Profiling substrate promiscuity of wild-type sugar kinases for multifluorinated 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-fluoro galactose, giving insight into changes in the active site that may account for the specificity of the enzyme towards certain substrate analogues

Towards the total synthesis of Chrysophaentin F

This thesis describes the synthetic work towards the natural product Chrysophaentin F which has been extracted from the alga Chrysophaeum taylori. This bisdiarylbutene macrocycle exhibits antimicrobial properties against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) (MIC50 (MRSA) = 4.2 ± 1.3 ?g/mL).Investigation of the key steps on an unchlorinated analogue allowed us to determine what strategy would be best to access the natural product. The formation of the desired core structure relied on a Chan-Lam-Evans coupling reaction, a Pd catalysed coupling reaction and a RCAM reaction to create the pivotal bonds of the complex scaffold. The investigation on a model system to form the vinyl chloride bridge has also been performed bringing insight on the possible regioselectivity of the necessary late stage hydrochlorination reaction.Finally the synthesis towards the chlorinated macrocycle was started and advanced enough to access some key intermediates which despite some unforeseen difficulties proved that the natural product is now at reach following the strategy developed for the unchlorinated analogue

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

Chemoenzymatic synthesis of an unnatural Manb1,4GlcNAc library using a glycoside phosphorylase with "reverse thiophosphorylase" activity

b-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manb1,4GlcNAc a constituent of the core N-glycan in eukaryotes, was recently identified as a STING immune pathway activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of b-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manb1,4GlcNAc analogues using the b-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 -mannan-like glycans. We also pioneer a reverse thiophosphorylase enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorylysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases

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 timescale. Here we show the application of direct infusion mass spectrometry (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ~40 seconds 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), enhancing its activity towards 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. </p

Molecular insights into DC-SIGN binding to self-antigens: the interaction with the blood group A/B antigens

12 p.-7 fig.-1 chart.-1 graph. abst.-1 schem.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.We thank Agencia Estatal de Investigacion of Spain and the European Research Council for financial support. B.L., J.-B.V., and J.M. thank the Industrial Biotechnology Catalyst (Innovate UK, BBSRC, EPSRC, BB/M028941/1) and the EPSRC (core capability EP/K039466/1) for funding.Peer reviewe

Reverse thiophosphorylase activity of a glycoside phosphorylase in the synthesis of an unnatural Manβ1,4GlcNAc library

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

Reverse thiophosphorylase activity of a glycoside phosphorylase in the synthesis of an unnatural Manβ1,4GlcNAc library

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

Reverse thiophosphorylase activity of a glycoside phosphorylase in the synthesis of an unnatural Manβ1,4GlcNAc library †

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