A Computational and Chemical Design Strategy for Manipulating Glycan-Protein Recognition.

Glycans are complex biomolecules that encode rich information and regulate various biological processes, such as fertilization, host-pathogen binding, and immune recognition, through interactions with glycan-binding proteins. A key driving force for glycan-protein recognition is the interaction between the π electron density of aromatic amino acid side chains and polarized C─H groups of the pyranose (termed the CH-π interaction). However, the relatively weak binding affinity between glycans and proteins has hindered the application of glycan detection and imaging. Here, computational modeling and molecular dynamics simulations are employed to design a chemical strategy that enhances the CH-π interaction between glycans and proteins by genetically incorporating electron-rich tryptophan derivatives into a lectin PhoSL, which specifically recognizes core fucosylated N-linked glycans. This significantly enhances the binding affinity of PhoSL with the core fucose ligand and enables sensitive detection and imaging of core fucosylated glycans in vitro and in xenograft tumors in mice. Further, the study showed that this strategy is applicable to improve the binding affinity of GafD lectin for N-acetylglucosamine-containing glycans. The approach thus provides a general and effective way to manipulate glycan-protein recognition for glycoscience applications.

Chemoenzymatic Synthesis of Tri-antennary N-Glycans Terminating in Sialyl-Lewisx Reveals the Importance of Glycan Complexity for Influenza A Virus Receptor Binding.

Sialyl-Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty-two isomeric asymmetrical tri-antennary N-glycans presenting SLex-Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high-affinity ligands for E-selectin. The N-glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5'-diphospho-N-trifluoracetylglucosamine (UDP-GlcNHTFA) that can be transferred by branching N-acetylglucosaminyltransferases to give, after base treatment, GlcNH2-containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E-selectin bound equally well to linear glycans and tri-antennary N-glycans presenting SLex-Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri-antennary N-glycans. Furthermore, several H5 HAs preferentially bound to N-glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N-glycans presenting SLex.

Stereoconvergent and Chemoenzymatic Synthesis of Tumor-Associated Glycolipid Disialosyl Globopentaosylceramide for Probing the Binding Affinity of Siglec-7.

Disialosyl globopentaosylceramide (DSGb5) is a tumor-associated complex glycosphingolipid. However, the accessibility of structurally well-defined DSGb5 for precise biological functional studies remains challenging. Herein, we describe the first total synthesis of DSGb5 glycolipid by an efficient chemoenzymatic approach. A Gb5 pentasaccharide-sphingosine was chemically synthesized by a convergent and stereocontrolled [2 + 3] method using an oxazoline disaccharide donor to exclusively form ß-anomeric linkage. After investigating the substrate specificity of different sialyltransferases, regio- and stereoselective installment of two sialic acids was achieved by two sequential enzyme-catalyzed reactions using α2,3-sialyltransferase Cst-I and α2,6-sialyltransferase ST6GalNAc5. A unique aspect of the approach is that methyl-ß-cyclodextrin-assisted enzymatic α2,6-sialylation of glycolipid substrate enables installment of the challenging internal α2,6-linked sialoside to synthesize DSGb5 glycosphingolipid. Surface plasmon resonance studies indicate that DSGb5 glycolipid exhibits better binding affinity for Siglec-7 than the oligosaccharide moiety of DSGb5. The binding results suggest that the ceramide moiety of DSGb5 facilitates its binding by presenting multivalent interactions of glycan epitope for the recognition of Siglec-7.

Chemical Synthesis Creates Single Glycoforms of the Ectodomain of Herpes Simplex Virus-1 Glycoprotein D.

Herpes simplex virus-1 (HSV-1) utilizes multiple viral surface glycoproteins to trigger virus entry and fusion. Among these glycoproteins, glycoprotein D (gD) functions as a receptor-binding protein, which makes it an attractive target for the development of vaccines against HSV-1 infection. Several recombinant gD subunit vaccines have been investigated in both preclinical and clinical phases with varying degrees of success. It is fundamentally critical to explore the functions of gD glycans. In light of this, we report an efficient synthetic platform to construct glycosylated gDs bearing homogeneous glycans at N94 and N121. The oligosaccharides were prepared by enzymatic synthesis and conjugated to peptidyl sectors. The glycoproteins were constructed via a combination of 7-(piperazin-1-yl)-2-(methyl)quinolinyl (PPZQ)-assisted expressed protein ligation and ß-mercapto amino acid-assisted-desulfurization strategies. Biological studies showed that synthetic gDs exhibited potent in vivo activity in mice.

One-step synthesis of site-specific antibody-drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates.

Glycosite-specific antibody‒drug conjugatess (gsADCs), harnessing Asn297 N-glycan of IgG Fc as the conjugation site for drug payloads, usually require multi-step glycoengineering with two or more enzymes, which limits the substrate diversification and complicates the preparation process. Herein, we report a series of novel disaccharide-based substrates, which reprogram the IgG glycoengineering to one-step synthesis of gsADCs, catalyzed by an endo-N-acetylglucosaminidase (ENGase) of Endo-S2. IgG glycoengineering via ENGases usually has two steps: deglycosylation by wild-type (WT) ENGases and transglycosylation by mutated ENGases. But in the current method, we have found that disaccharide LacNAc oxazoline can be efficiently assembled onto IgG by WT Endo-S2 without hydrolysis of the product, which enables the one-step glycoengineering directly from native antibodies. Further studies on substrate specificity revealed that this approach has excellent tolerance on various modification of 6-Gal motif of LacNAc. Within 1 h, one-step synthesis of gsADC was achieved using the LacNAc-toxin substrates including structures free of bioorthogonal groups. These gsADCs demonstrated good homogeneity, buffer stability, in vitro and in vivo anti-tumor activity. This work presents a novel strategy using LacNAc-based substrates to reprogram the multi-step IgG glycoengineering to a one-step manner for highly efficient synthesis of gsADCs.

Convergent Synthesis and Anti-Pancreatic Cancer Cell Growth Activity of a Highly Branched Heptadecasaccharide from Carthamus tinctorius.

Bioactive polysaccharides from natural resources target various biological processes and are increasingly used as potential target molecules for drug development. However, the accessibility of branched and long complex polysaccharide active domains with well-defined structures remains a major challenge. Herein we describe an efficient first total synthesis of a highly branched heptadecasaccharide moiety of the native bioactive galectin-3-targeting polysaccharide from Carthamus tinctorius L. as well as shorter fragments of the heptadecasaccharide. The key feature of the approach is that a photo-assisted convergent [6+4+7] one-pot coupling strategy enables rapid assembly of the heptadecasaccharide, whereby a photoremovable o-nitrobenzyl protecting group is used to generate the corresponding acceptor for glycosylation in situ upon ultraviolet radiation. Biological activity tests suggest that the heptadecasaccharide can target galectin-3 and inhibit pancreatic cancer cell growth.

Integrated Chemoenzymatic Approach to Streamline the Assembly of Complex Glycopeptides in the Liquid Phase.

Glycosylation of proteins is a complicated post-translational modification. Despite the significant progress in glycoproteomics, accurate functions of glycoproteins are still ambiguous owing to the difficulty in obtaining homogeneous glycopeptides or glycoproteins. Here, we describe a streamlined chemoenzymatic method to prepare complex glycopeptides by integrating hydrophobic tag-supported chemical synthesis and enzymatic glycosylations. The hydrophobic tag is utilized to facilitate peptide chain elongation in the liquid phase and expeditious product separation. After removal of the tag, a series of glycans are installed on the peptides via efficient glycosyltransferase-catalyzed reactions. The general applicability and robustness of this approach are exemplified by efficient preparation of 16 well-defined SARS-CoV-2 O-glycopeptides, 4 complex MUC1 glycopeptides, and a 31-mer glycosylated glucagon-like peptide-1. Our developed approach will open up a new range of easy access to various complex glycopeptides of biological importance.

Chemoenzymatic synthesis of the oligosaccharide moiety of the tumor-associated antigen disialosyl globopentaosylceramide.

Disialosyl globopentaosylceramide (DSGb5) is often expressed by renal cell carcinomas. To investigate properties of DSGb5, we have prepared its oligosaccharide moiety by chemically synthesizing Gb5 which was enzymatically sialylated using the mammalian sialyltransferases ST3Gal1 and ST6GalNAc5. Glycan microarray binding studies indicate that Siglec-7 does not recognize DSGb5, and preferentially binds Neu5Acα(2,8)Neu5Ac containing glycans.

Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins.

Glycan-binding proteins (GBPs) play critical roles in diverse cellular functions such as cell adhesion, signal transduction and immune response. Studies of the interaction between GBPs and glycans have been hampered by the availability of high throughput and high-content technologies. Here we report multiplex glycan bead array (MGBA) that allows simultaneous analyses of 384 samples and up to 500 glycans in a single assay. The specificity, sensitivity and reproducibility of MGBA are evaluated using 39 plant lectins, 13 recombinant anti-glycan antibodies, and mammalian GBPs. We demonstrate the utility of this platform by the analyses of natural anti-glycan IgM and IgG antibodies in 961 human serum samples and the discovery of anti-glycan antibody biomarkers for ovarian cancer. Our data indicate that the MGBA platform is particularly suited for large population-based studies that require the analyses of large numbers of samples and glycans.

Chemoenzymatic Approach for the Preparation of Asymmetric Bi-, Tri-, and Tetra-Antennary N-Glycans from a Common Precursor.

Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and unnatural Galα(1,4)-GlcNAc and Manß(1,4)-GlcNAc appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a substrate, and thus this structure can be uniquely extended. Next, the ß-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural α-Gal and ß-Man terminating antennae can sequentially be decaged by an appropriate glycosidase to liberate a terminal ß-GlcNAc moiety, which can be converted into LacNAc and then elaborated by a panel of glycosyltransferases. Asymmetric bi- and triantennary glycans could be obtained by removal of a terminal ß-GlcNAc moiety by treatment with ß-N-acetylglucosaminidase and selective extension of the other arms. The power of the methodology is demonstrated by the preparation of an asymmetric tetra-antennary N-glycan found in human breast carcinoma tissue, which represents the most complex N-glycan ever synthesized. Multistage mass spectrometry of the two isomeric triantennary glycans uncovered unique fragment ions that will facilitate identification of exact structures of glycans in biological samples.

Divergent Chemoenzymatic Synthesis of Asymmetrical-Core-Fucosylated and Core-Unmodified N-Glycans.

A divergent chemoenzymaytic approach for the preparation of core-fucosylated and core-unmodified asymmetrical N-glycans from a common advances precursor is described. An undecasaccharide was synthesized by sequential chemical glycosylations of an orthogonally protected core fucosylated hexasaccharide that is common to all mammalian core fucosylated N-glycans. Antennae-selective enzymatic extension of the undecasaccharide using a panel of glycosyl transferases afforded core fucosylated asymmetrical triantennary N-glycan isomers, which are potential biomarkers for breast cancer. A unique aspect of our approach is that a fucosidase (FucA1) has been identified that selectively can cleave a core-fucoside without affecting the fucoside of a sialyl LewisX epitope to give easy access to core-unmodified compounds.

Chemoenzymatic synthesis of ADP-d-glycero-ß-d-manno-heptose and study of the substrate specificity of HldE.

An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-d-glycero-ß-d-manno-heptose (ADP-d, d-heptose) was reported using chemically synthesized d, d-heptose-7-phosphate and the ADP-d, d-heptose biosynthetic enzymes HldE and GmhB. Moreover, the result of investigating substrate specificity of the kinase action of HldE revealed that HldE had highly restricted substrate specificity towards structurally modified heptose-7-phosphate analogs.

Elevation of cellular O-GlcNAcylation level by a potent and selective O-GlcNAcase inhibitor based on tetrahydroimidazopyridine scaffold.

Protein O-GlcNAc glycosylation is a ubiquitous post-translational modification in metazoans. O-GlcNAcase (OGA), which is responsible for removing O-GlcNAc from serine or threonine residues, plays a key role in O-GlcNAc metabolism. Potent and selective O-GlcNAcase (OGA) inhibitors are useful tools for investigating the role of this modification in a broad range of cellular processes, and may also serve as drug candidates for treatment of neurodegenerative diseases. Biological screening of the gluco-configured tetrahydroimidazopyridine derivatives identified a compound as a potent and competitive inhibitor of human O-GlcNAcase (OGA) with a K(i) of 5.9 µM, and it also displayed 28-fold selectivity for human OGA over human lysosomal ß-hexosaminidase A (Hex A, K(i)=163 µM). In addition, cell-based assay revealed that this compound was cell-permeant and effectively induced cellular hyper-O-GlcNAcylation at 10 µM concentration.

Structure-activity relationships in a series of C2-substituted gluco-configured tetrahydroimidazopyridines as ß-glucosidase inhibitors.

Inhibition of glycoside hydrolases has widespread application in treatment of diabetes, viral infections, lysosomal storage diseases and cancers. Gluco-configured tetrahydroimidazopyridines are the most potent ß-glucosidase inhibitors reported to date. Using transition state mimic strategy, a series of C2-substituted gluco-configured tetrahydroimidazopyridines were designed and synthesized. Compounds 3 (K(i)=0.64 nM) and 5 (K(i)=0.58 nM) showed stronger inhibitory potency against ß-glucosidase. Maestro 9.1 was used to study the structure-activity relationships by docking the compounds into the ß-glucosidase active sites.

Multivalent interaction-based carbohydrate biosensors for signal amplification.

Multivalent interaction between boronic acids immobilized on quartz crystal microbalance (QCM) sensor surface and the carbohydrates modified Au-nanoparticle (AuNP) has been demonstrated for the development of a sensitive carbohydrate biosensor. Briefly, a boronic acid-containing polymer (boropolymer) as multivalent carbohydrate receptor was oriented immobilized on the cysteamine coated electrode through isourea bond formation. Carbohydrates were conjugated to AuNPs to generate a multivalent carbohydrates moiety to amplify the response signal. Thus, the binding of the carbohydrate conjugated AuNPs to the boropolymer surface are multivalent which could simultaneously increase the binding affinity and specificity. We systematically studied the binding between five carbohydrates conjugated AuNPs and the boropolymer. Our studies show that the associate constant (K(a)) was in the order of fucose