Quantitative Analysis of 2D EXSY NMR Spectra of Strongly Coupled Spin Systems in Transmembrane Exchange.

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 CF2 and CF2 CF2 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.

De Novo Enantioselective Synthesis of Hexafluorinated d-Glucose.

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.

Conformational Analysis of 1,3-Difluorinated Alkanes.

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-CH2-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.

Molecular dynamics and NMR reveal the coexistence of H-bond-assisted and through-space JFH coupling in fluorinated amino alcohols.

The JFH coupling constants in fluorinated amino alcohols were investigated through experimental and theoretical approaches. The experimental JFH 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 JFH 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 JFH coupling. The contribution of TS to the JFH 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 JFH coupling observed for the molecules studied should be represented as 4TS/1hJFH coupling.

The Synthesis and Glycoside Formation of Polyfluorinated Carbohydrates.

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.

Obtaining Pure 1H NMR Spectra of Individual Pyranose and Furanose Anomers of Reducing Deoxyfluorinated Sugars.

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 1H 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.

Lipophilicity Modulations by Fluorination Correlate with Membrane Partitioning.

Bioactive compounds generally need to cross membranes to arrive at their site of action. The octanol-water partition coefficient (lipophilicity, logPOW ) has proven to be an excellent proxy for membrane permeability. In modern drug discovery, logPOW 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 logPOW values with the corresponding membrane molar partitioning coefficients (logKp ); 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.

Relating Conformational Equilibria to Conformer-Specific Lipophilicities: New Opportunities in Drug Discovery.

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.

Skipped Fluorination Motifs: Synthesis of Building Blocks and Comparison of Lipophilicity Trends with Vicinal and Isolated Fluorination Motifs.

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-CFnH2-n-CFmH2-m-X versus X-CFnH2-n-CH2-CFmH2-m-X, with n,m ≠ 0 and X = CH2OH) to compounds with isolated fluorination (n ≠ 0; m = 0, and including X-CH2-CFnH2-n-CH2-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.

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.

Fluorinated carbohydrates as chemical probes for molecular recognition studies. Current status and perspectives.

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.

Cubane Electrochemistry: Direct Conversion of Cubane Carboxylic Acids to Alkoxy Cubanes Using the Hofer-Moest Reaction under Flow Conditions.

The highly strained cubane system is of great interest as a scaffold and rigid linker in both pharmaceutical and materials chemistry. The first electrochemical functionalisation of cubane by oxidative decarboxylative ether formation (Hofer-Moest reaction) was demonstrated. The mild conditions are compatible with the presence of other oxidisable functional groups, and the use of flow electrochemical conditions allows straightforward upscaling.

Enzymatic glycosylation involving fluorinated carbohydrates.

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.

Lipophilicity trends upon fluorination of isopropyl, cyclopropyl and 3-oxetanyl groups.

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.

Synthesis and Conformational Properties of 3,4-Difluoro-l-prolines.

Fluorinated proline derivatives have found diverse applications in areas ranging from medicinal chemistry over structural biochemistry to organocatalysis. Depending on the stereochemistry of monofluorination at the proline 3- or 4-position, different effects on the conformational properties of proline (ring pucker, cis/ trans isomerization) are introduced. With fluorination at both 3- and 4-positions, matching or mismatching effects can occur depending on the relative stereochemistry. Here we report, in full, the syntheses and conformational properties of three out of the four possible 3,4-difluoro-l-proline diastereoisomers. The yet unreported conformational properties are described for (3 S,4 S)- and (3 R,4 R)-difluoro-l-proline, which are shown to bias ring pucker and cis/ trans ratios on the same order of magnitude as their respective monofluorinated progenitors, although with significantly faster amide cis/ trans isomerization rates. The reported analogues thus expand the scope of available fluorinated proline analogues as tools to tailor proline's distinct conformational and dynamical properties, allowing for the interrogation of its role in, for instance, protein stability or folding.

Synthesis of 2,3,4-Trideoxy-2,3,4-trifluoroglucose.

There is an increasing interest in investigating how polyfluorination of carbohydrates modifies their physical and biological properties. An example that has caught much attention is 2,3,4-trideoxy-2,3,4-trifluoroglucose. Four syntheses of this compound have been reported, which are either low yielding or long (13 or more steps). We report a 6-step synthesis of 2,3,4-trideoxy-2,3,4-trifluoroglucose starting from levoglucosan. The solution-phase structure of an intermediate, 1,6-anhydro-2,4-dideoxy-2,4-difluoroallose, features a rare example of a bifurcated F···H(O)···F hydrogen bond and is compared to its crystal structure.

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.

Unraveling Sugar Binding Modes to DC-SIGN by Employing Fluorinated Carbohydrates.

A fluorine nuclear magnetic resonance (19F-NMR)-based method is employed to assess the binding preferences and interaction details of a library of synthetic fluorinated monosaccharides towards dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), a lectin of biomedical interest, which is involved in different viral infections, including HIV and Ebola, and is able to recognize a variety of self- and non-self-glycans. The strategy employed allows not only screening of a mixture of compounds, but also obtaining valuable information on the specific sugar-protein interactions. The analysis of the data demonstrates that monosaccharides Fuc, Man, Glc, and Gal are able to bind DC-SIGN, although with decreasing affinity. Moreover, a new binding mode between Man moieties and DC-SIGN, which might have biological implications, is also detected for the first time. The combination of the 19F with standard proton saturation transfer difference (1H-STD-NMR) data, assisted by molecular dynamics (MD) simulations, permits us to successfully define this new binding epitope, where Man coordinates a Ca2+ ion of the lectin carbohydrate recognition domain (CRD) through the axial OH-2 and equatorial OH-3 groups, thus mimicking the Fuc/DC-SIGN binding architecture.

Transmembrane Exchange of Fluorosugars: Characterization of Red Cell GLUT1 Kinetics Using 19F NMR.

We have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carrier-mediated transmembrane transport of fluorinated substrates. The method is based on adapted kinetic theory that describes the concentration dependence of the transmembrane exchange rates of two competing, simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogs in human erythrocytes (red blood cells) using 19F NMR exchange spectroscopy. An inosine-based glucose-free medium was shown to promote survival and stable metabolism of red blood cells over the duration of the experiments (several hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we could categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 toward them. Differences in the membrane permeability of the three glucose analogs were qualitatively interpreted in terms of local perturbations in the bonding of substrates to key amino acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results can be applied to the design of probes of glucose transport or inhibitors of glucose metabolism in cells, including those exhibiting the Warburg effect.

Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy-D-manno-oct-2-ulosonic acid.

In plants, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N3 demonstrated that incorporation of Kdo-N3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo-N3 and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth.