Heavily fluorinated carbohydrates as enzyme substrates: oxidation of tetrafluorinated galactose by galactose oxidase

Galactose oxidase (GOase) was shown to oxidise several C2/C3 fluorinated galactose analogues. Interestingly, the enzyme was able to distinguish between the 2,3-tetrafluorinated galactose and its epimeric glucose analogue, and this represents the first reported biotransformation of a heavily fluorinated suga

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

Development of a solid phase array assay for the screening of galactose oxidase activity and for Fast identification of inhibitors

Background: Galactose oxidase (GOase) catalyses the highly selective oxidation of terminal galactosides on a wide range of natural glycoconjugates and has found wide applications in biotechnology – particularly in biocatalysis. GOase is copper dependent and uses oxygen to oxidise the C6-primary alcohol of galactose and produces hydrogen peroxide. The enzyme activity can be conveniently assessed by a colorimetric assay. Objectives: The objective of the present study was to develop an assay system, which is independent of the hydrogen peroxide formation to identify possible fluorinated GOase inhibitors. In case that the inhibitor bears a primary or secondary alcohol, it could also be oxidised by the enzyme. In such case, the colorimetric assay is not able to distinguish between substrate and inhibitor, since oxidation of both molecules would result in the formation of hydrogen peroxide. Methods: D-galactose (D-Gal) was immobilised onto a gold surface functionalised by selfassembled monolayers (SAMs,). A GOase solution was then added to the surface in a droplet for a certain period of time and thereafter washed away. The activity of GOase on the immobilised D-Gal can then be quantified by MALDI-ToF MS. Results: For inhibition studies, GOase was incubated together with 62.5 mM of deoxy-fluorinated monosaccharides on the D-Gal displaying platform. Five deoxy-fluorinated D-Gal showed a >50% inhibition of its activity. The array system has been moreover utilised to determine the apparent IC50 value of 3-F-Gal 15 as a proof of principle. Conclusion: The developed array platform allows the fast identification of GOase substrates and inhibitors from a library of deoxy-fluorinated sugars using MALDI-ToF MS as a label–free readout method. In addition, the enzymatic reaction enables for the in situ activation of sugar-coated surfaces to bioorthogonal aldehydes, which can be utilised for subsequent chemical modifications

Heavily fluorinated carbohydrates as enzyme substrates: Oxidation of tetrafluorinated galactose by galactose oxidase

Galactose oxidase (GOase) was shown to oxidise several C2/C3 fluorinated galactose analogues. Interestingly, the enzyme was able to distinguish between the 2,3-tetrafluorinated galactose and its epimeric glucose analogue, and this represents the first reported biotransformation of a heavily fluorinated sugar. © 2011 The Royal Society of Chemistry

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

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

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/β anomeric ratios were identified. Increasing fluorine content always increased the α/β ratio, with very little difference between regio- or stereoisomers, except when 4,6-difluorinated