites of some potent broadly neutralizing antibodies, and some lectins that can bind Man5GlcNAc2 show HIV-neutralizing activity. Therefore, Man5GlcNAc2 is a potential target for HIV-1 vaccine development. Herein, a highly convergent and effective strategy was developed for the synthesis of Man5 and its monofluoro-modified, trifluoro-modified, and S-linked analogues. We coupled these haptens to carrier protein CRM197 and evaluated the immunogenicity of the glycoconjugates in mice. The serological assays showed that the native Man5 conjugates failed to induce Man5-specific antibodies in vivo, while the modified analogue conjugates induced stronger antibody responses. However, these antibodies could not bind the native gp120 antigen. These results demonstrated that the immune tolerance mechanism suppressed the immune responses to Man5-related structures and the conformation of glycan epitopes on the synthesized glycoconjugates was distinct from that of native glycan epitopes on gp120.

Iterative Synthesis of 2-Deoxyoligosaccharides Enabled by Stereoselective Visible-Light-Promoted Glycosylation.

The photoinitiated intramolecular hydroetherification of alkenols has been used to form C-O bonds, but the intermolecular hydroetherification of alkenes with alcohols remains an unsolved challenge. We herein report the visible-light-promoted 2-deoxyglycosylation of alcohols with glycals. The glycosylation reaction was completed within 2 min in a high quantum yield (ϕ=28.6). This method was suitable for a wide array of substrates and displayed good reaction yields and excellent stereoselectivity. The value of this protocol was further demonstrated by the iterative synthesis of 2-deoxyglycans with α-2-deoxyglycosidic linkages up to a 20-mer in length and digoxin with ß-2-deoxyglycosidic linkages. Mechanistic studies indicated that this reaction involved a glycosyl radical cation intermediate and a photoinitiated chain process.

Glycan Assembly Strategy: From Concept to Application.

Glycans have been hot topics in recent years due to their exhibition of numerous biological activities. However, the heterogeneity of their natural source and the complexity of their chemical synthesis impede the progress in their biological research. Thus, the development of glycan assembly strategies to acquire plenty of structurally well-defined glycans is an important issue in carbohydrate chemistry. In this review, the latest advances in glycan assembly strategies from concepts to their applications in carbohydrate synthesis, including chemical and enzymatic/chemo-enzymatic approaches, as well as solution-phase and solid-phase/tag-assisted synthesis, are summarized. Furthermore, the automated glycan assembly techniques are also outlined.

Electrochemical Trifluoromethylation of Glycals.

Carbohydrates play essential roles in various physiological and pathological processes. Trifluoromethylated compounds have wide applications in the field of medicinal chemistry. Herein, we report a practical and efficient trifluoromethylation of glycals by an electrochemical approach using CF3SO2Na as the trifluoromethyl source and MnBr2 as the redox mediator. A variety of trifluoromethylated glycals bearing different protective groups are obtained in 60-90% yields with high regioselectivity. The successful capture of a CF3 radical indicates that a radical mechanism is involved in this reaction.

Carbocyclic Ring Closure of Aryl C-Glycosides Promoted by Fluoroboric Acid.

A novel transformation from rhamnose-type C-glycosides to 2-cyclopentenones is described. With the promotion of fluoroboric acid, C-glycosides underwent ring opening and subsequent Nazarov cyclization to afford 2-cyclopentenones in good to excellent yields. The solvent and the concentration of acid are crucial to the yield of this transformation.

Engineering a bacterial sialyltransferase for di-sialylation of a therapeutic antibody.

Terminal α-2,6-sialylation of N-glycans is a humanized glycosylation that affects the properties and efficacy of therapeutic glycoproteins. Fc di-sialylation (a biantennary N-glycan with two α-2,6-linked sialic acids) of IgG antibodies imparts them with enhanced anti-inflammatory activity and other roles. However, the microheterogeneity of N-glycoforms presents a challenge for therapeutic development. Therefore, controlled sialylation has drawn considerable attention, but direct access to well-defined di-sialylated antibodies remains limited. Herein, a one-pot three-enzyme protocol was developed by engineering a bacterial sialyltransferase to facilitate the modification of therapeutic antibodies with N-acetylneuraminic acid or its derivatives towards optimized glycosylation. To overcome the low proficiency of bacterial sialyltransferase in antibody remodeling, the Photobacterium sp. JT-ISH-224 α-2,6-sialyltransferase (Psp2,6ST) was genetically engineered by terminal truncation and site-directed mutagenesis based on its protein crystal structure. With the optimized reaction conditions and using activity-based screening of various Psp2,6ST variants, a truncated mutant Psp2,6ST (111-511)-His6 A235M/A366G was shown to effectively improve the catalytic efficiency of antibody di-sialylation. Herceptin and the donor substrate promiscuity allow the introduction of bioorthogonal modifications of N-acetylneuraminic acid into antibodies for site-specific conjugation. 2-AB hydrophilic interaction chromatography analysis of the released N-glycans and intact mass characterization confirmed the high di-sialylation of Herceptin via the optimized one-pot three-enzyme reaction. This study established a versatile enzymatic approach for producing highly di-sialylated IgG antibodies. It provides new insights into engineering bacterial sialyltransferase for adaptation to the enzymatic glycoengineering of therapeutic antibodies and the glycosite-specific conjugation of antibodies.

C-Glycosylation enabled by N-(glycosyloxy)acetamides.

The C-glycosylation of C-nucleophiles including allyltrimethylsilane, silyl enol ethers and phenols with N-(glycosyloxy)acetamides as glycosyl donors has been realized. This protocol provides a convenient and practical route for the synthesis of alkyl C-glycosides and aryl 2-deoxy-ß-C-glycosides under mild reaction conditions.

Stereoselective Electro-2-deoxyglycosylation from Glycals.

We report a novel and highly stereoselective electro-2-deoxyglycosylation from glycals. This method features excellent stereoselectivity, scope, and functional-group tolerance. This process can also be applied to the modification of a wide range of natural products and drugs. Furthermore, a scalable synthesis of glycosylated podophyllotoxin and a one-pot trisaccharide synthesis through iterative electroglycosylations were achieved.

Fluorine-modified sialyl-Tn-CRM197 vaccine elicits a robust immune response.

Even though a vaccine that targets tumor-associated carbohydrate antigens on epithelial carcinoma cells presents an attractive therapeutic approach, relatively poor immunogenicity limits its development. In this study, we investigated the immunological activity of a fluoro-substituted Sialyl-Tn (F-STn) analogue coupled to the non-toxic cross-reactive material of diphtheria toxin197 (CRM197). Our results indicate that F-STn-CRM197 promotes a greater immunogenicity than non-fluorinated STn-CRM197. In the presence or absence of adjuvant, F-STn-CRM197 remarkably enhances both cellular and humoral immunity against STn by increasing antigen-specific lymphocyte proliferation and inducing a mixed Th1/Th2 response leading to production of IFN-γ and IL-4 cytokines, as well as STn-specific antibodies. Furthermore, antisera produced from F-STn-CRM197 immunization significantly recognizes STn-positive tumor cells and increases cancer cell lysis induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) pathways. Our data suggest that this F-STn vaccine may be useful for cancer immunotherapy and possibly for prophylactic prevention of cancer.

N-Alkyl-1,5-dideoxy-1,5-imino-l-fucitols as fucosidase inhibitors: Synthesis, molecular modelling and activity against cancer cell lines.

1,5-Dideoxy-1,5-imino-l-fucitol (1-deoxyfuconojirimycin, DFJ) is an iminosugar that inhibits fucosidases. Herein, N-alkyl DFJs have been synthesised and tested against the α-fucosidases of T. maritima (bacterial origin) and B. taurus (bovine origin). The N-alkyl derivatives were inactive against the bacterial fucosidase, while inhibiting the bovine enzyme. Docking of inhibitors to homology models, generated for the bovine and human fucosidases, was carried out. N-Decyl-DFJ was toxic to cancer cell lines and was more potent than the other N-alkyl DFJs studied.

Validation, Identification, and Biological Consequences of the Site-specific O-GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP).

O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.

Carbohydrate-based vaccines for oncotherapy.

Cancer is still one of the most serious threats to human worldwide. Aberrant patterns of glycosylation on the surface of cancer cells, which are correlated with various cancer development stages, can differentiate the abnormal tissues from the healthy ones. Therefore, tumor-associated carbohydrate antigens (TACAs) represent the desired targets for cancer immunotherapy. However, these carbohydrate antigens may not able to evoke powerful immune response to combat with cancer for their poor immunogenicity and immunotolerance. Different approaches have been developed to address these problems. In this review, we want to summarize the latest advances in TACAs based anticancer vaccines.

Synthetic Glycans and Glycomimetics: A Promising Alternative to Natural Polysaccharides.

A large quantity of polysaccharide-derived conjugate vaccines have been developed to combat various pathogenic infections. Another prominent polysaccharide, heparin, is listed as an essential drug by the World Health Organization (WHO) to treat thrombus. One of their common problems is that they all derive from natural polysaccharides. Specifically, capsular polysaccharides are mainly obtained from bacterial fermentation and unfractionated heparin is extracted from animal tissues such as porcine mucosa. The quality of natural polysaccharides is inconsistent and traces of contamination would cause a disaster. By contrast, the use of chemical or chemoenzymatic methods could provide structurally homogeneous and quality-controlled glycans. To date, large numbers of polysaccharide fragments and their analogues have been synthesized and evaluated. Some of them even showed comparable activities to their corresponding natural polysaccharides. Here, the latest advances in these synthetic glycan analogues ranging from carbohydrate-based vaccines, heparin-related therapeutics and glycomimetics of polysaccharides are summarized.

O-Glycosylation Enabled by N-(Glycosyloxy)acetamides.

A novel glycosylation protocol has been established by using N-(glycosyloxy)acetamides as glycosyl donors. The N-oxyacetamide leaving group in donors could be rapidly activated in the presence of Cu(OTf)2 or SnCl4 under microwave irradiation. This glycosylation process afforded the coupled products in high yields, and the reaction enjoyed a broad substrate scope, even for disarmed donors and hindered acceptors. The easy availability of the donors, the high stability of N-(glycosyloxy)acetamides, and the small leaving group make this method very practical.

Synthesis and Antigenic Evaluation of Oligosaccharide Mimics of Vi Antigen from Salmonella typhi.

Salmonella typhi is responsible for typhoid fever, which is a serious health threat in developing countries. As a virulent factor of Salmonella typhi, the purified Vi polysaccharide (Vi PS) has become an effective vaccine to combat typhoid fever. The chemical synthesis can provide homogeneous and well-defined molecules for the development of Vi-based vaccines. However, the synthesis of Vi oligosaccharides in high yields and with exclusive α-stereoselectivities remains very challenging. In this paper, a series of Vi pseudooligosaccharides, including pseudo tetra-, hexa-, and octa-saccharides were efficiently synthesized. These oligosaccharide analogues were conjugated by carbon chain tether through olefin cross metathesis or by the 1,2,3-triazole moiety through copper (I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC). The binding affinities of these oligosaccharide mimics to anti-Vi antibodies were investigated. These results will be beneficial to the further development of Vi-based oligosaccharide vaccines.

Synthesis and biological evaluation of N-arylated-lactam-type iminosugars as potential immunosuppressive agents.

Since the immunosuppressive agents currently used in clinics have significant side effects, it is very important to search for new effective and safe immunosuppressants. Iminosugars as a new class of immunosuppressants are less explored. In this report, 24 new N-arylated iminosugar derivatives, including d-talo and d-galacto epimers, were designed and synthesized, and their immunosuppressive effects were evaluated by MTT assay. The experimental data demonstrated that compound 20 showed the strongest inhibition effect (IC50 = 6.94 µM). Further studies revealed that the inhibitory effects on splenocyte proliferation may come from the suppression of both IFN-γ and IL-4 cytokines. The preliminary structure-activity relationship (SAR) analysis suggested that N-arylated d-galacto-type iminosugars showed better inhibitory activities than d-talo-type analogues. The SAR analysis also showed that the inhibition effect of iminosugars can be improved by decreasing the polarity or increasing the hydrophobicity. These results may be beneficial to the discovery of new iminosugar derivatives as immunosuppressive agents.

Correction: Synthesis and immunological evaluation of MUC1 glycopeptide conjugates bearing N-acetyl modified STn derivatives as anticancer vaccines.

Correction for 'Synthesis and immunological evaluation of MUC1 glycopeptide conjugates bearing N-acetyl modified STn derivatives as anticancer vaccines' by An Xiao et al., Org. Biomol. Chem., 2016, 14, 7226-7237.