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The excessive hydrophilicity of carbohydrates hampers their application in drug discovery. Deoxyfluorination is one of the strategies to increase sugar lipophilicity. However, lipophilicities of dideoxy-difluorinated monosaccharides are still well below the desired range for oral drug candidates. Here we investigate the power of deoxychlorination to increase sugar lipophilicities. A series of dideoxygenated chloro-fluorosugars was synthesized and for these substrates it was shown that deoxychlorination increased the log  P by an average of 1.37 log  P units, compared to 0.83 log  P units for analogous deoxyfluorination. This shows the potential of deoxychlorination of carbohydrates to increase lipophilicity while limiting the number of potentially important hydrogen bond donating groups to be sacrificed, and will be of interest for glycomimetic development.

We report a de novo enantioselective synthesis of 2,3,4-trideoxy-2,2,3,3,4,4-hexafluoro-d- glycero -hexopyranose (hexafluorinated d-glucose), an iconic polar hydrophobic glycomimetic. The 12-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 crystallization procedure and the absolute confirmation was ascertained by X-ray analysis. The synthetic route also allowed access to the novel hexafluorinated heptose derivative 2,3,4-trideoxy-2,2,3,3,4,4-hexafluoro-l- threo -heptopyranose.

Glycans play a major role in biological cell-cell recognition and signal transduction but have found limited application in biosensors due to glycan/lectin promiscuity; multiple proteins are capable of binding to the same native glycan. Here, site-specific fluorination is used to introduce protein-glycan selectivity, and this is coupled with an electrochemical detection method to generate a novel biosensor platform. 3F-lacto- N -biose glycofluoroform is installed onto polymer tethers, which are subsequently immobilised onto gold screen printed electrodes, providing a non-fouling surface. The impedance biosensing platform is shown to selectively bind cancer-associated galectin-3 compared to control glycans and proteins. To improve the analytical capability, Bayesian statistical analysis was deployed in the equivalent circuit fitting of electrochemical impedance spectroscopy data. It is shown that Markov Chain Monte Carlo (MCMC) analysis is a helpful method for visualising experimental irreproducibility, and we apply this as a quality control step., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Glycan-mediated interactions play a crucial role in biology and medicine, influencing signalling, immune responses, and disease pathogenesis. However, the use of glycans in biosensing and diagnostics is limited by cross-reactivity, as certain glycan motifs can be recognised by multiple biologically distinct protein receptors. To address this specificity challenge, we report the enzymatic synthesis of a 150-member library of site-specifically fluorinated Lewis x analogues ('glycofluoroforms') using naturally occurring enzymes and fluorinated monosaccharides. Subsequent incorporation of a subset of these glycans into nanoparticles or a microarray revealed a striking spectrum of distinct binding intensities across different proteins that recognise Lewis x . Notably, we show that for two proteins with unique binding sites for Lewis x , glycofluoroforms exhibited enhanced binding to one protein, whilst reduced binding to the other, with selectivity governed by fluorination patterns. We finally showcase the potential diagnostic utility of this approach in glycofluoroform-mediated bacterial toxin detection by lateral flow., (© 2024. The Author(s).)

Fluorine substitution can have a profound impact on molecular conformation. Here, we present a detailed conformational analysis of how the 1,3-difluoropropylene motif (-CHF-CH 2 -CHF-) determines the conformational profiles of 1,3-difluoropropane, anti - and syn -2,4-difluoropentane, and anti - and syn -3,5-difluoroheptane. It is shown that the 1,3-difluoropropylene motif strongly influences alkane chain conformation, with a significant dependence on the polarity of the medium. The conformational effect of 1,3-fluorination is magnified upon chain extension, which contrasts with vicinal difluorination. Experimental evidence was obtained from NMR analysis, where polynomial complexity scaling simulation algorithms were necessary to enable J -coupling extraction from the strong second-order spectra, particularly for the large 16-spin systems of the difluorinated heptanes. These results improve our understanding of the conformational control toolkit for aliphatic chains, yield simple rules for conformation population analysis, and demonstrate quantum mechanical time-domain NMR simulations for liquid state systems with large numbers of strongly coupled spins.

The J FH coupling constants in fluorinated amino alcohols were investigated through experimental and theoretical approaches. The experimental J FH couplings were only reproduced theoretically when explicit solvation through molecular dynamics (MD) simulations was conducted in DMSO as the solvent. The combination of MD conformation sampling and DFT NMR spin-spin coupling calculations for these compounds reveals the simultaneous presence of through-space (TS) and hydrogen bond (H-bond) assisted J FH coupling between fluorine and hydrogen of the NH group. Furthermore, MD simulations indicate that the hydrogen in the amino group participates in both an intermolecular bifurcated H-bond with DMSO and in transmitting the observed J FH coupling. The contribution of TS to the J FH coupling is due to the spatial proximity of the fluorine and the NH group, aided by a combination of the non-bonding transmission pathway and the hydrogen bonding pathway. The experimental J FH coupling observed for the molecules studied should be represented as 4TS/1h J FH coupling.

The NLRP3 inflammasome plays a central role in various human diseases. Despite significant interest, most clinical-grade NLRP3 inhibitors are derived from sulfonylurea inhibitor CRID3 (also called MCC950). Here, we describe a novel chemical class of NLRP3-inhibiting compounds (NIC) that exhibit potent and selective NLRP3 inflammasome inhibition in human monocytes and mouse macrophages. BRET assays demonstrate that they physically interact with NLRP3. Structural modeling further reveals they occupy the same binding site of CRID3 but in a critically different conformation. Furthermore, we show that NIC-11 and NIC-12 lack the off-target activity of CRID3 against the enzymatic activity of carbonic anhydrases I and II. NIC-12 selectively reduces circulating IL-1ß levels in the LPS-endotoxemia model in mice and inhibits NLRP3 inflammasome activation in CAPS patient monocytes and mouse macrophages with about tenfold increased potency compared with CRID3. Altogether, this study unveils a new chemical class of highly potent and selective NLRP3-targeted inhibitors with a well-defined molecular mechanism to complement existing CRID3-based NLRP3 inhibitors in pharmacological studies and serve as novel chemical leads for the development of NLRP3-targeted therapies., (© 2024 Vande Walle et al.)

Solute 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 19 F 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 CF 2 and CF 2 CF 2 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., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

1,4-Disubstituted cubyl aryl thioethers were generated from the corresponding iodocubanes and aryl thiolates upon UV irradiation in dimethyl sulfoxide at room temperature. This simple procedure was found to be compatible with a variety of substituted aryl thiolates. This finding paved the way to a synthesis of the monocubyl analogue of dapsone, a key molecule in the treatment of leprosy, also known as Hansen's disease, and of acne.

Due to tautomeric equilibria, NMR spectra of reducing sugars can be complex with many overlapping resonances. This hampers coupling constant determination, which is required for conformational analysis and configurational assignment of substituents. Given that mixtures of interconverting species are physically inseparable, easy-to-use techniques that enable facile full 1 H NMR characterization of sugars are of interest. Here, we show that individual spectra of both pyranoside and furanoside forms of reducing fluorosugars can be obtained using 1D FESTA. We discuss the unique opportunities offered by FESTA over standard sel-TOCSY and show how it allows a more complete characterization. We illustrate the power of FESTA by presenting the first full NMR characterization of many fluorosugars, including of the important fluorosugar 2-deoxy-2-fluoroglucose. We discuss in detail all practical considerations for setting up FESTA experiments for fluorosugars, which can be extended to any mixture of fluorine-containing species interconverting slowly on the NMR frequency-time scale.

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., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

The acid-catalysed formation of glycosidic bonds is more difficult when glycosyl donors are fluorinated, especially at the 2-position. Here we report high-yielding glycosidation and glycosylation reactions of 2,3-difluorinated- and 2,3,4-trifluorinated gluco- and galactopyranoside donors with a variety of acceptors under conventional trichloroacetimidate/TMSOTf activation in moderate to high anomeric selectivities. This methodology allows access to highly fluorinated glycans, illustrated with the synthesis of a pentafluorinated disaccharide.

Bioactive compounds generally need to cross membranes to arrive at their site of action. The octanol-water partition coefficient (lipophilicity, logP OW ) has proven to be an excellent proxy for membrane permeability. In modern drug discovery, logP OW and bioactivity are optimized simultaneously, for which fluorination is one of the relevant strategies. The question arises as to which extent the often subtle logP modifications resulting from different aliphatic fluorine-motif introductions also lead to concomitant membrane permeability changes, given the difference in molecular environment between octanol and (anisotropic) membranes. It was found that for a given compound class, there is excellent correlation between logP OW values with the corresponding membrane molar partitioning coefficients (logK p ); a study enabled by novel solid-state 19 F NMR MAS methodology using lipid vesicles. Our results show that the factors that cause modulation of octanol-water partition coefficients similarly affect membrane permeability., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Fluorinated carbohydrates have found many applications in the glycosciences. Typically, these contain fluorination at a single position. There are not many applications involving polyfluorinated carbohydrates, here defined as monosaccharides in which more than one carbon has at least one fluorine substituent directly attached to it, with the notable exception of their use as mechanism-based inhibitors. The increasing attention to carbohydrate physical properties, especially around lipophilicity, has resulted in a surge of interest for this class of compounds. This review covers the considerable body of work toward the synthesis of polyfluorinated hexoses, pentoses, ketosugars, and aminosugars including sialic acids and nucleosides. An overview of the current state of the art of their glycosidation is also provided.

Efficient drug discovery is based on a concerted effort in optimizing bioactivity and compound properties such as lipophilicity, and is guided by efficiency metrics that reflect both aspects. While conformation-activity relationships and ligand conformational control are known strategies to improve bioactivity, the use of conformer-specific lipophilicities (logp) is much less explored. Here we show how conformer-specific logp values can be obtained from knowledge of the macroscopic logP value, and of the equilibrium constants between the individual species in water and in octanol. This is illustrated with fluorinated amide rotamers, with integration of rotamer 19 F NMR signals as a facile, direct method to obtain logp values. The difference between logp and logP optimization is highlighted, giving rise to a novel avenue for lipophilicity control in drug discovery., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Proline homopolymer motifs are found in many proteins; their peculiar conformational and dynamic properties are often directly involved in those proteins' functions. However, the dynamics of proline homopolymers is hard to study by NMR due to a lack of amide protons and small chemical shift dispersion. Exploiting the spectroscopic properties of fluorinated prolines opens interesting perspectives to address these issues. Fluorinated prolines are already widely used in protein structure engineering - they introduce conformational and dynamical biases - but their use as 19 F NMR reporters of proline conformation has not yet been explored. In this work, we look at model peptides where C γ -fluorinated prolines with opposite configurations of the chiral C γ centre have been introduced at two positions in distinct polyproline segments. By looking at the effects of swapping these (4 R )-fluoroproline and (4 S )-fluoroproline within the polyproline segments, we were able to separate the intrinsic conformational properties of the polyproline sequence from the conformational alterations instilled by fluorination. We assess the fluoroproline 19 F relaxation properties, and we exploit the latter in elucidating binding kinetics to the SH3 (Src homology 3) domain., Competing Interests: The contact author has declared that neither they nor their co-authors have any competing interests., (Copyright: © 2021 Davy Sinnaeve et al.)

The cubane ring has received intense attention as a 3D benzene isostere and scaffold. Mono- and 1,4-disubstituted cubanes are well-described. Here we report a practical procedure for a direct radical-mediated chlorocarbonylation process initially reported by Bashir-Hashemi, to access a range of 2-substituted 1,4-cubanedicarboxylic ester derivatives. A subsequent regioselective ester hydrolysis to give fully differentiated 1,2,4-trisubstituted cubanes is demonstrated.

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.

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., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Given there is an optimal lipophilicity range for orally bioavailable drugs, structural modifications applied in the drug development process are not only focused on optimizing bioactivity but also on fine-tuning lipophilicity. Fluorine introduction can be used for both purposes. Insights into how fluorine introduction affects lipophilicity are thus of importance, and systematic series of fluorinated compounds with measured octanol-water partition coefficients are a powerful way to enhance our qualitative understanding in this regard and are essential as input for computational log  P estimation programs. Here, we report a detailed comparison of all possible vicinal and skipped (1,3-substituted) fluorination motifs when embedded in structurally equivalent environments (X- CF n H 2- n - CF m H 2- m -X versus X- CF n H 2- n -CH 2 - CF m H 2- m -X, with n,m ≠ 0 and X = CH 2 OH) to compounds with isolated fluorination ( n ≠ 0; m = 0, and including X-CH 2 - CF n H 2- n -CH 2 -X, n = 0-2). It is shown that skipped fluorination is more powerful for log  P reduction purposes compared to single or vicinal fluorination. Efficient stereoselective syntheses of the compounds with skipped fluorination motifs are reported, which where relevant can be made enantioselective using known chiral building blocks. These compounds, and some intermediates, will be of interest as advanced fluorinated building blocks.

Galectins are potential biomarkers and therapeutic targets. However, galectins display broad affinity towards β-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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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., Competing Interests: Declaration of Interests The authors declare no competing financial interests. Prozomix is a commercial enzyme producer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A systematic comparison of lipophilicity modulations upon fluorination of isopropyl, cyclopropyl and 3-oxetanyl substituents, at a single carbon atom, is provided using directly comparable, and easily accessible model compounds. In addition, comparison with relevant linear chain derivatives is provided, as well as lipophilicity changes occurring upon chain extension of acyclic precursors to give cyclopropyl containing compounds. For the compounds investigated, fluorination of the isopropyl substituent led to larger lipophilicity modulation compared to fluorination of the cyclopropyl substituent., (Copyright © 2020, Jeffries et al.; licensee Beilstein-Institut.)

This review provides an extensive summary of the effects of carbohydrate fluorination with regard to changes in physical, chemical and biological properties with respect to regular saccharides. The specific structural, conformational, stability, reactivity and interaction features of fluorinated sugars are described, as well as their applications as probes and in chemical biology.

Fluorinated carbohydrates, where one (or more) fluorine atom(s) have been introduced into a carbohydrate structure, typically through deoxyfluorination chemistry, have a wide range of applications in the glycosciences. Fluorinated derivatives of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, talose, fucose and sialic acid have been employed as either donor or acceptor substrates in glycosylation reactions. Fluorinated donors can be synthesised by synthetic methods or produced enzymatically from chemically fluorinated sugars. The latter process is mediated by enzymes such as kinases, phosphorylases and nucleotidyltransferases. Fluorinated donors produced by either method can subsequently be used in glycosylation reactions mediated by glycosyltransferases, or phosphorylases yielding fluorinated oligosaccharide or glycoconjugate products. Fluorinated acceptor substrates are typically synthesised chemically. Glycosyltransferases are most commonly used in conjunction with natural donors to further elaborate fluorinated acceptor substrates. Glycoside hydrolases are used with either fluorinated donors or acceptors. The activity of enzymes towards fluorinated sugars is often lower than towards the natural sugar substrates irrespective of donor or acceptor. This may be in part attributed to elimination of the contribution of the hydroxyl group to the binding of the substrate to enzymes. However, in many cases, enzymes still maintain a significant activity, and reactions may be optimised where necessary, enabling enzymes to be used more successfully in the production of fluorinated carbohydrates. This review describes the current state of the art regarding chemoenzymatic production of fluorinated carbohydrates, focusing specifically on examples of the enzymatic production of activated fluorinated donors and enzymatic glycosylation involving fluorinated sugars as either glycosyl donors or acceptors.

Optimization of compound lipophilicity is a key aspect of drug discovery. The aim of this work was to compare the lipophilicity modulations induced by 16 distinct known and novel fluoroalkyl motifs on three parent models. Fifty fluorinated compounds, with 28 novel experimental aliphatic log  P values, are involved in discussing various lipophilicity trends. As well as confirming known trends, a number of novel lipophilicity-reducing motifs are introduced. Tactics to reduce lipophilicity are discussed, such as "motif extensions" and "motif rearrangements", including with concomitant extension of the carbon chain, as well as one- and two-fluorine 'deletions' within perfluoroalkyl groups. Quantum chemical log  P calculations (SMD-MN15) based on solvent-dependent three-dimensional (3D) conformational analysis gave excellent correlations with experimental values, superior to C log  P predictions based on 2D structural motifs. The availability of a systematic collection of data based on a small number of parent molecules illustrates the relative lipophilicity modulations of aliphatic fluorination motifs.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.