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Due to the high variation in viral surface properties, a platform method for virus purification is still lacking. A potential alternative to the high-cost conventional methods is aqueous two-phase systems (ATPSs). However, optimizing virus purification in ATPS requires a large experimental design space, and the optimized systems are generally found to operate at high ATPS component concentrations. The high concentrations capitalize on hydrophobic and electrostatic interactions to obtain high viral particle yields. This study investigated using osmolytes as driving force enhancers to reduce the high concentration of ATPS components while maintaining high yields. The partitioning behavior of porcine parvovirus (PPV), a nonenveloped mammalian virus, and human immunodeficiency virus-like particle (HIV-VLP), a yeast-expressed enveloped VLP, were studied in a polyethylene glycol (PEG) 12 kDa-citrate system. The partitioning of the virus modalities was enhanced by osmoprotectants glycine and betaine, while trimethylamine N-oxide was ineffective for PPV. The increased partitioning to the PEG-rich phase pertained only to viruses, resulting in high virus purification. Recoveries were 100% for infectious PPV and 92% for the HIV-VLP, with high removal of the contaminant proteins and more than 60% DNA removal when glycine was added. The osmolyte-induced ATPS demonstrated a versatile method for virus purification, irrespective of the expression system.

Funding: We thank EPSRC (EP/R013799/1) for a grant (DB) and the University of St Andrews for a Studentship (XF). The isolation of three adenosine based metabolites 6–8 from Streptomyces calvus is reported. The metabolites are structurally related to the fluorine containing antibiotic nucleocidin 1 and two recently identified glycosylated fluoroadenosines 2 and 3, however in this case the three metabolites do not contain a fluorine, suggesting that the biosynthetic enzymes to the fluorometabolites also process their non-fluorinated counterparts. Publisher PDF

We thank EPSRC for a grant (EP/R013799/1) and for a Studentship (JC) through the CRITICAT Centre for Doctoral training (CDT). FAPESP is also gratefully acknowledged for a studentship (BAP, #2019/03855-3), and a Young Research Award (RAC, #2018/03910-1). CENAPAD-SP, CESUP and SDumont are acknowledged for computational clusters used in theory calculations. This study uses X-ray crystallography, theory and Langmuir isotherm analysis to explore the conformations and molecular packing of alkyl all-cis 2,3,4,5,6-pentafluorocyclohexyl motifs, which are prepared by direct aryl hydrogenations from alkyl- or vinyl- pentafluoroaryl benzenes. Favoured conformations retain the more polar triaxial C-F bond arrangement of the all-cis 2,3,4,5,6-pentafluorocyclohexyl ring systems with the alkyl substituent adopting an equatorial orientation, and accommodating strong supramolecular interactions between rings. Langmuir isotherm analysis on a water subphase of a long chain fatty acid and alcohol carrying terminal all-cis 2,3,4,5,6-pentafluorocyclohexyl rings do not show any indication of monolayer assembly relative to their cyclohexane analogues, instead the molecules appear to aggregate and form higher molecular assemblies prior to compression. The study indicates the power and potential of this ring system as a motif for ordering supramolecular assembly. Publisher PDF

We report the synthesis of all‐cis 1,2,4,5‐tetrakis (trifluoromethyl)‐ and all‐cis 1,2,3,4,5,6‐hexakis (trifluoromethyl)‐ cyclohexanes by direct hydrogenation of precursor tetrakis‐ or hexakis‐ (trifluoromethyl)benzenes. The resultant cyclohexanes have a stereochemistry such that all the CF3 groups are on the same face of the cyclohexyl ring. All‐cis 1,2,3,4,5,6‐hexakis(trifluoromethyl)cyclohexane is the most sterically demanding of the all‐cis hexakis substituted cyclohexanes prepared to date, with a barrier (ΔG) to ring inversion calculated at 27 kcal mol−1. The X‐ray structure of all‐cis 1,2,3,4,5,6‐hexakis(trifluoromethyl)cyclohexane displays a flattened chair conformation and the electrostatic profile of this compound reveals a large diffuse negative density on the fluorine face and a focused positive density on the hydrogen face. The electropositive hydrogen face can co‐ordinate chloride (K≈103) and to a lesser extent fluoride and iodide ions. Dehydrofluorination promoted decomposition occurs with fluoride ion acting as a base., All‐cis 1,2,4,5‐tetrakis (trifluoromethyl)‐ and all‐cis 1,2,3,4,5,6‐hexakis (trifluoromethyl)‐ cyclohexanes were prepared by direct aryl hydrogenation. All‐cis 1,2,3,4,5,6‐hexakis(trifluoromethyl)cyclohexane has a high barrier to ring inversion (ΔG≠=27 kcal mol−1) and a Janus face profile with a large diffuse negative density on the fluorine face, and a focused positive density on the hydrogen face which coordinates carbonyls and halides.

We report counter‐intuitive axial preferences in non‐stereochemically biased, selectively fluorinated methoxycyclohexanes. These pseudo‐anomeric effects are apparent when electronegative CF2 groups are placed at the C‐2, C‐4 and C‐6 positions of the cyclohexane ring to render the C‐3/5 axial hydrogen atoms electropositive. The electrostatic interaction between these axial hydrogen atoms and the ‐OMe oxygen is stabilising. The effect is explored using high‐level ab initio and DFT calculations in the framework of NBO, QTAIM and NCI analysis across a range of derivatives, and experimentally (19F{1H}‐NMR at −80 °C) for some illustrative examples. The effect is significant in energy terms for a weak interaction, and illustrates a new stereoelectronic aspect attributed to selective fluorine substitution in organic chemistry., Fluorine chemistry: Selective fluorination in methoxycyclohexanes induces an electrostatic 1,3‐diaxial interaction favouring a counter‐intuitive axial preference.

Authors thank the Chinese Scholarship Council for a studentship (ZF) and the EPSRC Mass Spectroscopy Service at Swansea for analysis. This paper describes the synthesis of a series of organic liquid crystals (LC) containing selectively fluorinated cyclopropanes at their termini. The syntheses used difluorocarbene additions to olefin precursors, an approach which proved straightforward such that these liquid crystal candidates could be efficiently prepared. Their physical and thermodynamic properties were evaluated and depending on individual structures, they either displayed positive or negative dielectric anisotropy. The study gives some guidance into effective structure property relationships for the design of LCs containing selectively fluorinated cyclopropane motifs. Publisher PDF

Authors thank the Leverhulme Trust, EPSRC and the Sinfonia consortium of the EU for grants as well as the Chinese Scholarship Council for a Studentship (YC), all of which helped to support this work. Genome homology and the presence of a putative biosynthetic gene cluster identified Streptomyces aureorectus DSM 41692 and Streptomyces virens DSM 41465 as candidate producers of the antibiotic nucleocidin 1. Indeed when these bacterial strains were cultured in a medium supplemented with fluoride (4 mM) they each produced nucleocidin 1 and the previously identified 4'-fluoro-3'-O-β-glucosylated adenosine 2 and its sulfamylated derivative 3. In both of these cases 4'-fluoroadenosine 9 is also identified as a natural product although it has never been observed during fermentations of Streptomyces calvus, the original source of nucleocidin 1. The identity of 4'-fluoroadenosine 9 was confirmed by a total synthesis as well as by its in vitro enzymatic conversion to metabolite 2 using the glucosyl transferase enzyme, NucGT. Postprint Postprint

We thank EPSRC for a grant (EP/R013799/1) and Studentships (JLC) through the EPSRC CRITICAT CDT programme and also the Chinese Scholarship Council (CY). Monoalkylated derivatives of the unusually polar all- cis 2,3,4,5,6- pentafluorocyclohexyl (Janus face) motif are prepared starting from an aryl hydrogenation of 2,3,4,5,6- pentafluorophenylacetate methyl ester 15 . The method used Zeng's Rh(CAAC) carbene catalyst 4 in the hydrogenation following the protocol developed by Glorius. The resultant Janus pentafluorocyclohexylacetate methyl ester 16 was converted to the corresponding alcohol 18 , aldehyde 13 , bromide 29 and azide 14 through functional group manipulations, and some of these building blocks were used in Ugi-multicomponent and Cu-catalysed click reactions. NBoc protected pentafluoroarylphenylalanine methyl ester 35 was also subject to an aryl hydrogenation, and then deprotection to generate the Janus face ß-pentafluorocyclohexyl-alanine amino acid 15 , which was incorporated into representative members of an emerging class of candidate antiviral compounds. Log P measurements demonstrate that the all- cis 2,3,4,5,6-pentafluorocyclohexyl ring system is more polar than a phenyl ring. In overview the paper introduces new building blocks containing this Janus ring and demonstrates their progression to molecules typically used in bioactives discovery programmes. Publisher PDF

Authors thank the Engineering and Physical Sciences Research Council (EPSRC) for funding and we are grateful to the EPSRC Mass Spectroscopy Service at the University of Swansea for carrying out some of the accurate mass analysis of prepared compounds. A series of five 12-dodecanolides have been synthesised containing CF2 groups at C5, C6, C7, C8 and in one case a double substitution at C5 & C8, as a strategy to bias the conformational space accessed by these macrocycles, and to assess if the analogues may act as mimetics for 12-decenolide pheromones associated with the Emerald Ash Borer. Accordingly individual syntheses of 5,5-difluoro- 5, 6,6-difluoro- 6, 7,7-difluoro- 7, 8,8-difluoro- 8 and 5,5,8,8-tetrafluoro- 9, 12-decanolides is outlined and X-ray structural data was obtained for three (5, 8 and 9) of these compounds. The structures show clearly that the CF2 groups occupy ‘corner’ locations in the macrocycle consistent with their ability to bias accessible conformations. The fluorine containing 12-dodecanolides all generated an electro-antennogram response in female beetles. Postprint

Funding: This research was funded by JSPS KAKENHI through a Grant-in-Aid for Scientific Research (C), grant number JP18K05262. Photoluminescentliquid crystals (PLLCs) have attracted significant attention owing to their broad applicabilityin thermosensing and PL switching. Extensive efforts have been madeto develop bistolane-based PLLCs containing flexible units at both molecular terminals, and it has been revealed that their PL behavior can switch with the phase transition between the crystallineand LC phases. Although slight modulation of the flexible unit structure dramatically alters the LC and PL behaviors, few studies into modification of the flexible units have been conducted. With the aim of achieving dynamic changes in their physical behaviors, we developed a family of bistolane derivatives containing a simple alkyl or a fluoroalkyl flexible chain, and carried out a detailed systematic evaluationof their physical behaviors. Bistolanes containing a simple alkyl chain showed a nematic LC phase, whereas switching the flexible chain in the bistolane to a fluoroalkyl moiety significantly altered the phase to generate a smectic phase. The fluoroalkyl-containing bistolanes displayed a stronger deep blue PL than their corresponding non-fluorinated counterparts, even in the crystalline phase, which was attributed to the construction of rigid molecular aggregates through intermolecular F···H and F···F interactions to suppress non-radiative deactivation. Publisher PDF

This study uses X-ray crystallography, theory and Langmuir isotherm analysis to explore the conformations and molecular packing of alkyl all-cis 2,3,4,5,6-pentafluorocyclohexyl motifs, which are prepared by direct aryl hydrogenations from alkyl- or vinyl-pentafluoroaryl benzenes. Favoured conformations retain the more polar triaxial C–F bond arrangement of the all-cis 2,3,4,5,6-pentafluorocyclohexyl ring systems with the alkyl substituent adopting an equatorial orientation, and accommodating strong supramolecular interactions between rings. Langmuir isotherm analysis on a water subphase of a long chain fatty acid and alcohol carrying terminal all-cis 2,3,4,5,6-pentafluorocyclohexyl rings do not show any indication of monolayer assembly relative to their cyclohexane analogues, instead the molecules appear to aggregate and form higher molecular assemblies prior to compression. The study indicates the power and potential of this ring system as a motif for ordering supramolecular assembly., Theory and Langmuir isotherm analysis was used to explore the conformations and molecular packing of alkyl all-cis 2,3,4,5,6-pentafluorocyclohexyl motifs, which are prepared by direct aryl hydrogenations from alkyl- or vinyl-pentafluoroaryl benzenes.

Authors thank FAPESP, CONFAP and The UK Academies FAPESP for a São Paulo International Research Collaboration (FAPESP #2019/05028-7). FAPESP is also gratefully acknowledge for an undergraduate fellowship to BAP (#2019/03855-3), and a Young Research Award to RAC (#2018/03910-1). CENAPAD-SP, CESUP and SDumont are acknowledged for computational resources used in the theory calculations. We also thank EPSRC for a grant (EP/S030506/1). In this theory study we demonstrate the dominance of non-classical 1,3-diaxial CHax∙∙∙OC hydrogen bonds (NCHBs) dictating a ‘pseudo‘ anomeric effect in selectively fluorinated methoxycyclohexanes and also influencing the axial preference in the classical anomeric exhibitor 2-methoxytetrahydropyran, a phenomenon which is most often described as a consequence of hyperconjugation. Analogues of methoxycyclohexane where ring CH2’s are replaced by CF2 can switch to an axial preference and theory methods (NBO, QTAIM, NCI) indicate the dominance of 1,3- CHax∙∙∙OMe interactions over hyperconjugation. For 2-methoxytetrahydropyran, it is revealed that the global contribution to the anomeric effect is from electrostatic interactions including NCHBs, not hyperconjugation, although hyperconjugation (nO→σ *CO or nO →σ*CC) remains the main contributor to the exo-anomeric phenomenon. When two and three ether oxygens are introduced into the ring, then both the NCHB interactions and hyperconjugative contributions become weaker, not stronger as might have been anticipated, and the equatorial anomers progressively dominate. Postprint

M.M. thanks SULSA for a PhD studentship. We gratefully acknowledge financial support from the EPSRC (grant EP/I034793/1). We report the synthesis of five radiotracers incorporating different oxyamine spacers between the hypoxia‐reactive 2‐nitroimidazole moiety and the 5‐[18F]‐fluorodeoxyribose ([18F]FDR, 12 ) prosthetic group: three linear alkyl chains with 3, 5, 7 carbon atoms ( 15 a – c ), a cyclopropyl ring ( 15 d ) and a 1,4‐disubstituted‐1,2,3‐triazole ( 15 e ). Experiments in hypoxic cells showed that 15 d displays superior uptake kinetics – and similar selectivity for hypoxic cells – relative to the gold standard hypoxia tracers [18F]fluoroazomycin arabinoside ([18F]FAZA) and [18F]fluoromisonidazole ([18F]FMISO). Lipophilicity and structural rigidity have strong influence on the selectivity of tracers 15 towards hypoxic cells: the lead tracer 15 d displays a logP=0.38 and the most rigid spacer. A sixth radiotracer ( 15 f ), with a 2‐H‐imidazole replacing the 2‐nitroimidazole moiety of 15 d , was used to demonstrate that the cyclopropyl group does not play a meaningful role in the sensitivity towards hypoxia. Postprint

Fluorine is a key element in the synthesis of molecules broadly used in medicine, agriculture and materials. Addition of fluorine to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to integrate fluorometabolites into the biochemistry of living cells are scarce. In this work, synthetic gene circuits for organofluorine biosynthesis are implemented in the platform bacterium Pseudomonas putida. By harnessing fluoride-responsive riboswitches and the orthogonal T7 RNA polymerase, biochemical reactions needed for in vivo biofluorination are wired to the presence of fluoride (i.e. circumventing the need of feeding expensive additives). Biosynthesis of fluoronucleotides and fluorosugars in engineered P. putida is demonstrated with mineral fluoride both as only fluorine source (i.e. substrate of the pathway) and as inducer of the synthetic circuit. This approach expands the chemical landscape of cell factories by providing alternative biosynthetic strategies towards fluorinated building-blocks., Addition of fluorine to organic structures is a unique strategy for tuning molecular properties, but approaches to integrate fluorometabolites into the biochemistry of living cells are scarce. Here, the authors develop a fluoride-responsive genetic circuit to enable in vivo biofluorination in engineered Pseudomonas putida.

An unexpected and highly efficient reaction of dibenzo[a,e]cyclooctatetraene with Ag(i)F and N-bromosuccinamide in dichloromethane results in a sequential ring contraction pathway to generate bridged bicyclic α,α'-difluoroether isomers 10 and 11 where the ether oxygen is isotopically labelled from oxygen-18 water. In diethyl ether the same reaction generates the more classical vicinal difluoride isomers 13 and 14.

We thank the EPSRC for financial support. M. B. thanks the School of Chemistry and EaStCHEM for support. This study extends our interest in the synthesis and conformational behaviour of all-syn multivicinal fluoro alkane motifs. Specifically an all-syn 1,2,3,6,7,8-hexafluorooctane chain was assembled with a run of three fluorines, of the same stereochemical sense (syn) to the direction of the chain, on each side of an ethylene (–CH2CH2–) spacer to explore if the helical sense of the chain crosses the ethylene bridge. The solid state (X-ray) structure indicated a continuous helix however in solution (NMR) and by DFT computation, although the individual all-syn 1,2,3-trifluoro motifs maintain good helical integrity, the molecule is much more dynamic across the ethylene bridge. It was notable however that a low energy, non-helical conformer has a high molecular dipole (μ = 7.15 D) indicating a role for this skipped motif in soft materials such as liquid crystals or polar polymers. Postprint

Selectively fluorinated hydrocarbons continue to attract attention for tuning pharmacokinetic properties in agrochemical and pharmaceutical discovery programmes. This study identifies benzylic bromination of phenyl all-cis-2,3,5,6-tetrafluorocyclohexane 2 as a key reaction for accessing building blocks containing the all-cis-2,3,5,6-tetrafluorocyclohexane ring system. These cyclohexanes are of interest as the fluorines are only on one face of the cyclohexane, and this imparts an unusual polar aspect, very different to an otherwise hydrophobic cyclohexane. Ritter type reactions of benzyl bromide 4 with DMF and acetonitrile generated the corresponding benzyl alcohol 6 and benzylacetamide 7 respectively. Benzylacetamide 7 was hydrolysed to benzyl amine 8 and syn-amino-alcohol 9, and separately the phenyl ring was oxidatively cleaved to furnish carboxylic acid acetamide 10, which after hydrolysis gave the tetrafluorocyclohexyl amino acid 11. A trans-halogenation of benzylbromide 4 with AgF2 gave benzyl fluoride 13. Oxidative cleavage of the aryl ring then gave pentafluorocyclohexyl carboxylic acid 14. This carboxylic acid was readily converted to amides 23-26 and the preferred conformations of these α-fluoroamides were explored by DFT, X-ray structure and 1 H-19 F HOESY NMR analysis.

NK and DOH acknowledge support from the University of St Andrews, Engineering and Physical Sciences Research Council (EPSRC, Grant No.: EP/L017911/1), and the EPSRC UK National Mass Spectrometry Facility at Swansea University. This work was also supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the FRQNT Centre in Green Chemistry and Catalysis (CGCC), and the Université Laval. In recent years, the highly polar C-F bond has been utilised in activation chemistry despite its low reactivity to traditional nucleophiles, when compared to other C-X halogen bonds. Paquin's group has reported extensive studies on the C-F activation of benzylic fluorides for nucleophilic substitutions and Friedel-Crafts reactions, using a range of hydrogen bond donors such as water, triols or hexafluoroisopropanol (HFIP) as the activators. This study examines the stereointegrity of the C-F activation reaction through the use of an enantiopure isotopomer of benzyl fluoride to identify whether the reaction conditions favour a dissociative (SN1) or associative (SN2) pathway. [2H]-Isotopomer ratios in the reactions were assayed using the Courtieu 2H NMR method in a chiral liquid crystal (poly-γ-benzyl-L-glutamate) matrix and demonstrated that both associative and dissociative pathways operate to varying degrees, according to the nature of the nucleophile and the hydrogen bond donor. Publisher PDF

Meeting the increasing demand for vaccines throughout the world is key to decrease the spread of infectious diseases. The switch to a fully continuous vaccine manufacturing process would increase productivity and the supply of crucial vaccines. To aid in this switch, we have developed a novel, continuous downstream purification technique based on an aqueous two-phase system (ATPS). The system has the potential to be used as a platform system for viral product purification. A 12 kDa poly(ethylene glycol) (PEG) and trisodium citrate ATPS was able to purify porcine parvovirus (PPV) and human immunodeficiency virus type-1 group antigen virus-like particles (HIV VLPs) from cell supernatant. PPV was recovered in the PEG-rich phase at 90 ± 16% with a DNA removal of 96 ± 3% and ≥89% protein removal. The system was also able to recover 99 ± 2% of HIV VLPs in the PEG-rich phase with a 73 ± 1% DNA removal and high protein removal shown by SDS-PAGE. Continuous ATPS recovered virus at the same amount as batch recovery. This demonstrates that continuous ATPS can be scaled up and runrun continuously without a loss in purity or recovery. Mixing and settling time both played an important role in developing a continuous ATPS for viral particles.

The use of fluorine-18 as a radionuclide for positron emission tomography (PET) has become increasingly popular over the last two decades and cancer and neurology clinical centres worldwide are increasingly establishing competence in this modality for diagnostic imaging. Progress has been particularly impressive for small molecule pharmaceutical candidates and low molecular weight affinity peptides, where clearance rates of the peptides in the body are compatible with the half-life of fluorine-18 (t 1/2 = 110 min). However high molecular weight proteins present challenges as they circulate and clear much more slowly. This review focuses on the methods used to radiolabel antibodies and their derivatives with fluorine-18 as tracers for PET. The very high specificity of these biomolecules for disease indicators at the molecular level makes labelling them attractive, however antibodies can circulate for days within the blood, with slow clearance times due to their high molecular weights, and this is inconsistent with the relatively short half-life of fluorine-18. Thus, lower molecular weight fragments of antibodies present more realistic targets for labelling. This review describes the approaches and protocols which have been successfully used to radiolabel antibodies and particularly antibody fragments with fluorine-18, and highlights this challenging aspect of fluorine-18 labelling for PET imaging.

This paper reports the synthesis of three amine stereoisomers 5a–c of the tetrafluorocyclohexyl ring system, as building blocks for discovery chemistry programmes. The synthesis starts from a Birch reduction of benzonitrile, followed by an in situ methyl iodide quench. The resultant 2,5-cyclohexadiene was progressed via double epoxidations and then hydrofluorination ring opening reactions. The resultant fluorohydrin moieties were then converted to different stereoisomers of the tetrafluorocyclohexyl ring system, and then reductive hydrogenation of the nitrile delivered three amine stereoisomers. It proved necessary to place a methyl group on the cyclohexane ring in order to stabilise the compound against subsequent HF elimination. The two all-cis tetrafluorocyclohexyl isomers 5a and 5b constitute facially polarized cyclohexane rings, with fluorines on the electronegative face and hydrogens on the electropositive face.

This Review outlines the progression, primarily of our own work, but with important contributions from other labs, on the synthesis and properties of multiple vicinally fluorinated alkyl chains and rings. Chain conformations of individual diastereoisomers with -CHF- at adjacent carbons are influenced by stereoelectronic factors associated with the polar C-F bond and the polarised geminal hydrogens. Generally, the chain will prefer a conformation which acts to minimise overall molecular polarity, and where the C-F bonds orient away from each other. However, when vicinal fluorine atoms are positioned on a ring then conformations are more constrained. The ring will adopt optimal conformations such as a chair in cyclohexane and then C-F bonds can be introduced with a stereochemistry that forces parallel (axial) orientations. In the case of cyclohexane, 1,3-diaxial arrangements of C-F bonds impart considerable polarity to the ring, resulting in an electronegative 'fluorine face' and an electropositive 'hydrogen face'. For all-syn 1,2,3,4,5,6-hexafluorocyclohexane, this arrangement generates an unusually polar aliphatic ring system. Most recently the concept has been extended to the preparation of all-syn 1,2,3-trifluorocyclopropanes, a rigid ring system with fluorine atoms on one face and hydrogens on the other. Lipophilicity Log P values of such compounds indicate that they are significantly more polar than their parent alicyclic hydrocarbons and give some positive indication for a future role of such substituents in medicinal chemistry. Expanding to such a role will require access to improved synthesis methods to these motifs and consequently access to a broader a range of building blocks, however some exciting new methods have emerged recently and these are briefly reviewed.

Authors thank the EPSRC Catalysis HUB (University of Manchester) for financial support. We also thank Roger Howard, Usa Reilly, Jeffery Janso and Alessandra Eustaquio at the Biocatalysis Technologies Laboratory, Pfizer, Groton, USA, for financial support, a producing strain of S. calvus, and supporting the research with genome sequencing. The antibiotic nucleocidin is a product of the soil bacterium Streptomyces calvus T-3018. It is among the very rare fluorine containing natural products but is distinct from the other fluorometabolites in that it is not biosynthesised from 5′-fluorodeoxyadenosine via the fluorinase. It seems to have a unique enzymatic fluorination process. We disclose here the structures of two 4′-fluoro-3′-O-β-glucosylated metabolites (F-Mets I and II) which appear and then disappear before nucleocidin production in batch cultures of S. calvus. Full genome sequencing of S. calvus T-3018 and an analysis of the putative biosynthetic gene cluster for nucleocidin identified UDP-glucose dependent glucosyl transferase (nucGT) and glucosidase (nucGS) genes within the cluster. We demonstrate that these genes express enzymes that have the capacity to attach and remove glucose from the 3′-O-position of adenosine analogues. In the case of F-Met II, deglucosylation with the NucGS glucosidase generates nucleocidin suggesting a role in its biosynthesis. Gene knockouts of nucGT abolished nucelocidin production. Publisher PDF

We thank the EPSRC for financial support (GR: EP/R013799/1) and we are grateful to the Chinese Scholarship Council (CSC) for Ph.D Studentship support (ZF). The synthesis of substituted all-cis-1,2,3-trifluorocylopropanes are described for the first time. The three fluorines located on each of the cyclopropyl carbons with a stereochemistry where they are all on the same face of the cyclopropane, imparts a significant polarity to the molecule, and the inherent conformational rigidity and lowering of Log P makes this motif attractive for exploration as a substituent for pharmaceuticals and agrochemicals research. Postprint

We thank the Engineering and Physical Sciences Research Council, UK, for a research grant. The fluorinase enzyme from Streptomyces cattleya is shown to catalyse a direct displacement of bromide and iodide by fluoride ion from 5′-bromodeoxyadenosine (5′-BrDA) and 5′-iododeoxyadenosine (5′-IDA) respectively to form 5′-fluorodeoxyadenosine (5′-FDA) in the absence of L-methionine (L-Met) or S-adenosyl-L-methionine (SAM). 5′-BrDA is the most efficient substrate for this enzyme catalysed Finkelstein reaction. Postprint

We report the metabolism of the recently introduced α,α-difluoroethyl thioether motif to explore further its potential as a substituent for bioactives discovery chemistry. Incubation of two aryl–SCF2CH3 ethers with the model yeast organism Cunninghamella elegans, indicates that the sulfur of the thioether is rapidly converted to the corresponding sulfoxide, and then significantly more slowly to the sulfone. When the substrate was (p-OMe)PhSCF2CH3, then the resultant (demethylated) phenol sulfoxide had an enantiomeric excess of 60%, and when the substrate was the β-substituted-SCF2CH3 naphthalene, then the enantiomeric excess of the resultant sulfoxide was 54%. There was no evidence of defluorination, unlike the corresponding oxygen ether (p-OMe)PhOCF2CH3, which was converted to the (demethylated) phenol acetate ester during C. elegans incubation. We conclude that the aryl–S–CF2CH3 motif is metabolised in a similar manner to aryl–SCF3, a motif that is being widely explored in discovery chemistry. It is however, significantly less lipophilic than aryl-SCF3 which may offer a practical advantage in tuning overall pharmacokinetic profiles of molecules in development.

The metabolic relationship between littorine and hyoscyamine has been monitored in transformed roots of Datura stramonium. Quantification by GC of unlabelled littorine and by GCMS of 13C-labelled littorine demonstrated that exogenously added littorine (0.1 mM) was significantly metabolised (35%) to hyoscyamine. In contrast, exogenously added hyoscyamine was not metabolised to littorine, indicating that this conversion is irreversible. The conversion of littorine to hyoscyamine was suppressed by P-450 oxidase inhibitors (particularly clotrimazole), implicating the involvement, at least in part of a cytochrome P-450 activity operating hyoscyamine biosynthesis.

Clusters of all-cis 1,2,3,4,5,6-hexafluorocyclohexane and the dodecafluorododecaboron dianion, [C6F6H6]n[B12F12]2– (n = 0–4), are investigated in a combined experimental and computational study. DFT calculations and IRMPD spectra in the region of 800–2000 cm–1 indicate that C6H6F6 binds to open trigonal faces of B12F122– via a three-point interlocking binding motif. Calculated binding interactions reveal substantial contributions from C–H···F hydrogen bonding and binding energies that are among the strongest observed for a neutral-anion system.

This work was supported by the Engineering and Physical Sciences Research Council (EPSRC) and the European Research Council (ERC). The authors acknowledge the EPSRC National Mass Spectrometry Facility (Swansea). D.O’H. thanks the Royal Society for a Wolfson Research Merit Award. 2,2-Dimethyl-5-phenyl-1,1,3,3-tetrafluororocyclohexane has been prepared and characterised as an example of a facially polarised cyclohexane containing 1,3 related CF2 groups. The dipolar nature of the ring arises from the axial orientation of two of the C-F bonds pointing in the same direction, and set by the chair conformation of the cyclohexane. This electrostatic profile is revealed experimentally both in the solid-state (X-ray) packing of the rings and by solution (NMR) in different solvents. A computationally derived electrostatic profile of this compound is consistent with a more electronegative and a more electropositive face of the cyclohexane ring. This placing of CF2 groups 1,3 to each other in a cyclohexane ring is introduced as a new design strategy which could be applicable to the preparation of polar hydrophobic cyclohexane motifs. Publisher PDF

We thank the Engineering and Physical Sciences Research Council, UK, for a research grant. We report a last-step fluorinase-catalyzed [18F]-fluorination of a cysteine-containing RGD peptide. The peptide was attached through sulfur to a modified and more hydrophilic variant of the recently disclosed Barbas linker which was itself linked to a chloroadenosine moiety via a PEGylated chain. The fluorinase was able to use this construct as a substrate for a transhalogenation reaction to generate [18F]-radiolabeled RGD peptides, which retained high affinity to cancer-cell relevant αvβ3 integrins. Postprint

This work was supported by the Engineering and Physical Sciences Research Council (EPSRC) and the European Research Council (ERC). The authors acknowledge the EPSRC National Mass Spectrometry Facility (Swansea). M.Y. thanks the China Scholarship Council for financial support. D.O'H. thanks the Royal Society for a Wolfson Research Merit Award. The difluoromethylene (CF2) group has a strong tendency to adopt corner over edge locations in aliphatic macrocycles. In this study, the CF2 group has been introduced into musk relevant macrocyclic ketones. Nine civetone and five muscone analogues have been prepared by synthesis for structure and odour comparisons. X-ray studies indeed show that the CF2 groups influence ring structure and they give some insight into the preferred ring conformations, triggering a musk odour as determined in a professional perfumery environment. The historical conformational model of Bersuker and co-workers for musk fragrance generally holds, and structures that become distorted from this consensus, by the particular placement of the CF2 groups, lose their musk fragrance and become less pleasant. Postprint

The role of organo-fluorine compounds in modern health, food and energy related industries is widely-appreciated. The unique properties that fluorine imparts to organic molecules, stemming from its high electronegativity and stability when bound to carbon, finds it increasingly being used in the development of new bioactivities. Around 25 % of the current blockbuster drugs contain fluorine and this number is increasing to well above 30 % for recent FDA approvals. In this Review we highlight a selection of the most successful organo-fluorine drugs, that have achieved blockbuster status, namely, sitagliptin (diabetes), sofosbuvir (hepatitis C), emtricitabine (HIV), glecaprevir/pibrentasvir (hepatitis C), elvitegravir (HIV), dolutegravir (HIV), bictegravir (HIV), efavirenz (HIV), enzalutamide (prostate cancer), aubagio (immunomodulatory) and paliperidone palmitate (schizophrenia). For each compound we discuss their discovery, their relevant disease state and how they are made, emphasizing the source of fluorine-containing moieties, and where known, their mode of action.

Prostate cancer represents a major public health threat as it is one of the most common male cancers worldwide. The prostate-specific membrane antigen (PSMA) is highly over-expressed in prostatic cancer cells in a manner that correlates with both tumour stage and clinical outcome. As such, PSMA has been identified as an attractive target for both imaging and treatment of prostate cancer. In recent years the focus on urea-based peptidomimetic inhibitors of the PSMA (representing low molecular weight/high affinity binders) has intensified as they have found use in the clinical imaging of prostate tumours. Reported herein are the design, synthesis and evaluation of a new fluorinated PSMA targeting small-molecule, FDA-PEG-GUL, which possesses the Glu-NH-CO-NH-Lys pharmacophore conjugated to a 5'-fluorodeoxy- adenosine unit. Inhibition assays were performed with FDA-PEG-GUL which revealed that it inhibits the PSMA in the nanomolar range. Additionally, it has been purposely designed so that it can be produced using the fluorinase enzyme from its chlorinated precursor, allowing for the enzymatic synthesis of radiolabelled [F-18]FDA-PEG-GUL via a nucleophilic reaction that takes place in experimentally advantageous conditions (in water at neutral pH and at ambient temperature). Specific binding of [F-18]FDA-PEG-GUL to PSMA expressing cancer cells was demonstrated, validating it as a promising PSMA diagnostic tool. This work establishes a successful substrate scope expansion for the fluorinase and demonstrates its first application towards targeting the PSMA.

Fluorine has been used widely to tune the pharmacokinetic properties of bioactive compounds as they progress through development. Accordingly, selective fluorination has had a major impact in the pharmaceuticals and agrochemicals industries and therefore on human health and well being, though in this context it is notable that fluorine has no presence at all in the flavours and fragrance industries. There does not appear to be an obvious molecular basis for this exclusion. It may be that costs have become prohibitive in this market and it may also be that there are negative perceptions of safety for products that will have intimate contact with healthy individuals, though given the impresssive track record of fluorine to influence the properties of bioactives for health improvement it is a striking omission. It seemed timely therefore to bring to the surface the literature that does exist in this area, if for no other purpose than to begin to explore prospects. We extend the survey to literature in the related area of insect pheromone and semiochemical analogues, as such analogues have the lucrative potential to be used in the field in traps and in small quantities to monitor insect populations. On the face of it, it would appear that there are genuine prospects to develop improved molecules for these purposes using fluorine, however the area remains underdeveloped. In this review we collate those studies which have prepared fluorinated analogues of flavours, fragrance and pheromone molecules and, where known, we discuss their efficacy.

Authors thank the EPSRC (Grant EP/N03001X/1) for financial support. In this study, we probed the inhibition of pig heart citrate synthase (E.C. 4.1.3.7) by synthesising seven analogues either designed to mimic the proposed enolate intermediate in this enzyme reaction or developed from historical inhibitors. The most potent inhibitor was fluorovinyl thioether 9 (Ki=4.3 μm), in which a fluorine replaces the oxygen atom of the enolate. A comparison of the potency of 9 with that of its non‐fluorinated vinyl thioether analogue 10 (Ki=68.3 μm) revealed a clear “fluorine effect” favouring 9 by an order of magnitude. The dethia analogues of 9 and 10 proved to be poor inhibitors. A methyl sulfoxide analogue was a moderate inhibitor (Ki=11.1 μm), thus suggesting hydrogen bonding interactions in the enolate site. Finally, E and Z propenoate thioether isomers were explored as conformationally constrained carboxylates, but these were not inhibitors. All compounds were prepared by the synthesis of the appropriate pantetheinyl diol and then assembly of the coenzyme A structure according to a three‐enzyme biotransformation protocol. A quantum mechanical study, modelling both inhibitors 9 and 10 into the active site indicated short CF ⋅⋅⋅ H contacts of ≈2.0 Å, consistent with fluorine making two stabilising hydrogen bonds, and mimicking an enolate rather than an enol intermediate. Computation also indicated that binding of 9 to citrate synthase increases the basicity of a key aspartic acid carboxylate, which becomes protonated. Postprint

The authors are grateful to the EPSRC for supporting this work. The authors acknowledge the EPSRC National Mass Spectrometry Facility (Swansea) and D.O’H. thanks the Royal Society for a Wolfson Research Merit Award. A general route to aryl α,β,β-trifluorocyclopropanes is reported and aryl oxidation gave the corresponding α,β,β-trifluorocyclopropane carboxylic acid. Reactions of the corresponding amides with phenol/thiophenol resulted in HF elimination and then conjugate addition. The partially fluorinated cyclopropane has a similar lipophilicity to –CF3 despite three carbon atoms, and it emerges as a novel motif for drug discovery. Postprint Postprint

The use of radiolabelled antibodies and antibody-derived recombinant constructs has shown promise for both imaging and therapeutic use. In this context, the biotin-avidin/streptavidin pairing, along with the inverse-electron-demand Diels-Alder (iEDDA) reaction, have found application in pretargeting approaches for positron emission tomography (PET). This study reports the fluorinase-mediated transhalogenation [5'-chloro-5'-deoxyadenosine (ClDA) substrates to 5'-fluoro-5'-deoxyadenosine (FDA) products] of two antibody pretargeting tools, a FDA-PEG-tetrazine and a [18 F]FDA-PEG-biotin, and each is assessed either for its compatibility towards iEDDA ligation to trans-cyclooctene or for its affinity to avidin. A protocol to avoid radiolytically promoted oxidation of biotin during the synthesis of [18 F]FDA-PEG-biotin was developed. The study adds to the repertoire of conjugates for use in fluorinase-catalysed radiosynthesis for PET and shows that the fluorinase will accept a wide range of ClDA substrates tethered at C-2 of the adenine ring with a PEGylated cargo. The method is exceptional because the nucleophilic reaction with [18 F]fluoride takes place in water at neutral pH and at ambient temperature.

We acknowledge support from NSF (CHE-1265679) and NIH (5R01DC014423 subaward) (EB), NIH (5R01 DC014423) (HM), the European Reasearch Council (ERC) and the Engineering Science Research Council (EPSRC) (DO'H), FAPESP and CNPq (RAC), the Chinese Scholarship Council (CSC) for studentship support (MY), National Science Foundation (31070972) (HZ), Science and Technology Commission of Shanghai Municipality (16ZR1418300) (HZ), the Shanghai Eastern Scholar Program (J50201) (HZ). VSB thanks NIH grant 1R01GM106121-01A1 and computational time from NERSC. Understanding olfaction at the molecular level is challenging due to the lack of crystallographic models of odorant receptors (ORs). To better understand the molecular mechanism of OR activation, we focused on chiral (R)-muscone and other musk smelling odorants due to their great importance and widespread use in perfumery and traditional medicine, as well as environmental concerns associated with bioaccumulation of musks with estrogenic/antiestrogenic properties. We experimentally and computationally examined the activation of human receptors OR5AN1 and OR1A1, recently identified as specifically responding to musk compounds. OR5AN1 responds at nanomolar concentrations to musk ketone and robustly to macrocyclic sulfoxides and fluorine-substituted macrocyclic ketones; OR1A1 responds only to nitromusks. Structural models of OR5AN1 and OR1A1 based on quantum mechanics/molecular mechanics (QM/MM) hybrid methods were validated through direct comparisons with activation profiles from site-directed mutagenesis experiments and analysis of binding energies for 35 musk-related odorants. The experimentally found chiral selectivity of OR5AN1 to (R)- over (S)-muscone was also computationally confirmed for muscone and fluorinated (R)-muscone analogs. Structural models show that OR5AN1, highly responsive to nitromusks over macrocyclic musks, stabilizes odorants by hydrogen bonding to Tyr260 of transmembrane a-helix 6 and hydrophobic interactions with surrounding aromatic residues Phe105, Phe194 and, Phe207. The binding of OR1A1 to nitromusks is stabilized by hydrogen bonding to Tyr258 along with hydrophobic interactions with surrounding aromatic residues Tyr251 and Phe206. Hydrophobic/nonpolar and hydrogen bonding interactions contribute, respectively, 77% and 13% to the odorant binding affinities, as shown by an atom-based quantitative structure-activity relationship model. Postprint

The authors thank the Marie Curie ITN programme for Funding (AR). A method for the preparation of aryl α,α-difluoroethyl thioethers (ArSCF2CH3) is reported and the synthesis approach is extended to aryl alpha,alpha-difluoroethyl oxygen ethers. Selected building blocks are further elaborated in cross-coupling reactions and are incorporated into analogues of established trifluoromethyl ether drugs. Conformations are explored and log P studies of these motifs indicate that they are significantly more polar than their trifluoromethyl ether analogues rendering them attractive for bioactives discovery. Postprint Postprint

This work was supported by the Initial Training Network, FLUOR21, funded by the FP7 Marie Curie Actions of the European Commission (FP7-PEOPLE-2013-ITN-607787). JM is supported by a University Research Fellowship from the Royal Society. The research leading to these results has received funding from the European Research Council under the EU 7th Framework Programme (FP7/2007-2013)/ERC No 336289. The metabolism and polarity of the all-cis tetra-fluorocyclohexane motif is explored in the context of its potential as a motif for inclusion in drug discovery programmes. Biotransformations of phenyl all-cis tetra-, tri- and di- fluoro cyclohexanes with the human metabolism model organism Cunninghamella elegans illustrates various hydroxylated products, but limited to benzylic hydroxylation for the phenyl all-cis tetrafluorocyclohexyl ring system. Evaluation of the lipophilicities (Log P) indicate a significant and progressive increase in polarity with increasing fluorination on the cyclohexane ring system. Molecular dynamics simulations indicate that water associates much more closely with the hydrogen face of these Janus face cyclohexyl rings than the fluorine face owing to enhanced hydrogen bonding interactions with the polarised hydrogens and water. Publisher PDF

The authors thank the European Research Council for and Advanced Grant and DO’H acknowledges the Royal Society for a Wolfson Merit Award. This paper explores the synthesis of a series of prototype negative dielectric liquid crystalline (LC) compounds which contain fluorinated cyclohexane motifs. The series are analogues and differ only in that they contain between one to four fluorine atoms. The stereochemistry is such that all of the fluorines/C-F bonds are on one face of the cyclohexane ring. This follows from the recent recognition that cyclohexanes with fluorines orientated in one direction, perpendicular to the ring have a strong molecular dipole, a characteristic that might be an advantage in the design of negative dielectric materials. However it is found that the increased polarity, particularly with two or more oriented C-F bonds, leads to higher melting points and poorer solubility in test matrix formulations, relative to hydrocarbon liquid crystals. This arises due to electrostatic intermolecular interactions between the polarised cyclohexyl and aryl rings. The study highlights that in order to take advantage of these polar cyclohexane motifs for liquid crystal design, appropriate scaffolds are required which are free of aromatic rings and contain peripheral solubilising motifs. Postprint Postprint

The authors thank EPSRC for a grant. CNvH also thanks the Fluorine Division of the American Chemical Society for a Moissan Summer Undergraduate Fellowship. RAC thank FAPESP for a fellowship (#2015/00975-7). The CF2 group is incorporated into specific positions within the lactone ring of the natural musk lactone, (12R)-(+)-12-methyl-13-tridecanolide, a constituent of Angelica root oil, Angelica archangelica L. The approach is taken as it was anticipated that CF2 groups would dictate corner locations in the macrocycle and limit the conformational space available to the lactone. Three fluorine containing lactones are prepared by organic synthesis. One (8) has CF2 groups located at the C-6 and C-9 positions, another (9) with CF2 groups at the C-5 and C-9 positions, and a third (10) with a CF2 group at C-8. Two of the fluorine containing lactones (8 and 10) were sufficiently crystalline to obtain X-ray crystal structures which revealed that the CF2 groups do adopt corner locations. All three lactones were subject to computational analysis at the B3LYP-D3/6-311+G** level to assess the relative energies of different conformers. In all cases, the global minima and most of the lowest energy minima have squared /rectangular geometries and located the CF2 groups at the corners. The lowest energy structures for 8 and 10 closely approximated the observed X-ray structures, suggesting good convergence of theory and experiment in determining relevant low energy conformations. All three compounds retained a pleasant odour suggesting the rings retained sufficient conformational flexibility to access relevant olfactory conformations. Publisher PDF

MA thanks the Egyptian Government for a Scholarship under the Channel Scheme and we are grateful to the support of Professor L. Fathey of the University of Alexandria, Department of Chemistry, Egypt. DO'H thanks the Royal Society for a Wolfson Research Merit Award and we also thank the EPSRC Mass Spectrometry Service at Swansea University, UK for analyses. Palladium catalysed carbonylation reactions using the meta- and para-iodo derivatives of all-cis-3-phenyl-1,2,4,5-tetrafluorocyclohexane ( 4 ) are illustrated as the start point for a variety of functional group interconversions. The resultant benzaldehyde and benzoic acids offer novel building blocks for further derivatisation and facilitate the incorporation of the facially polarised all-cis-1,2,4,5-tetrafluorocyclohexane motif into more advanced molecular scaffolds. Publisher PDF

The authors thank EPSRC for supporting this work and DO’H is grateful for a Royal Society Wolfson Research Merit Award. A new method for the preparation of alpha-fluorovinyl thioethers is reported which involves the hydrofluorination of alkynyl sulfides with 3HF center dot Et3N, a process that requires Lewis acid activation using BF3 center dot Et2O and TiF4. The method gives access to a range of α-fluorovinyl thioethers, some in high stereoselectivity with the Z-isomer predominating over the E-isomer. The α-fluorovinyl thioether motif has prospects as a steric and electronic mimetic of thioester enols and enolates, important intermediates in enzymatic C-C bond forming reactions. The method opens access to appropriate analogues for investigations in this direction. Publisher PDF

Polar exploration: Recently, Linclau and co-workers disclosed a straightforward (19)F-NMR method for determining the log P values of fluorocarbons. The method is particularly useful for most polar compounds and provides a quantitative way to rationalize the more subtle stereoelectronic consequences of fluorine introduction.

The metabolism and polarity of the all-cis tetra-fluorocyclohexane motif is explored in the context of its potential as a motif for inclusion in drug discovery programmes., The metabolism and polarity of the all-cis tetra-fluorocyclohexane motif is explored in the context of its potential as a motif for inclusion in drug discovery programmes. Biotransformations of phenyl all-cis tetra-, tri- and di- fluoro cyclohexanes with the human metabolism model organism Cunninghamella elegans illustrates various hydroxylated products, but limited to benzylic hydroxylation for the phenyl all-cis tetrafluorocyclohexyl ring system. Evaluation of the lipophilicities (log P) indicates a significant and progressive increase in polarity with increasing fluorination on the cyclohexane ring system. Molecular dynamics simulations indicate that water associates much more closely with the hydrogen face of these Janus face cyclohexyl rings than the fluorine face owing to enhanced hydrogen bonding interactions with the polarised hydrogens and water.

The authors thank EPSRC and the Scottish Imaging Network (SINAPSE) for grants. DO’H thanks the Royal Society for a Wolfson Research Merit Award and ST is grateful to the John and Kathleen Watson Scholarship for financial support. A two-step radiolabelling protocol of a cancer relevant cRGD peptide is described where the fluorinase enzyme is used to catalyse a transhalogenation reaction to generate [18F]-5'-fluoro-5'-deoxy-2-ethynyladenosine, [18F]FDEA, followed by a ‘click’ reaction to an azide tethered cRGD peptide. This protocol offers an efficient radiolabelling of a biologically relevant peptide construct in water at pH 7.8, 37oC in 2 hours, that was metabolically stable in rats and retained high affinity for the αVβ3 integrin. Publisher PDF