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Identification of bacterial lectins offers an attractive route to the development of new diagnostics, but the design of specific sensors is complicated by the low selectivity of carbohydrate-lectin interactions. Here we describe a glycopolymer-based sensor array which can identify a selection of lectins with similar carbohydrate recognition preferences through a pattern-based approach. Receptors were generated using a polymer scaffold functionalized with an environmentally sensitive fluorophore, along with simple carbohydrate motifs. Exposure to lectins induced changes in the emission profiles of the receptors, enabling the discrimination of analytes using linear discriminant analysis. The resultant algorithm was used for lectin identification across a range of concentrations and within complex mixtures of proteins. The sensor array was shown to discriminate different strains of pathogenic bacteria, demonstrating its potential application as a rapid diagnostic tool to characterize bacterial infections and identify bacterial virulence factors such as production of adhesins and antibiotic resistance.

Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies., (© 2024. The Author(s).)

Pseudaminic acid is a non-mammalian sugar found in the surface glycoconjugates of many bacteria, including several human pathogens, and is a virulence factor thought to facilitate immune evasion. The final step in the biosynthesis of the nucleotide activated form of the sugar, CMP-Pse5Ac7Ac is performed by a CMP-Pse5Ac7Ac synthetase (PseF). Here we present the biochemical and structural characterization of PseF from Aeromonas caviae (AcPseF), with AcPseF displaying metal-dependent activity over a broad pH and temperature range. Upon binding to CMP-Pse5Ac7Ac, AcPseF undergoes dynamic movements akin to other CMP-ulosonic acid synthetases. The enzyme clearly discriminates Pse5Ac7Ac from other ulosonic acids, through active site interactions with side-chain functional groups and by positioning the molecule in a hydrophobic pocket. Finally, we show that AcPseF binds the CMP-Pse5Ac7Ac side chain in the lowest energy conformation, a trend that we observed in the structures of other enzymes of this class., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Colicins are antimicrobial proteins produced by certain strains of Escherichia coli that function as offensive weapons against closely-related competitor strains. Their bactericidal properties and narrow bacterial targeting range has made them of therapeutic interest. Furthermore, the applications of engineered non-bactericidal colicins are of interest as a cell surface-directed protein anchor for decorating E. coli with biomolecules. We previously demonstrated that an engineered non-bacteriocidal colicin E9 could be used to label bacterial cells with multiple biomolecules including glycans. Herein we extend our approach to colicin Ia, constructing mannose-presenting colicin la neoglycoproteins, through N-terminal organocatalyst-mediated protein aldol ligation (OPAL), or maleimide ligation targeting an internal cysteine. This work further highlights the potential utility of engineered colicins for non-genetic glyco-engineering of the E. coli cell surface., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Glycans play a major role in biological cell-cell recognition and signal transduction but have found limited application in biosensors due to glycan/lectin promiscuity; multiple proteins are capable of binding to the same native glycan. Here, site-specific fluorination is used to introduce protein-glycan selectivity, and this is coupled with an electrochemical detection method to generate a novel biosensor platform. 3F-lacto- N -biose glycofluoroform is installed onto polymer tethers, which are subsequently immobilised onto gold screen printed electrodes, providing a non-fouling surface. The impedance biosensing platform is shown to selectively bind cancer-associated galectin-3 compared to control glycans and proteins. To improve the analytical capability, Bayesian statistical analysis was deployed in the equivalent circuit fitting of electrochemical impedance spectroscopy data. It is shown that Markov Chain Monte Carlo (MCMC) analysis is a helpful method for visualising experimental irreproducibility, and we apply this as a quality control step., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Glycan-mediated interactions play a crucial role in biology and medicine, influencing signalling, immune responses, and disease pathogenesis. However, the use of glycans in biosensing and diagnostics is limited by cross-reactivity, as certain glycan motifs can be recognised by multiple biologically distinct protein receptors. To address this specificity challenge, we report the enzymatic synthesis of a 150-member library of site-specifically fluorinated Lewis x analogues ('glycofluoroforms') using naturally occurring enzymes and fluorinated monosaccharides. Subsequent incorporation of a subset of these glycans into nanoparticles or a microarray revealed a striking spectrum of distinct binding intensities across different proteins that recognise Lewis x . Notably, we show that for two proteins with unique binding sites for Lewis x , glycofluoroforms exhibited enhanced binding to one protein, whilst reduced binding to the other, with selectivity governed by fluorination patterns. We finally showcase the potential diagnostic utility of this approach in glycofluoroform-mediated bacterial toxin detection by lateral flow., (© 2024. The Author(s).)

Prostate cancer is the most common cancer in men in the UK with over 50 000 new cases diagnosed each year and although therapeutic advances in surgery, anti-androgens, radio- and chemotherapy have increased survival rates, there still remains a need for new treatments to combat the most aggressive forms of the disease. Gene therapy offers promise as an alternative approach but is reliant on selective targeting to the cancer cell surface. Herein we describe the novel construction of a prostate specific membrane antigen (PSMA) binding bioconjugate-polyplex, based on a glutamate-urea peptide scaffold using 'click' chemistry, which we demonstrate is capable of targeted delivery of a GFP gene to PSMA overexpressing prostate cancer cells, and therefore may have potential future application as part of a prostate cancer gene delivery therapy., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Nucleoside analogues are established treatments for cancer and viral infection. Gemcitabine is a commonly employed nucleoside analogue displaying anticancer properties against a range of tumor types but is rapidly inactivated in vivo . Efforts to bolster its pharmaceutical profile include investigating prodrug forms. Herein, we explore the synthesis of a novel glucose-gemcitabine glycoconjugate, targeting uptake via glucose transport. We select a redox-reactive disulfide linker for conjugation of gemcitabine (through N 4-cytosine) with glucose. Evaluation of this glycoconjugate reveals increased toxicity against androgen insensitive PC3 prostate cancer cells compared to LNCaP (which have lower levels of glucose transporter GLUT1). These preliminary results suggest that glycoconjugation of nucleosides may be an effective approach to targeting cells which display increased uptake and metabolism of glucose., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Biofilm formation is integral to the pathogenesis of numerous adherent bacteria and contributes to antimicrobial resistance (AMR). The rising threat of AMR means the need to develop novel nonbactericidal antiadhesion approaches against such bacteria is more urgent than ever. Both adherent-invasive Escherichia coli (AIEC, implicated in inflammatory bowel disease) and uropathogenic E. coli (UPEC, responsible for ∼80% of urinary tract infections) adhere to terminal mannose sugars on epithelial glycoproteins through the FimH adhesin on their type 1 pilus. Although mannose-based inhibitors have previously been explored to inhibit binding of adherent bacteria to epithelial cells, this approach has been limited by monovalent carbohydrate-protein interactions. Herein, we pioneer a novel approach to this problem through the preparation of colicin E9 bioconjugates that bind to the abundant BtuB receptor in the outer membrane of bacteria, which enables multivalent presentation of functional motifs on the cell surface. We show these bioconjugates label the surface of live E. coli and furthermore demonstrate that mannose-presenting "glyco-colicins" induce E. coli aggregation, thereby using the bacteria, itself, as a multivalent platform for mannose display, which triggers binding to adjacent FimH-presenting bacteria., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Organic synthesis provides an accessible route to preparative scale biological glycans, although schemes to access these complex structures are often complicated by preparation of multiple monosaccharide building blocks. Bimodal glycosyl donors capable of forming both α- and β-anomers selectively, are an emerging tactic to reduce the required number of individual synthetic components in glycan construction. This review discusses examples of bimodal donors in the literature, and how they achieve their stereocontrol for both anomers. Notable examples include a bespoke O-2 benzyl protecting group, a strained glycal for reaction using organometallic catalysis, and a simple perbenzylated donor optimised for stereoselective glycosylation through extensive reaction tuning., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Despite an array of hypothesised implications for health, disease, and therapeutic development, antibodies against the non-human sialic acid N -glycolylneuraminic acid (Neu5Gc) remain a subject of much debate. This systematic review of 114 publications aimed to generate a comprehensive overview of published studies in this field, addressing both the reported prevalence of anti-Neu5Gc antibodies in the human population and whether experimental variation accounts for the conflicting reports about the extent of this response. Absolute titres of anti-Neu5Gc antibodies, the reported prevalence of these antibodies, and the individual variation observed within experiments were analysed and grouped according to biological context ('inflammation', 'xenotransplantation', 'biotherapeutic use', 'cancer', and 'healthy populations'), detection method, target epitope selection, and choice of blocking agent. These analyses revealed that the experimental method had a notable impact on both the reported prevalence and absolute titres of anti-Neu5Gc antibodies in the general population, thereby limiting the ability to ascribe reported trends to genuine biological differences or the consequence of experimental design. Overall, this review highlights important knowledge gaps in the study of antibodies against this important xenoautoantigen and the need to establish a standardised method for their quantification if the extent of the importance of Neu5Gc in human health is to be fully understood., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hutton, Scott, Robson, Signoret and Fascione.)

New methods are described that expand the scope of the Successive Ring Expansion (SuRE) with respect to synthetically challenging lactams. A protocol has been developed for use with 'unreactive' lactams, enabling SuRE reactions to be performed on subsrates that fail under previously established conditions. Ring expansion is also demonstarted on 'reactive' lactams derived from iminosugars for the first time. The new SuRE methods were used to prepare a diverse array of medium-sized and macrocyclic lactams and lactones, which were evaluted in an anti-bacterial assay against E. coli BW25113WT.

Cell surface sugar 5,7-diacetyl pseudaminic acid (Pse5Ac7Ac) is a bacterial analogue of the ubiquitous sialic acid, Neu5Ac, and contributes to the virulence of a number of multidrug resistant bacteria, including ESKAPE pathogens Pseudomonas aeruginosa, and Acinetobacter baumannii. Despite its discovery in the surface glycans of bacteria over thirty years ago, to date no glycosyltransferase enzymes (GTs) dedicated to the synthesis of a pseudaminic acid glycosidic linkage have been unequivocally characterised in vitro. Herein we demonstrate that A. baumannii KpsS1 is a dedicated pseudaminyltransferase enzyme (PseT) which constructs a Pse5Ac7Ac-α(2,6)-Glcp linkage, and proceeds with retention of anomeric configuration. We utilise this PseT activity in tandem with the biosynthetic enzymes required for CMP-Pse5Ac7Ac assembly, in a two-pot, seven enzyme synthesis of an α-linked Pse5Ac7Ac glycoside. Due to its unique activity and protein sequence, we also assign KpsS1 as the prototypical member of a previously unreported GT family (GT118)., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Due to the variety of roles served by the cell membrane, its composition and structure are complex, making it difficult to study. Bioorthogonal reactions, such as the strain promoted azide-alkyne cycloaddition (SPAAC), are powerful tools for exploring the function of biomolecules in their native environment but have been largely unexplored within the context of lipid bilayers. Here, we developed a new approach to study the SPAAC reaction in liposomal membranes using azide- and strained alkyne-functionalized Förster resonance energy transfer (FRET) dye pairs. This study represents the first characterization of the SPAAC reaction between diffusing molecules inside liposomal membranes. Potential applications of this work include in situ bioorthogonal labeling of membrane proteins, improved understanding of membrane dynamics and fluidity, and the generation of new probes for biosensing assays., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Truncated thioester N , S -diacetylcysteamine (SNAc) was utilised as a co-factor mimic for PseH, an acetyl-coA dependent aminoglycoside N -acetyltransferase, in the biosynthesis of the bacterial sugar, pseudaminic acid. Additionally, an azido-SNAc analogue was used to smuggle N 7-azide functionality into the pseudaminic acid backbone, facilitating its use as a reporter of pseudaminyltransferase activity.

Novel methods to construct small molecule-protein bioconjugates are integral to the development of new biomedicines for a variety of diseases. C-C linked bioconjugates are increasingly desirable in this application due to their in vivo stability and can be accessed through cross aldol bioconjugation of reactive α-oxo aldehyde handles easily introduced at the N-terminus of proteins by periodate oxidation. We previously developed an organocatalyst-mediated protein aldol ligation (OPAL) for chemical modification of these reactive aldehydes, but the efficiency of this method was limited when a proline residue was directly adjacent to the N-terminus due to intramolecular hemiaminal formation. Herein we explore the competition between this cyclisation and the OPAL modification and demonstrate bioconjugation can be favoured through use of acidic pH for both oxidation and OPAL, and optimisation of reaction conditions and organocatalyst. We then showcase the utility of this acidic-OPAL in modification of the cholera toxin B-subunit (CTB), a homo-pentameric protein of biomedical promise., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

The Sulfo-NHS ester is a mainstay reagent for facilitating amide bond formation between carboxylic acids and amine functionalities in water. However, the preparation of Sulfo-NHS esters currently requires hydrophobic carboxylic acids, which are poorly water-soluble, to first be reacted with the N -hydroxysulfosuccinimide sodium salt, which is insoluble in organic solvents. The mutually incompatible solvation requirements thus complicate the synthesis of Sulfo-NHS esters. As a simple, rapid, and cost-effective solution to this problem, we report that the use of 15-crown-5 to complex the sodium cation of N -hydroxysulfosuccinimide sodium salt circumnavigates these solvation incompatibility issues by rendering the N -hydroxysulfosuccinimide salt soluble in organic solvents, resulting in a cleaner esterification reaction and thus improved yields of activated ester product. We also demonstrate that the resultant "crowned" Sulfo-NHS-ester remains water-soluble and is no less reactive than its classic "uncrowned" Sulfo-NHS counterpart when used in bioconjugation reactions between protein amine-functionalities and hydrophobic carboxylic acids.

β-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manβ1,4GlcNAc a constituent of the core N -glycan in eukaryotes was recently identified as an immune activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of β-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manβ1,4GlcNAc analogues using the β-1,4-d-mannosyl- N -acetyl-d-glucosamine phosphorylase, BT1033. We show that the presence of fluorine in the GlcNAc acceptor facilitates the formation of longer β-mannan-like glycans. We also pioneer a "reverse thiophosphorylase" enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorolysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Aryl diazonium cations are versatile bioconjugation reagents due to their reactivity towards electron-rich aryl residues and secondary amines, but historically their usage has been hampered by both their short lifespan in aqueous solution and the harsh conditions required to generate them in situ. Triazabutadienes address many of these issues as they are stable enough to endure multiple-step chemical syntheses and can persist for several hours in aqueous solution, yet upon UV-exposure rapidly release aryl diazonium cations under biologically-relevant conditions. This paper describes the synthesis of a novel maleimide-functionalized triazabutadiene suitable for site-selectively installing aryl diazonium cations into proteins at neutral pH; we show reaction with this molecule and a surface-cysteine of a thiol disulfide oxidoreductase. Through photoactivation of the site-selectively installed triazabutadiene motifs, we generate aryl diazonium functionality, which we further derivatize via azo-bond formation to electron-rich aryl species, showcasing the potential utility of this strategy for the generation of photoswitches or protein-drug conjugates., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Protein N-termini provide uniquely reactive motifs for single site protein modification. Though a number of reactions have been developed to target this site, the selectivity, generality, and stability of the conjugates formed has not been studied. We have therefore undertaken a comprehensive comparative study of the most promising methods for N-terminal protein modification, and find that there is no 'one size fits all' approach, necessitating reagent screening for a particular protein or application. Moreover, we observed limited stability in all cases, leading to a need for continued innovation and development in the bioconjugation field., Competing Interests: The authors declare no competing financial interests., (This journal is © The Royal Society of Chemistry.)

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function., (© 2022. The Author(s).)

Site-selective chemical methods for protein bioconjugation have revolutionized the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic, or even toxic cargos. Herein, we report two new methods for the bioconjugation of α-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The "α-oxo-Mannich" and "catalyst-free aldol" ligations both compete for the electrophilic α-oxo aldehyde, which displays pH divergent reactivity proceeding through the "Mannich" pathway at acidic pH to afford bifunctionalized bioconjugates, and the "catalyst-free aldol" pathway at neutral pH to afford monofunctionalized bioconjugates. We explore the substrate scope and utility of both of these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH's.

Pseudaminic acids present on the surface of pathogenic bacteria, including gut pathogens Campylobacter jejuni and Helicobacter pylori, are postulated to play influential roles in the etiology of associated infectious diseases through modulating flagella assembly and recognition of bacteria by the human immune system. Yet they are underexplored compared to other areas of glycoscience, in particular enzymes responsible for the glycosyltransfer of these sugars in bacteria are still to be unambiguously characterised. This can be largely attributed to a lack of access to nucleotide-activated pseudaminic acid glycosyl donors, such as CMP-Pse5Ac7Ac. Herein we reconstitute the biosynthesis of Pse5Ac7Ac in vitro using enzymes from C. jejuni (PseBCHGI) in the process optimising coupled turnover with PseBC using deuterium wash in experiments, and establishing a method for co-factor regeneration in PseH tunover. Furthermore we establish conditions for purification of a soluble CMP-Pse5Ac7Ac synthetase enzyme PseF from Aeromonas caviae and utilise it in combination with the C. jejuni enzymes to achieve practical preparative synthesis of CMP-Pse5Ac7Ac in vitro, facilitating future biological studies.

During their lifetime almost half of women will experience a symptomatic urinary tract infection (UTI) with a further half experiencing a relapse within six months. Currently UTIs are treated with antibiotics, but increasing antibiotic resistance rates highlight the need for new treatments. Uropathogenic Escherichia coli (UPEC) is responsible for the majority of symptomatic UTI cases and thus has become a key pathological target. Adhesion of type one pilus subunit FimH at the surface of UPEC strains to mannose-saturated oligosaccharides located on the urothelium is critical to pathogenesis. Since the identification of FimH as a therapeutic target in the late 1980s, a substantial body of research has been generated focusing on the development of FimH-targeting mannose-based anti-adhesion therapies. In this review we will discuss the design of different classes of these mannose-based compounds and their utility and potential as UPEC therapeutics., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Galectins are potential biomarkers and therapeutic targets. However, galectins display broad affinity towards β-galactosides meaning glycan-based (nano)biosensors lack the required selectivity and affinity. Using a polymer-stabilized nanoparticle biosensing platform, we herein demonstrate that the specificity of immobilised lacto- N -biose towards galectins can be 'turned on/off' by using site-specific glycan fluorination and in some cases reversal of specificity can be achieved. The panel of fluoro-glycans were obtained by a chemoenzymatic approach, exploiting BiGalK and BiGalHexNAcP enzymes from Bifidobacterium infantis which are shown to tolerate fluorinated glycans, introducing structural diversity which would be very laborious by chemical methods alone. These results demonstrate that integrating non-natural, fluorinated glycans into nanomaterials can encode unprecedented selectivity with potential applications in biosensing., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs., Competing Interests: Declaration of Interests The authors declare no competing financial interests. Prozomix is a commercial enzyme producer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The α-oxo aldehyde is a highly reactive aldehyde for which many protein bioconjugation strategies exist. Here, we explore the genetic incorporation of a threonine-lysine dipeptide into proteins, harbouring a "masked"α-oxo aldehyde that is rapidly unveiled in four minutes. The reactive aldehyde could undergo site-specific protein modification by SPANC ligation.

We report a diazonium electro-grafting method for the covalent modification of conducting surfaces with aldehyde-reactive hydroxylamine functionalities that facilitate the wiring of redox-active (bio)molecules to electrode surfaces. Hydroxylamine near-monolayer formation is achieved via a phthalimide-protection and hydrazine-deprotection strategy that overcomes the multilayer formation that typically complicates diazonium surface modification. This surface modification strategy is characterized using electrochemistry (electrochemical impedance spectroscopy and cyclic voltammetry), X-ray photoelectron spectroscopy, and quartz crystal microbalance with dissipation monitoring. Thus-modified glassy carbon, boron-doped diamond, and gold surfaces are all shown to ligate to small molecule aldehydes, yielding surface coverages of 150-170, 40, and 100 pmol cm -2 , respectively. Bioconjugation is demonstrated via the coupling of a dilute (50 μM) solution of periodate-oxidized horseradish peroxidase enzyme to a functionalized gold surface under biocompatible conditions (H 2 O solvent, pH 4.5, 25 °C).

Pseudaminic acids (Pses) are a group of non-mammalian nonulosonic acids (nulOs) that have been shown to be an important virulence factor for a number of pathogenic bacteria, including emerging multidrug-resistant ESKAPE pathogens. Despite their discovery over 30 years ago, relatively little is known about the biological significance of Pse glycans compared with their sialic acid analogues, primarily due to a lack of access to the synthetically challenging Pse architecture. Recently, however, the Pse backbone has been subjected to increasing synthetic exploration by carbohydrate (bio)chemists, and the total synthesis of complex Pse glycans achieved with inspiration from the biosynthesis and subsequent detailed study of chemical glycosylation by using Pse donors. Herein, context is provided for these efforts by summarising recent synthetic approaches pioneered for accessing Pse glycans, which are set to open up this underexplored area of glycoscience to the wider scientific community., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The non-mammalian nonulosonic acid sugar pseudaminic acid (Pse) is present on the surface of a number of human pathogens including Campylobacter jejuni and Helicobacter pylori and other bacteria such as multidrug resistant Acinetobacter baumannii. It is likely important for evasion of the host immune sysyem, and also plays a role in bacterial motility through flagellin glycosylation. Herein we review the mechanistic and structural characterisation of the enzymes responsible for the biosynthesis of the Pse parent structure, Pse5Ac7Ac in bacteria.

Herein we report synthesis of complex heparan sulfate oligosaccharide precursors by automated glycan assembly using disaccharide donor building blocks. Rapid access to a hexasaccharide was achieved through iterative solid phase glycosylations on a photolabile resin using Glyconeer™, an automated oligosaccharide synthesiser, followed by photochemical cleavage and glycan purification using simple flash column chromatography.

The immobilization of redox proteins or enzymes onto conductive surfaces has application in the analysis of biological processes, the fabrication of biosensors, and in the development of green technologies and biochemical synthetic approaches. This review evaluates the methods through which redox proteins can be attached to electrode surfaces in a "wired" configuration, that is, one that facilitates direct electron transfer. The feasibility of simple electroactive adsorption onto a range of electrode surfaces is illustrated, with a highlight on the recent advances that have been achieved in biotechnological device construction using carbon materials and metal oxides. The covalent crosslinking strategies commonly used for the modification and biofunctionalization of electrode surfaces are also evaluated. Recent innovations in harnessing chemical biology methods for electrically wiring redox biology to surfaces are emphasized., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing biological mechanisms. The general use of chemical methods for protein functionalisation can be limited however by the requirement for complicated reaction partners to be present in large excess, and harsh conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH. OPAL enables rapid modification of proteins using simple aldehyde probes in minimal excess, and is utilised here in the affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the β-hydroxy aldehyde OPAL product can be functionalised again at neutral pH in a tandem organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the 'chemical mimicry' of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis.

Protein bioconjugation frequently makes use of aldehydes as reactive handles, with methods for their installation being highly valued. Here a new, powerful strategy to unmask a reactive protein aldehyde is presented. A genetically encoded caged glyoxyl aldehyde, situated in solvent-accessible locations, can be rapidly decaged through treatment with just one equivalent of allylpalladium(ii) chloride dimer at physiological pH. The protein aldehyde can undergo subsequent oxime ligation for site-selective protein modification. Quick yet mild conditions, orthogonality and powerful exposed reactivity make this strategy of great potential in protein modification.

The pyrrolysine tRNA synthetase-tRNA pair is probably one of the most promiscuous tRNA-synthetase pairs found in nature, capable of genetically encoding a plethora of noncanonical amino acids through stop codon reassignment. Proteins containing reactive handles, post-translational modification mimics or both can be produced in practical quantities, allowing inter alia the probing of biological pathways, generating antibody-drug conjugates and enhancing protein function. This Minireview summarises the development of pyrrolysine amber stop-codon suppression, presents some of the considerations required to utilise this technique to its greatest potential, and showcases the creative ways in which this technique has led to a better understanding of biological systems., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe single-chain polymer nanoparticles (SCNPs) possessing intramolecular dynamic covalent crosslinks that can transform into polymer films through a molecular recognition-mediated crosslinking process. The SCNPs utilise molecular recognition with surface-immobilised proteins to concentrate upon a substrate, bringing the SCNPs into close spatial proximity with one another and allowing their dynamic covalent crosslinkers to undergo intra- to interpolymer chain crosslinking leading to the formation of polymeric film. SCNPs must possess both the capacity for specific molecular recognition and a dynamic nature to their intramolecular crosslinkers to form polymer films, and an investigation of the initial phase of film formation indicates it proceeds from features which form upon the surface then grow predominantly in the xy directions. This approach to polymer film formation presents a potential method to "wrap" surfaces displaying molecular recognition motifs-which could potentially include viral, cellular and bacterial surfaces or artificial surfaces displaying multivalent recognition motifs-within a layer of polymer film., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The incorporation of aldehyde handles into proteins, and subsequent chemical reactions thereof, is rapidly proving to be an effective way of generating homogeneous, covalently linked protein constructs that can display a vast array of functionality. In this review, we discuss methods for introducing aldehydes into target proteins, and summarise the ligation strategies for site-selective modification of proteins containing this class of functional handles.

A highly efficient cap-exchange approach for preparing compact, dense polyvalent mannose-capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC-SIGN and DC-SIGNR (collectively termed as DC-SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC-SIGN, but not its closely related receptor DC-SIGNR, which is further confirmed by its specific blocking of DC-SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC-SIGN binds more efficiently to densely packed mannosides. A FRET-based thermodynamic study reveals that the binding is enthalpy-driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein-ligand interactions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The utility of sulfoxides in a diverse range of transformations in the field of carbohydrate chemistry has seen rapid growth since the first introduction of a sulfoxide as a glycosyl donor in 1989. Sulfoxides have since developed into more than just anomeric leaving groups, and today have multiple roles in glycosylation reactions. These include as activators for thioglycosides, hemiacetals, and glycals, and as precursors to glycosyl triflates, which are essential for stereoselective β-mannoside synthesis, and bicyclic sulfonium ions that facilitate the stereoselective synthesis of α-glycosides. In this review we highlight the mechanistic investigations undertaken in this area, often outlining strategies employed to differentiate between multiple proposed reaction pathways, and how the conclusions of these investigations have and continue to inform upon the development of more efficient transformations in sulfoxide-based carbohydrate synthesis., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

A conceptually new approach to the design of macromolecular receptors for lectins is outlined. Carbohydrate-functionalised Polymer-Scaffolded Dynamic Combinatorial Libraries (PS-DCLs) have been prepared in aqueous solution by the reversible conjugation of carbohydrates possessing acylhydrazide functionalities in their aglycone on to an aldehyde-functionalised polymer scaffold. PS-DCLs have been shown to undergo compositional change in response to the addition of lectin templates, with polymer scaffolds preferentially incorporating carbohydrate units which recognise the lectin added. This compositional change has been shown to generate polymers of significantly enhanced affinity for the lectin added, with enhancements in free energy of binding in the range of 5.2-8.8 kJ mol(-1) observed. Experiments indicate that these enhancements are not only as a consequence of increased display of the preferred carbohydrate upon the polymer scaffold, but that templation also reorganises key residues into strategic positions in order to interact more strongly with the target.

Protein toxins produced by bacteria are the cause of many life-threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site-specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC50) of 104 pM for the CT B-subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies., (© 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Sulfoxides are frequently used in organic synthesis as chiral auxiliaries and reagents to mediate a wide variety of chemical transformations. For example, diphenyl sulfoxide and triflic anhydride can be used to activate a wide range of glycosyl donors including hemiacetals, glycals and thioglycosides. In this way, an alcohol, enol or sulfide is converted into a good leaving group for subsequent reaction with an acceptor alcohol. However, reaction of diphenyl sulfoxide and triflic anhydride with oxathiane-based thioglycosides, and other oxathianes, leads to a different process in which the thioglycoside is oxidised to a sulfoxide. This unexpected oxidation reaction is very stereoselective and proceeds under anhydrous conditions in which the diphenyl sulfoxide acts both as oxidant and as the source of the oxygen atom. Isotopic labelling experiments support a reaction mechanism that involves the formation of oxodisulfonium (S-O-S) dication intermediates. These intermediates undergo oxygen-exchange reactions with other sulfoxides and also allow interconversion of axial and equatorial sulfoxides in oxathiane rings. The reversibility of the oxygen-exchange reaction suggests that the stereochemical outcome of the oxidation reaction may be under thermodynamic control, which potentially presents a novel strategy for the stereoselective synthesis of sulfoxides., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Novel oxathiane spiroketal donors have been synthesised and activated via an umpolung S-arylation strategy using 1,3,5-trimethoxybenzene and 1,3-dimethoxybenzene. The comparative reactivity of the resulting 2,4,6-trimethoxyphenyl (TMP)- and 2,4-dimethoxyphenyl (DMP)-oxathiane spiroketal sulfonium ions is discussed, and their α-stereoselectivity in glycosylation reactions is compared to the analogous TMP- and DMP-sulfonium ions derived from an oxathiane glycosyl donor bearing a methyl ketal group. The results show that the stereoselectivity of the oxathiane glycosyl donors is dependent on the structure of the ketal group and reactivity can be tuned by varying the substituent on the sulfonium ion., (Copyright © 2011 Elsevier Ltd. All rights reserved.)