Conformational Analysis Explores the Role of Electrostatic Nonclassical CF···HC Hydrogen Bonding Interactions in Selectively Halogenated Cyclohexanes.

The conformational equilibria of selectively halogenated cyclohexanes are explored both experimentally (VT-NMR) for 1,1,4,-trifluorocyclohexane 7 and by computational analysis (M06-2X/aug-cc-pVTZ level), with the latter approach extending to a wider range of more highly fluorinated cyclohexanes. Perhaps unexpectedly, 7ax is preferred over the 7eq conformation by ΔG = 1.06 kcal mol-1, contradicting the accepted norm for substituents on cyclohexanes. The axial preference is stronger again in 1,1,3,3,4,5,5,-heptafluorocyclohexane 9 (ΔG = 2.73 kcal mol-1) as the CF2 groups further polarize the isolated CH2 hydrogens. Theoretical decomposition of electrostatic and hyperconjugative effects by natural bond orbital analysis indicated that nonclassical hydrogen bonding (NCHB) between the C-4 fluorine and the diaxial hydrogens at C-2 and C-6 in cyclohexane 7 and 9 largely accounts for the observed bias. The study extended to changing fluorine (F) for chlorine (Cl) and bromine (Br) at the pseudoanomeric position in the cyclohexanes. Although these halogens do not become involved in NCHBs, they polarize the geminal -CHX- hydrogen at the pseudoanomeric position to a greater extent than fluorine, and consequent electrostatic interactions influence conformer stabilities.

4'-Fluoro-nucleosides and nucleotides: from nucleocidin to an emerging class of therapeutics.

The history and development of 4'-fluoro-nucleosides is discussed in this review. This is a class of nucleosides which have their origin in the discovery of the rare fluorine containing natural product nucleocidin. Nucleocidin contains a fluorine atom located at the 4'-position of its ribose ring. From its early isolation as an unexpected natural product, to its total synthesis and bioactivity assessment, nucleocidin has played a role in inspiring the exploration of 4'-fluoro-nucleosides as a privileged motif for nucleoside-based therapeutics.

Identification of Genes Essential for Fluorination and Sulfamylation within the Nucleocidin Gene Clusters of Streptomyces calvus and Streptomyces virens.

The gene cluster in Streptomyces calvus associated with the biosynthesis of the fluoro- and sulfamyl-metabolite nucleocidin was interrogated by systematic gene knockouts. Out of the 26 gene deletions, most did not affect fluorometabolite production, nine abolished sulfamylation but not fluorination, and three precluded fluorination, but had no effect on sulfamylation. In addition to nucI, nucG, nucJ, nucK, nucL, nucN, nucO, nucQ and nucP, we identified two genes (nucW, nucA), belonging to a phosphoadenosine phosphosulfate (PAPS) gene cluster, as required for sulfamyl assembly. Three genes (orf(-3), orf2 and orf3) were found to be essential for fluorination, although the activities of their protein products are unknown. These genes as well as nucK, nucN, nucO and nucPNP, whose knockouts produced results differing from those described in a recent report, were also deleted in Streptomyces virens - with confirmatory outcomes. This genetic profile should inform biochemistry aimed at uncovering the enzymology behind nucleocidin biosynthesis.

The Emergence and Properties of Selectively Fluorinated 'Janus' Cyclohexanes.

This account describes the evolution of a research programme that started by linking fluoromethylene (-CHF-) groups along aliphatic chains and then progressing to alicyclic rings with contiguous fluorine atoms. Different stereoisomers of aliphatic chains tend to adopt low polarity conformations. In order to force polar conformations, the programme began to address ring systems and in particular cyclohexanes, to restrain conformational freedom and co-aligned C-F bonds. The flagship molecule, all-cis-1,2,3,4,5,6-hexafluorocyclohexane 7, emerged to be the most polar aliphatic compound recorded. The polarity arises because there are three co-aligned triaxial C-F bonds and the six fluorines occupy one face of the ring. Conversely the electropositive hydrogens occupy the other face. These have been termed Janus face cyclohexanes after the Roman god with two faces. The review outlines progress by our group and others in preparing derivatives of the parent cyclohexane 7, in order to explore properties and potential applications of these Janus cyclohexanes.

Contribution of Hyperconjugation and Inductive Effects to the Pseudo-anomeric Effect in 4-Substituted Methoxycyclohexanes.

The importance of electrostatic nonconventional hydrogen bonds (NCHBs) to the pseudo-anomeric effect of 4-substituted methoxycyclohexanes is evaluated using theory [natural bond orbital (NBO)] to deconvolute electrostatic from other contributing effects. There is an interesting interplay between σCH → σCX* hyperconjugation and the electropositive charge on 3,5-axial hydrogens (Hax). In essence, better σCX* (or πCO*) acceptors increase the charge on 3,5-CHax, which in turn strengthens Cδ+Hax···Î´-OMe NCHB interactions.

3'-O-ß-Glucosyl-4',5'-didehydro-5'-deoxyadenosine Is a Natural Product of the Nucleocidin Producers Streptomyces virens and Streptomyces calvus.

3'-O-ß-Glucosyl-4',5'-didehydro-5'-deoxyadenosine 13 is identified as a natural product of Streptomyces calvus and Streptomyces virens. It is also generated in vitro by direct ß-glucosylation of 4',5'-didehydro-5'-deoxyadenosine 12 with the enzyme NucGT. The intact incorporation of oxygen-18 and deuterium isotopes from (±)[1-18O,1-2H2]-glycerol 14 into C-5' of nucleocidin 1 and its related metabolites precludes 3'-O-ß-glucosyl-4',5'-didehydro-5'-deoxyadenosine 13 as a biosynthetic precursor to nucleocidin 1.

Janus All-Cis 2,3,4,5,6-Pentafluorocyclohexyl Building Blocks Applied to Medicinal Chemistry and Bioactives Discovery Chemistry.

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.

Osmolyte enhanced aqueous two-phase system for virus purification.

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.

Streptomyces aureorectus DSM 41692 and Streptomyces virens DSM 41465 are producers of the antibiotic nucleocidin and 4'-fluoroadenosine is identified as a co-product.

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-ß-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.

The contribution of non-classical CHaxOC hydrogen bonds to the anomeric effect in fluoro and oxa-methoxycyclohexanes.

In this theory study we demonstrate the dominance of non-classical 1,3-diaxial CHaxOC 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-CHaxOMe 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.

Molecular mechanism of activation of human musk receptors OR5AN1 and OR1A1 by (R)-muscone and diverse other musk-smelling compounds.

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 α-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.

An Engineered E.†coli Strain for Direct in Vivo Fluorination.

Selectively fluorinated compounds are found frequently in pharmaceutical and agrochemical products where currently 25-30 % of optimised compounds emerge from development containing at least one fluorine atom. There are many methods for the site-specific introduction of fluorine, but all are chemical and they often use environmentally challenging reagents. Biochemical processes for C-F bond formation are attractive, but they are extremely rare. In this work, the fluorinase enzyme, originally identified from the actinomycete bacterium Streptomyces cattleya, is engineered into Escherichia coli in such a manner that the organism is able to produce 5'-fluorodeoxyadenosine (5'-FDA) from S-adenosyl-l-methionine (SAM) and fluoride in live E. coli cells. Success required the introduction of a SAM transporter and deletion of the endogenous fluoride efflux capacity in order to generate an E. coli host that has the potential for future engineering of more elaborate fluorometabolites.

Polar Organofluorine Substituents: Multivicinal Fluorines on Alkyl Chains and Alicyclic Rings.

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.

Fluorine-Induced Pseudo-Anomeric Effects in Methoxycyclohexanes through Electrostatic 1,3-Diaxial Interactions.

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 (19 F{1 H}-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.

Probing the helical integrity of multivicinal all-syn-fluoro alkanes.

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.

Janus Face All-cis 1,2,4,5-tetrakis(trifluoromethyl)- and All-cis 1,2,3,4,5,6-hexakis(trifluoromethyl)- Cyclohexanes.

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.

Synthesis of organic liquid crystals containing selectively fluorinated cyclopropanes.

This paper describes the synthesis of a series of organic liquid crystals (LCs) 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.

Acetyl Coenzyme†A Analogues as Rationally Designed Inhibitors of Citrate Synthase.

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.

An enzymatic Finkelstein reaction: fluorinase catalyses direct halogen exchange.

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.

Enzymatic radiosynthesis of a 18F-Glu-Ureido-Lys ligand for the prostate-specific membrane antigen (PSMA).

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 [18F]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 [18F]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.