1,720,969 research outputs found
Review of mutarotase in ‘Metabolic Subculture’ and analytical biochemistry: prelude to 19F NMR studies of its substrate specificity and mechanism
No full textThis is the first paper in a sequential pair devoted to the enzyme mutarotase (aldose 1-epimerase; EC 5.1.3.3). Here, the broader context of the physiological role of mutarotase, among those enzymes considered to be part of 'metabolic structure', is reviewed. We also summarise the current knowledge about the molecular mechanism and substrate specificity of the enzyme, which is considered in the context of the binding of fluorinated glucose analogues to the enzyme's active site. This was done as a prelude to our experimental studies of the anomerisation of fluorinated sugars by mutarotase that are described in the following paper.</p
Isolation and characterisation of an unexpected byproduct in the regioselective butane diacetal protection of α-methyl galactopyranoside
No full textThe regioselective protection of both methyl galactopyranoside anomers at the 2 and 3-positions as the butane diacetal (BDA) is well known. Here we describe the formation of an unexpected byproduct, which mainly occurs when α-methyl galactopyranoside is reacted with 2,3-butanedione under BF3•OEt2 catalysis. The structure of the byproduct, which did not arise from anomerisation to the β-anomer or from BDA formation at the galactopyranoside 3,4-positions, was elucidated by NMR and X-ray crystallographic analysis, and proved to be the expected BDA protected galactopyranoside, but in which the stereochemistry of both its BDA acetal centres are inverted. Interestingly, the conformation of the resulting six-membered BDA ring was distorted to a skew boat conformation in order to maintain anomeric stabilisation
Anomerisation of fluorinated sugars by mutarotase studied by 19F NMR two-dimensional exchange spectroscopy
No full textFive
19F-substituted glucose analogues were used to probe the activity and mechanism of the enzyme mutarotase by using magnetisation-exchange NMR spectroscopy. The sugars (2-fluoro-2-deoxy-d-glucose, FDG2; 3-fluoro-3-deoxy-d-glucose, FDG3; 4-fluoro-4-deoxy-d-glucose, FDG4; 2,3-difluoro-2,3-dideoxy-d-glucose, FDG23; and 2,2,3,3-tetrafluoro-2,3-dideoxy-d-glucose (2,3-dideoxy-2,2,3,3-tetrafluoro-d-erythro-hexopyranose), FDG2233) showed separate
19F NMR spectroscopic resonances from their respective α- and β-anomers, thus allowing two-dimensional exchange spectroscopy measurements of the anomeric interconversion at equilibrium, on the time scale of a few seconds. Mutarotase catalysed the rapid exchange between the anomers of FDG4, but not the other four sugars. This finding, combined with previous work identifying the mechanism of the anomerisation by mutarotase, suggests that the rotation around the C1-C2 bond of the pyranose ring is the rate-limiting reaction step. In addition to d-glucose itself, it was shown that all other fluorinated sugars inhibited the FDG4 anomerisation, with the tetrafluorinated FDG2233 being the most potent inhibitor. Inhibition of mutarotase by F-sugars paves the way for the development of novel fluorinated compounds that are able to affect the activity of this enzyme in vitro and in vivo.
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Quantitative analysis of 2D EXSY NMR spectra of strongly coupled spin systems in transmembrane exchange
No full textSolute translocation by membrane transport proteins is a vital biological process that can be tracked, on the sub-second timescale, using nuclear magnetic resonance (NMR). Fluorinated substrate analogues facilitate such studies because of high sensitivity of 19F NMR and absence of background signals. Accurate extraction of translocation rate constants requires precise quantification of NMR signal intensities. This becomes complicated in the presence of J-couplings, cross-correlations, and nuclear Overhauser effects (NOE) that alter signal integrals through mechanisms unrelated to translocation. Geminal difluorinated motifs introduce strong and hard-to-quantify contributions from non-exchange effects, the nuanced nature of which makes them hard to integrate into data analysis methodologies. With analytical expressions not being available, numerical least squares fitting of theoretical models to 2D spectra emerges as the preferred quantification approach. For large spin systems with simultaneous coherent evolution, cross-relaxation, cross-correlation, conformational exchange, and membrane translocation between compartments with different viscosities, the only available simulation framework is Spinach. In this study, we demonstrate GLUT-1 dependent membrane transport of two model sugars featuring CF2 and CF2CF2 fluorination motifs, with precise determination of translocation rate constants enabled by numerical fitting of 2D EXSY spectra. For spin systems and kinetic networks of this complexity, this was not previously tractable.</p
<i>De Novo</i> enantioselective synthesis of hexafluorinated d-glucose
No full textWe report a de novo enantioselective synthesis of 2,3,4-trideoxy-2,2,3,3,4,4-hexafluoro-Dglycero-hexopyranose (“hexafluorinated D-glucose”), an iconic polar hydrophobic glycomimetic. The12-step synthesis features robust and reproducible chemistry and was achieved by incorporating an asymmetric dihydroxylation step to install the stereogenic center with excellent enantioselectivity (95:5 er). Virtual enantiopurity (> 99.5% ee) was further reached using a simple crystallisation procedure and the absolute confirmation was ascertained by X-ray analysis. The synthetic route also allowed the access to the novel hexafluorinated heptose derivative 2,3,4-trideoxy-2,2,3,3,4,4- hexafluoro-L-threo-heptopyranose
Synthesis and structural characteristics of all mono- and difluorinated 4,6-dideoxy-d-xylo-hexopyranoses
No full textProtein–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
Investigating the influence of (Deoxy)fluorination on the lipophilicity of non-UV-active fluorinated alkanols and carbohydrates by a new log P determination method
No full textProperty tuning by fluorination is very effective for a number of purposes, and currently increasingly investigated for aliphatic compounds. An important application is lipophilicity (log?P) modulation. However, the determination of log?P is cumbersome for non-UV-active compounds. A new variation of the shake-flask log?P determination method is presented, enabling the measurement of log?P for fluorinated compounds with or without UV activity regardless of whether they are hydrophilic or lipophilic. No calibration curves or measurements of compound masses/aliquot volumes are required. With this method, the influence of fluorination on the lipophilicity of fluorinated aliphatic alcohols was determined, and the log?P values of fluorinated carbohydrates were measured. Interesting trends and changes, for example, for the dependence on relative stereochemistry, are reporte
Design of fluorinated 5-HT4R antagonists: influence of the basicity and lipophilicity toward the 5-HT4R binding affinities
No full textAnalogues of potent 5-HT4R antagonists possessing a fluorinated N-alkyl chain have been synthesized in order to investigate the effect of the resulting change in basicity and lipophilicity on the affinity and selectivity profile. We demonstrate that for this series, the affinity is decreased with decreased basicity of the piperidine’s nitrogen atom. In contrast, the resulting increase in lipophilicity has minimal impact on binding affinity and selectivity. 3,3,3-Trifluoropropyl and 4,4,4-trifluorobutyl derivatives 6d and 6e have shown to bind to the 5-HT4R while maintaining their pharmacological profile and selectivity toward other 5-HT receptors
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