Reverse thiophosphorylase activity of a glycoside phosphorylase in the synthesis of an unnatural Manß1,4GlcNAc library.

ß-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manß1,4GlcNAc 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 ß-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manß1,4GlcNAc analogues using the ß-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 ß-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.

Fast Identification and Quantification of Uropathogenic E. coli through Cluster Analysis.

Rapid diagnostic tools to detect, identify, and enumerate bacteria are key to maintaining effective antibiotic stewardship and avoiding the unnecessary prescription of broad-spectrum agents. In this study, a 15 min agglutination assay is developed that relies on the use of mannose-functionalized polymeric microspheres in combination with cluster analysis. This allows for the identification and enumeration of laboratory (BW25113), clinical isolate (NCTC 12241), and uropathogenic Escherichia coli strains (NCTC 9001, NCTC 13958, J96, and CFT073) at clinically relevant concentrations in tryptic soy broth (103-108 CFU/mL) and in urine (105-108 CFU/mL). This fast, simple, and efficient assay offers a step forward toward efficient point-of-care diagnostics for common urinary tract infections.

Rapid Screening of Diverse Biotransformations for Enzyme Evolution.

The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s per sample. The heat map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL) as a case study, enhancing its activity toward electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence.

Synthesis and Structural Characteristics of all Mono- and Difluorinated 4,6-Dideoxy-d-xylo-hexopyranoses.

Protein-carbohydrate interactions are implicated in many biochemical/biological processes that are fundamental to life and to human health. Fluorinated carbohydrate analogues play an important role in the study of these interactions and find application as probes in chemical biology and as drugs/diagnostics in medicine. The availability and/or efficient synthesis of a wide variety of fluorinated carbohydrates is thus of great interest. Here, we report a detailed study on the synthesis of monosaccharides in which the hydroxy groups at their 4- and 6-positions are replaced by all possible mono- and difluorinated motifs. Minimization of protecting group use was a key aim. It was found that introducing electronegative substituents, either as protecting groups or as deoxygenation intermediates, was generally beneficial for increasing deoxyfluorination yields. A detailed structural study of this set of analogues demonstrated that dideoxygenation/fluorination at the 4,6-positions caused very little distortion both in the solid state and in aqueous solution. Unexpected trends in α/ß anomeric ratios were identified. Increasing fluorine content always increased the α/ß ratio, with very little difference between regio- or stereoisomers, except when 4,6-difluorinated.

Profiling Substrate Promiscuity of Wild-Type Sugar Kinases for Multi-fluorinated Monosaccharides.

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.

Chemoenzymatic synthesis of 3-deoxy-3-fluoro-l-fucose and its enzymatic incorporation into glycoconjugates.

The first synthesis of 3-deoxy-3-fluoro-l-fucose is presented, which employs a d- to l-sugar translation strategy, and involves an enzymatic oxidation of 3-deoxy-3-fluoro-l-fucitol. Enzymatic activation (FKP) and glycosylation using an α-1,2 and an α-1,3 fucosyltransferase to obtain two fluorinated trisaccharides demonstrates its potential as a novel versatile chemical probe in glycobiology.

Introducing affinity and selectivity into galectin-targeting nanoparticles with fluorinated glycan ligands.

Galectins are potential biomarkers and therapeutic targets. However, galectins display broad affinity towards ß-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.

Molecular Insights into DC-SIGN Binding to Self-Antigens: The Interaction with the Blood Group A/B Antigens.

The dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is an important receptor of the immune system. Besides its role as pathogen recognition receptor (PRR), it also interacts with endogenous glycoproteins through the specific recognition of self-glycan epitopes, like LeX. However, this lectin represents a paradigmatic case of glycan binding promiscuity, and it also has been shown to recognize antigens with α1-α2 linked fucose, such as the histo blood group antigens, with similar affinities to LeX. Herein, we have studied the interaction in solution between DC-SIGN and the blood group A and B antigens, to get insights into the atomic details of such interaction. With a combination of different NMR experiments, we demonstrate that the Fuc coordinates the primary Ca2+ ion with a single binding mode through 3-OH and 4-OH. The terminal αGal/αGalNAc affords marginal direct polar contacts with the protein, but provides a hydrophobic hook in which V351 of the lectin perfectly fits. Moreover, we have found that αGal, but not αGalNAc, is a weak binder itself for DC-SIGN, which could endow an additional binding mode for the blood group B antigen, but not for blood group A.

Synthesis of vicinal dideoxy-difluorinated galactoses.

Fluorinated carbohydrates have been employed as probes for fundamental studies of protein-carbohydrate interactions, but also in the development of mechanism-based enzyme inhibitors. There is a continuing demand for novel fluorinated carbohydrate probes. Whereas most examples so far involved monodeoxyfluorinated sugars, multiply deoxyfluorinated sugars have gained much interest. Here we report the synthesis and characterisation of novel vicinal dideoxy-difluorinated d-galactoses with fluorination at the 3,4-positions, and at the 2,3-positions, the latter in both the pyranose and furanose forms. This includes a successful pyranose-into-furanose isomerisation protocol.

A synthetic approach to chrysophaentin F.

The chrysophaentins are a newly discovered natural product family displaying promising anti-infective activity. Herein we describe an approach to chrysophaentin F that uses an array of metal catalysed coupling reactions (Cu, Ni, Pd, W, Mo) to form key bonds.