Benjamin G. Davis

Glycoprotein Synthesis: An Update

2.2. Assembly Strategies 139 2.2.1. Linear Assembly 139 2.2.2. Convergent Assembly 139 2.2.3. Elaborative and Mixed Assembly Strategies 139 2.2.4. Native Ligation Assembly 140 3. Chemical Glycoprotein Synthesis 143 3.1. Indiscriminate Convergent Glycosylation 143 3.2. Chemoselective and Site-Specific Glycosylation 145 3.3. Site-Selective Glycosylation 147 3.4. Native Ligation Assembly 151 4. Enzymatic Glycoprotein and Glycopeptide Synthesis 151 4.1. Glycan Extension 151 4.2. Glycoprotein Remodeling (GPR) 152 4.3. Enzymatic Formation of the Glycan-Protein/ Peptide Link 153

Lectins: tools for the molecular understanding of the glycocode

Recent progress in glycobiology has revealed that cell surface oligosaccharides play an essential role in recognition events. More precisely, these saccharides may be complexed by lectins, carbohydrate-binding proteins other than enzymes and antibodies, able to recognise sugars in a highly specific manner. The ubiquity of lectin-carbohydrate interactions opens enormous potential for their exploitation in medicine. Therefore, extraordinary effort is made into the identification of new lectins as well as into the achievement of a deep understanding of their functions and of the precise mechanism of their association with specific ligands. In this review, a summary of the main features of lectins, particularly those found in legumes, will be presented with a focus on the mechanism of carbohydrate-binding. An overview of lectin-carbohydrate interactions will also be given, together with an insight into their energetics. In addition, therapeutic applications of lectins will be discussed.

Structure of a Flavonoid Glucosyltransferase Reveals the Basis for Plant Natural Product Modification.

Glycosylation is a key mechanism for orchestrating the bioactivity, metabolism and location of small molecules in living cells. In plants, a large multigene family of glycosyltransferases is involved in these processes, conjugating hormones, secondary metabolites, biotic and abiotic environmental toxins, to impact directly on cellular homeostasis. The red grape enzyme UDP‐glucose:flavonoid 3‐O‐glycosyltransferase (VvGT1) is responsible for the formation of anthocyanins, the health‐promoting compounds which, in planta, function as colourants determining flower and fruit colour and are precursors for the formation of pigmented polymers in red wine. We show that VvGT1 is active, in vitro, on a range of flavonoids. VvGT1 is somewhat promiscuous with respect to donor sugar specificity as dissected through full kinetics on a panel of nine sugar donors. The three‐dimensional structure of VvGT1 has also been determined, both in its ‘Michaelis’ complex with a UDP‐glucose‐derived donor and the acceptor kaempferol and in complex with UDP and quercetin. These structures, in tandem with kinetic dissection of activity, provide the foundation for understanding the mechanism of these enzymes in small molecule homeostasis.

Chemical Modification of Proteins at Cysteine: Opportunities in Chemistry and Biology

Chemical modification of proteins is a rapidly expanding area in chemical biology. Selective installation of biochemical probes has led to a better understanding of natural protein modification and macromolecular function. In other cases such chemical alterations have changed the protein function entirely. Additionally, tethering therapeutic cargo to proteins has proven invaluable in campaigns against disease. For controlled, selective access to such modified proteins, a unique chemical handle is required. Cysteine, with its unique reactivity, has long been used for such modifications. Cysteine has enjoyed widespread use in selective protein modification, yet new applications and even new reactions continue to emerge. This Focus Review highlights the enduring utility of cysteine in protein modification with special focus on recent innovations in chemistry and biology associated with such modifications.

Expanding the diversity of chemical protein modification allows post-translational mimicry

One of the most important current scientific paradoxes is the economy with which nature uses genes. In all higher animals studied, we have found many fewer genes than we would have previously expected. The functional outputs of the eventual products of genes seem to be far more complex than the more restricted blueprint. In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs). These alterations of amino-acid side chains lead to higher structural and functional protein diversity and are, therefore, a leading contender for an explanation for this seeming incongruity. Natural protein production methods typically produce PTM mixtures within which function is difficult to dissect or control. Until now it has not been possible to access pure mimics of complex PTMs. Here we report a chemical tagging approach that enables the attachment of multiple modifications to bacterially expressed (bare) protein scaffolds: this approach allows reconstitution of functionally effective mimics of higher organism PTMs. By attaching appropriate modifications at suitable distances in the widely-used LacZ reporter enzyme scaffold, we created protein probes that included sensitive systems for detection of mammalian brain inflammation and disease. Through target synthesis of the desired modification, chemistry provides a structural precision and an ability to retool with a chosen PTM in a manner not available to other approaches. In this way, combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein–PTM interactions. We therefore anticipate that this ability to build model systems will allow some of this gene product complexity to be dissected, with the aim of eventually being able to completely duplicate the patterns of a particular protein’s PTMs from an in vivo assay into an in vitro system.

Allyl Sulfides Are Privileged Substrates in Aqueous Cross-Metathesis: Application to Site-Selective Protein Modification

Allyl sulfides undergo efficient cross-metathesis in aqueous media with Hoveyda-Grubbs second generation catalyst 1. The high reactivity of allyl sulfides in cross-metathesis was exploited in the first examples of cross-metathesis on a protein surface. S-Allylcysteine was incorporated chemically into the protein, providing the requisite allyl sulfide handle. Preliminary efforts to genetically incorporate S-allylcysteine into proteins are also reported.

Filled and glycosylated carbon nanotubes for in vivo radioemitter localization and imaging

Functionalization of nanomaterials for precise biomedical function is an emerging trend in nanotechnology. Carbon nanotubes are attractive as multifunctional carrier systems because payload can be encapsulated in internal space whilst outer surfaces can be chemically modified. Yet, despite potential as drug delivery systems and radiotracers, such filled-and-functionalized carbon nanotubes have not been previously investigated in vivo. Here we report covalent functionalization of radionuclide-filled single-walled carbon nanotubes and their use as radioprobes. Metal halides, including Na(125)I, were sealed inside single-walled carbon nanotubes to create high-density radioemitting crystals and then surfaces of these filled-sealed nanotubes were covalently modified with biantennary carbohydrates, improving dispersibility and biocompatibility. Intravenous administration of Na(125)I-filled glyco-single-walled carbon nanotubes in mice was tracked in vivo using single-photon emission computed tomography. Specific tissue accumulation (here lung) coupled with high in vivo stability prevented leakage of radionuclide to high-affinity organs (thyroid/stomach) or excretion, and resulted in ultrasensitive imaging and delivery of unprecedented radiodose density. Nanoencapsulation of iodide within single-walled carbon nanotubes enabled its biodistribution to be completely redirected from tissue with innate affinity (thyroid) to lung. Surface functionalization of (125)I-filled single-walled carbon nanotubes offers versatility towards modulation of biodistribution of these radioemitting crystals in a manner determined by the capsule that delivers them. We envisage that organ-specific therapeutics and diagnostics can be developed on the basis of the nanocapsule model described here.

A Convenient Catalyst for Aqueous and Protein Suzuki−Miyaura Cross-Coupling

A phosphine-free palladium catalyst for aqueous Suzuki-Miyaura cross-coupling is presented. The catalyst is active enough to mediate hindered, ortho-substituted biaryl couplings but mild enough for use on peptides and proteins. The Suzuki-Miyaura couplings on protein substrates are the first to proceed in useful conversions. Notably, hydrophobic aryl and vinyl groups can be transferred to the protein surface without the aid of organic solvent since the aryl- and vinylboronic acids used in the coupling are water-soluble as borate salts. The convenience and activity of this catalyst prompts use in both general synthesis and bioconjugation.

Glyconanoparticles allow pre-symptomatic in vivo imaging of brain disease

Initial recruitment of leukocytes in inflammation associated with diseases such as multiple sclerosis (MS), ischemic stroke, and HIV-related dementia, takes place across intact, but activated brain endothelium. It is therefore undetectable to symptom-based diagnoses and cannot be observed by conventional imaging techniques, which rely on increased permeability of the blood–brain barrier (BBB) in later stages of disease. Specific visualization of the early-activated cerebral endothelium would provide a powerful tool for the presymptomatic diagnosis of brain disease and evaluation of new therapies. Here, we present the design, construction and in vivo application of carbohydrate-functionalized nanoparticles that allow direct detection of endothelial markers E-/P-selectin (CD62E/CD62P) in acute inflammation. These first examples of MRI-visible glyconanoparticles display multiple copies of the natural complex glycan ligand of selectins. Their resulting sensitivity and binding selectivity has allowed acute detection of disease in mammals with beneficial implications for treatment of an expanding patient population suffering from neurological disease.

Thiyl Glycosylation of Olefinic Proteins: S‐Linked Glycoconjugate Synthesis

Over half of all proteins in nature are estimated to be glycosylated, and these biomolecules play key roles in protein expression, folding and stability and are fundamental to various biological processes. In recent years, synthetic homogenous glycoforms of glycoproteins have been one of the primary targets in glycobiology, 21] not only to allow function determination, but also to create glycoprotein mimetics useful as, e.g., therapeutic agents. Beyond the preference for the more abundant native Oand N-linked glycoproteins, S-linked glycoproteins are also attractive synthetic targets as a result of their enhanced chemical stability and enzymatic resistance. Following the discovery of the first natural S-glycosidic linkage by Lote and Weiss in 1971, methods have been developed for the synthesis of S-linked glycopeptides and more recently S-linked glycoproteins. 27] Here, we describe the development of a convergent approach for the synthesis of a novel class of S-linked glyconjugate proteins through the site-specific ligation of 1glycosyl thiols to proteins (Scheme 1). The strategy exploits non-natural amino acid incorporation 29] for the introduction of l-homoallylglycine (l-Hag) into a protein and freeradical addition hydrothiolation reactions, under conditions mild enough to retain protein activity throughout (Scheme 1). In this way, Hag functions as a new “tag” combined here with a new modification as part of a general “tag–modify” strategy for synthetic-protein construction. Using this strategy previously we have, for example, been able to demonstrate the successful use of azide or alkyne tags. Prior, elegant conjugations have shown that radical-addition reactions may be successfully applied to proteins. Whilst 1-thioglycoside formation by the free-radical addition of 1-glycosyl thiols to alkenes has been reported for the synthesis of small molecules, to date this method has not been applied to the synthesis of S-linked glycoproteins or bioconjugates. The unique reactivity profile of l-Hag, with an olefinic side-chain compared to the natural amino acids characteristically found in proteins, allows for a chemoselective chemical reaction. Amongst these selectivity advantages is the inertness of Hag to almost all common protein modification reactions, thereby allowing the potential for orthogonal use in combined, multireaction protein chemistry strategies. This inertness to other reagents would not be shared by strategies that would use a protein-thiyl radical, perhaps derived from Cys, although this “reverse” approach would allow the potential for use of proteins containing only natural amino acids. In this context, during the final stages of this work, we became aware of usefully complementary methods employed by Dondoni, Massi and co-workers for glycosylation of proteinthiyl radicals. The free-radical reaction of glycosyl thiyls was investigated and optimized initially on representative amino acid model systems containing Hag (Table 1). As a prerequisite for later protein stability, solubility and viability, the reaction was developed to comply with aqueous solution chemistry. Significantly, all prior protocols for glycosyl thiyl generation have until now employed organic solvents. Since the stability and reactivity of glycosyl thiyls can vary, and to demonstrate substrate breadth and broad applicability of the method, we utilized a wide range of 1-glycosyl thiols, in both protected and unprotected form, as starting materials including 1-thio-b-d-glucose (b-GlcSH), 1-thio-a-d-glucose (aGlcSH), 2-acetamido-2-deoxy-1-thio-b-d-glucose (GlcNAcSH), 1-thio-b-d-galactose (GalSH), 1-thio-b-d-mannose (ManSH), and disaccharide 4-O(b-d-galactosyl)-1-thio-b-dglucose (Gal(b1,4)GlcSH). Together reactions (Table 1) probed the effect of bulk, differing configuration, protecting groups, and anomeric stereochemistry in the thiols; variation around the Nand C-termini of Hag and varying conditions, including alternative modes of initiation (using water soluble initiator Vazo44 (VA044, 2,2’-azobis[2-(2-imidazolin-2yl)propane]dihydrochloride) and/or photochemical Scheme 1. Summarized strategic approach.