Glycosaminoglycan (GAG)-binding lectins represent a rare class of carbohydrate-binding proteins with the ability to recognize and organize linear polysaccharide chains. Here, we describe XN-IL, a novel calcium-dependent lectin from the Gram-negative bacterium Xenorhabdus nematophila, which exhibits an unusual specificity for glycosaminoglycans. X. nematophila is an entomopathogenic bacterium and a symbiont of insect-parasitic Steinernema nematodes. Glycan array screening, analytical ultracentrifugation, and differential scanning fluorimetry revealed that XN-IL selectively binds hyaluronan and low-sulfated heparan sulfate, while showing negligible affinity for monosaccharides and galactosylated glycans. GAG binding is mediated exclusively by calcium ions, enabling the reversible crosslinking and precipitation of hyaluronan polymers. Crystal structures of the apo and ligand-bound forms reveal a conserved LecA-like fold with a widened, calcium-dependent binding pocket that accommodates extended GAG chains without major conformational rearrangements. XN-IL is the first member of the LecA family with defined GAG specificity and the first lectin identified in the genus Xenorhabdus. Its divergence from galactophilic LecA homologues reflects an evolutionary adaptation towards calcium-driven recognition and reversible assembly of linear polysaccharides. These findings expand the functional diversity of the LecA family and introduce XN-IL as a new tool for probing and manipulating GAG-based polymer systems.

We explore a synthesis of nucleotide-pyranose conjugates, successfully developing an H-phosphonate-mediated coupling of pyranoses with nucleosides that favors making the reactive phosphorus(III) species within the pyranose (C6-OH or C1-OH) and affords, after oxidation to the free phosphate, glycoconjugates in good overall yields (65-84%). Following deprotection and final purification, the materials are evaluated in U87-MG and PANC-1 cells, with activities comparable to the parent drugs, indicating such conjugates worthy of deeper validation as prodrugs.

ABSTRACT Fluorination is a powerful strategy for the editing of glycans, creating bespoke tools to probe carbohydrate‐protein recognition and processing. Positioning the site for fluorination within a glycan is critical, whether examining bonding interactions or installing benign reporter capability. Herein, we study the effect of C‐3 fluorination of mannuronic acids in the assembly of β‐1,4‐ d ‐mannuronic acid alginate fragments and the effect of these point mutations on the conformational preference of the generated oligosaccharides. We explore the synthesis and glycosylating properties of a 3‐deoxy‐3‐fluoro d ‐mannuronate donor to establish that the C‐3 fluoride does not thwart the high β‐stereoselectivity of the mannuronate system. Subsequent deployment of these building blocks in automated glycan assembly enables the preparation of a small library of β‐ d ‐mannuronate oligosaccharides containing the C‐3‐F modification. Comprehensive NMR analysis shows that even though the 3‐deoxy‐3‐fluoro units disrupt the native inter‐residue C3‐OH•••O5 hydrogen bonds, the overall conformation remains in line with that of the native β‐1,4 mannuronic acid alginate. This indicates tolerance for this modification in these structural tools and provides an exciting foundation to study self‐assembly and enzymatic processing of β‐1,4 mannuronic acid oligosaccharides.

Autophagy is a critical mechanism of cellular quality control, orchestrated by selective autophagy receptor (SAR) proteins. Pharmacologically enhancing the cargo-targeting capacity of SARs presents an attractive but underexplored strategy for the precise therapeutic activation of autophagy. Here, we characterize SQ-1, a small-molecule activator of autophagy that engages the prototypical SAR protein p62/sequestosome-1 (SQSTM1). We show that SQ-1 sensitizes p62 to oxidation and promotes its disulfide-mediated oligomerization in response to mitochondrial reactive oxygen species (ROS). This ROS-dependent activation of p62-mediated selective autophagy enhances the clearance of ROS-generating mitochondria and restores cell viability in models of Niemann-Pick type C1 disease, which is marked by impaired autophagic flux. In summary, the unique mode of action of SQ-1 enables self-regulated autophagy activation, offering a potential therapeutic strategy for lysosomal storage disorders and a broader spectrum of age-related diseases characterized by defective autophagy.

Mammalian cells receive signaling instructions through interactions on their surfaces. Proteoglycans are critical to these interactions, carrying long glycosaminoglycans that recruit signaling molecules. Biosynthetic redundancy in the first glycosylation step by two xylosyltransferases XT1/2 complicates annotation of proteoglycans. Here we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. Through a bump-and-hole tactic, we engineer the two isoenzymes XT1 and XT2 to specifically transfer the chemically tagged xylose analog 6AzGlc to target proteins. The tag contains a bioorthogonal functionality, allowing to visualize and profile target proteins in mammalian cells. Unlike xylose analogs, 6AzGlc is amenable to cellular nucleotide-sugar biosynthesis, establishing the XT1/2 bump-and-hole tactic in cells. The approach allows pinpointing glycosylation sites by mass spectrometry and exploiting the chemical handle to manufacture proteoglycans with defined glycosaminoglycan chains for cellular applications. Engineered XT enzymes permit an orthogonal view into proteoglycan biology through conventional techniques in biochemistry. The xylosyltransferase isoenzymes XT1 and XT2 catalyze the first glycosylation step in the biosynthesis of proteoglycans. Now, bump-and-hole engineering of XT1 and XT2 enables substrate profiling and modification of proteins as designer proteoglycans to modulate cellular behavior.

The catalytic activity of pristine UiO-67 has been explored for the hydroboration of a wide scope of aryl aldehydes, including both substituted and alpha,beta-unsaturated, in short time frames with high conversions and selectivity in the presence of HBpin. Tests for hidden borane catalysts amongst other constituents were carried out to prove no free BH3, zirconium or ligand were participating in the reaction, and that the MOF is the active catalyst in these reactions. Recyclability tests showed minimal reduction in activity after seven runs, but no complete degradation of the system.

Glycosyl 1-phosphates are key precursors to nucleotide sugars that drive glycosyltransferasecatalysed glycan assembly, yet stereocontrolled access to L-configured variants found across nature remains limited. Here we report a Design-of-Experiments (DoE)-guided optimisation of L-glycosyl 1-phosphate synthesis, enabling high isolated yields of anomerically pure protected and unprotected β-L-glycosyl 1-phosphates. These donors support a robust chemoenzymatic route to natural and non-natural nucleotide sugars, expanding the repertoire of activated building blocks available for in vitro glycan synthesis. Using β-L-sugar 1-phosphates prepared in enabling quantities, we demonstrate that native and engineered microbial nucleotidyltransferases convert rare and canonical L-sugar donors into diverse dTDP-, UDP-, and GDP-β-L-sugar nucleotides, including previously unreported products. Tag-free nanoscale differential scanning fluorimetry (nano-DSF) reveals substrate-dependent stabilisation and destabilisation effects that illuminate cooperative binding behaviour and highlight opportunities for evolving improved nucleotidyltransferase stability and turnover. Collectively, this integrated synthetic–biocatalytic platform enhances access to rare nucleotide sugars and provides mechanistic insight into nucleotidyltransferase activity, enabling catalytic strategies for exploiting these enzymes in glycan synthesis.

Multivalent lectin–glycan interactions (MLGIs) are vital for viral infection, cell-cell communication and regulation of immune responses. Their structural and biophysical data are thus important, not only for providing insights into their underlying mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information remains to be limited for some important MLGIs, significantly restricting the research progress. We have recently demonstrated that functional nanoparticles, including ∼4 nm quantum dots and varying sized gold nanoparticles (GNPs), densely glycosylated with various natural mono- and oligo- saccharides, are powerful biophysical probes for MLGIs. Using two important viral receptors, DC-SIGN and DC-SIGNR (together denoted as DC-SIGN/R hereafter), as model multimeric lectins, we have shown that α-mannose and α-manno-α-1,2-biose (abbreviated as Man and DiMan, respectively) coated GNPs not only can provide sensitive measurement of MLGI affinities but also reveal critical structural information (e.g., binding site orientation and mode) which are important for MLGI targeting. In this study, we produced mannuronic acid (ManA) coated GNPs (GNP-ManA) of two different sizes to probe the effect of glycan modification on their MLGI affinity and antiviral property. Using our recently developed GNP fluorescence quenching assay, we find that GNP-ManA binds effectively to both DC-SIGN/R and increasing the size of GNP significantly enhances their MLGI affinity. Consistent with this, increasing the GNP size also significantly enhances their ability to block DC-SIGN/R-augmented virus entry into host cells. Particularly, ManA coated 13 nm GNP potently block Ebola virus glycoprotein-driven entry into DC-SIGN/R-expressing cells with sub-nM levels of EC50. Our findings suggest that GNP-ManA probes can act as a useful tool to quantify the characteristics of MLGIs, where increasing the GNP scaffold size substantially enhances their MLGI affinity and antiviral potency.

Given the rise in prominence of nucleic acid therapeutics and technologies (e.g., gene editing, antisense, siRNA), the development of efficient, sustainable routes towards the discovery and manufacture of requisite nucleoside analogue building blocks is pressing. Adenosine deaminase (ADA) is a ubiquitous enzyme that catalyses an irreversible hydrolytic deamination of adenosine to inosine within purine metabolism. It has been exploited for biotechnological purposes, affording synthetic access to modified nucleoside analogues, through to applications in gene editing and therapeutic intervention. This review outlines structural modifications in purine substrates and their impact upon ADA activity. We also offer a perspective on future directions, highlighting a need for increased data towards enzyme engineering and promiscuous ADA activity for biotechnological applications.

We report a practical 5 g scale stereoselective synthesis of the valuable iminosugar DMDP from d-fructose in only 7 synthetic steps and in a 70% overall yield, which doubles previously reported yields. This process requires only two chromatographic purification steps, taking advantage of a regioselective Appel reaction. The regioselective reaction has also been applied on a similar scale to prepare the C-2 diastereomer of DMDP, DGDP, from l-sorbose in 7 steps (two purifications) and 56% overall yield, albeit with diminished diastereomeric purity (d.r. 90:10). The C-5 regioselectivity has also been illustrated on d-psicose and d-tagatose, making this an attractive method for preparing pyrrolidine iminosugars or 5-thiosugars from ketopyranoses.

The rapid emergence of RNA therapeutics has highlighted the need for more efficient, scalable and sustainable methods for their manufacture. Biocatalytic approaches hold particular promise, but rely on a secure, sustainable and low-cost supply of nucleoside triphosphate (NTP) building blocks, including those containing chemical modifications. Here we report the development of a biocatalytic approach and engineered enzymes to convert widely available nucleosides into NTPs featuring pharmaceutically relevant modifications using inexpensive phosphate donors. Importantly our strategy obviates the need for ATP as a phosphate donor that complicates NTP isolation using existing methods. To showcase the utility of our approach, we employ an engineered acid phosphatase, polyphosphate kinase and acetate kinase to produce 2'-O-methoxyethyl-ATP (2'-MOE-ATP) and 2'-fluoro-ATP, key building blocks of commercial therapeutics. Finally, we show that crude NTPs from our process can be used directly in enzymatic oligonucleotide synthesis, obviating the need for costly NTP isolation or purification steps.

The title compound was isolated as the unexpected reaction product from a reaction attempting to access a glycosyl 1-phosphate. The product was isolated in good yield, as one diastereoisomer, and was characterised by 1H, 13C, and 2D NMR, alongside HRMS analysis.

An O-glycosylation method for accessing coumarin glycosides is presented. We report the reaction of 6,8-difluoro-7-hydroxy-4-methylcoumarin and 4-methylumbelliferone with a variety of glycosyl imidate donors using BF3·Et2O as activator to access a series of coumarin glycosides in 64%-76% isolated yields. Several reaction parameters are evaluated including promotors, temperature and reagent equivalents. Following initial methodology development using simple D-glucose donors, D-galactosamino mono- and disaccharides are explored as substrates, showcasing applicability towards late-stage transformation of biologically relevant chondroitin sulfate glycosides. Glycosylation diastereoselectivity trends were also considered, proposing that the identity of the D-galactosamino N-protecting group and the coumarin acceptor contribute to observed anomeric product ratios. This methodology provides a convenient access to D-galactosamino-coumarin glycoconjugates and provides a benchmark for the development of related systems for biological evaluation.

We report here the continuous flow synthesis of a high-value sugar nucleotide. Immobilisation of enzymes onto solid carriers permitted transfer of the biocatalysts into packed bed reactors to realise a continuous biocatalytic platform for the synthesis of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) on 100 mg scale, with capacity for multiple reuses. The modular continuous flow approach described here represents a significant, up to 11-fold, improvement in space time yield (STY) when compared to batch studies, along with preventing product induced enzyme inhibition, reducing the need for an additional enzyme to break down inorganic pyrophosphate (PPi). The modular nature of the system has also allowed tailored conditions to be applied to each enzyme, overcoming issues relating to thermal stability. This development presents a platform approach towards a more efficient, continuous synthesis of important glycan targets including glycoproteins, specific oligosaccharide sequences and glycosylated drug targets.

Fluorinated glycans offer a prime opportunity to study the intricacies of their associated binding events with proteins, invoke resistance toward enzymatic hydrolysis, and modulate carbohydrate physicochemical properties. Sugar nucleotides are the key building blocks used by glycosyltransferases and associated enzymes to assemble glycans and, as such, represent a considerable landscape of opportunity to develop fluorinated motifs and enable structure-to-function understanding. Herein, we target the isosteric inclusion of fluorine within the nucleoside diphosphate sugar framework of GDP-mannose using a chemoenzymatic approach. Utilizing chemical synthesis to incorporate bespoke fluorine modifications and a promiscuous pyrophosphorylase, to assemble the sugar nucleotide, enables first-in-class access to GDP-mannoses containing fluorine within the nucleotide alongside double fluorination, within both the pyranose and nucleotide. These materials are utilized to probe a guanosine diphosphate mannose dehydrogenase critical to mucoid Pseudomonas aeruginosa alginate biosynthesis. This work provides an exemplar framework for incorporating fluorine within the nucleotide of derived sugar nucleotides and thus the capability to study glycosyltransferesase utilizing GDP-mannose more broadly.

Nucleoside analogue therapeutics have a proven capability within drug discovery as antimicrobial, antiviral and antineoplastic agents. However, their efficacy can be limited by poor cellular uptake, high off target toxicity and poor bioavailability. Prodrugs of such analogues contribute to an improved pharmacokinetic profile. Herein, we explore biocatalytic glycosylation of nucleoside analogues. The activity of the nucleoside-specific 3’-O-glycosyltransferase AvpGT from Streptomyces sp. AVP053U2 is investigated against a panel of both natural and clinically relevant purine and pyrimidine nucleoside analogues. AvpGT demonstrates broad substrate promiscuity, with 16 of 22 nucleosides tested showing glycosylation by HILIC-MS. Of these, 13 nucleosides were successfully glycosylated on 25 μmol scale in 39-91% yields, including four nucleoside analogue therapeutics. Furthermore, a novel β-glucosidase, AvpGS, was identified from the same Streptomyces sp. strain, heterologously expressed, purified and shown to display high substrate promiscuity in subsequently removing glucose from the glycoconjugates.

The photoinitiated thiol-ene reaction is emerging as a highly efficient methodology for thioglycoside synthesis. Herein, the radical-mediated hydrothiolation reaction of 4,5-unsaturated saccharides was extended, offering efficient access to C4-position, S-linked glycosides. A diverse range of 4,5-unsaturated saccharides were investigated with high-yields achieved for the thioether products with complete regioselectivity and good diastereoselectivity. 1,2-Ethanedithiol products furnished a thiol-residue suitable for tagging and fluorescent labelling of a disaccharide.

Sugar nucleotides represent the cornerstone building blocks for glycan biosynthesis. While methods to access these crucial biomolecules using traditional batch synthetic chemistry and enzymatic approaches have blossomed, uptake using flow-based synthesis is burgeoning. This perspective analyzes recent advances concerning enzyme immobilization and continuous flow biocatalysis for sugar nucleotide production and usage. Evaluation of related technologies is also discussed, highlighting new enzyme immobilization approaches, novel reactor design, and improved downstream processing as areas that must evolve to enable wider, scalable access to sugar nucleotides as commodity chemicals.

Analogues of the canonical nucleosides have a longstanding presence and proven capability within medicinal chemistry and drug discovery research. Herein we report chemical diversification of carbocyclic pyrimidine nucleosides, containing CF2 and CHF in place of furanose oxygen, to introduce ring unsaturation and 2’-epimers. Utilising gram-scale access to 6’-(R)-monofluoro- and 6’-gem-difluorouridine we explore provision of 2’,3’-didehydro-2’,3’-dideoxy and 1’,2’-didehydro-2’-deoxy analogues, alongside the first example of 6’-(R)-fluoroarabino carbauridine. Key stereochemistries and the presence of unsaturation are confirmed using X-ray crystallography and NMR, and an indicative conformational preference for a monofluoro 2’,3’-didehydro-2’,3’-dideoxy system is presented. This synthetic blueprint offers potential to explore biological activity for these hitherto unavailable materials, including for direct comparison to established nucleoside analogue drugs.