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.

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.

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.

Enzymatic Fluorination of Biotin and Tetrazine Conjugates for Pretargeting Approaches to Positron Emission Tomography Imaging.

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.

A New Class of Fluorinated A2A Adenosine Receptor Agonist with Application to Last-Step Enzymatic [18 F]Fluorination for PET Imaging.

The A2A adenosine receptor belongs to a family of G-coupled protein receptors that have been subjected to extensive investigation over the last few decades. Due to their prominent role in the biological functions of the heart, lungs, CNS and brain, they have become a target for the treatment of illnesses ranging from cancer immunotherapy to Parkinson's disease. The imaging of such receptors by using positron emission tomography (PET) has also been of interest, potentially providing a valuable tool for analysing and diagnosing various myocardial and neurodegenerative disorders, as well as offering support to drug discovery trials. Reported herein are the design, synthesis and evaluation of two new 5'-fluorodeoxy-adenosine (FDA)-based receptor agonists (FDA-PP1 and FDA-PP2), each substituted at the C-2 position with a terminally functionalised ethynyl unit. The structures enable a synthesis of 18 F-labelled analogues by direct, last-step radiosynthesis from chlorinated precursors using the fluorinase enzyme (5'-fluoro-5'-deoxyadenosine synthase), which catalyses a transhalogenation reaction. This delivers a new class of A2A adenosine receptor agonist that can be directly radiolabelled for exploration in PET studies.

Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.

Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch-ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening.

Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species.

Ligand-dependent control of gene expression is essential for gene functional analysis, target validation, protein production, and metabolic engineering. However, the expression tools currently available are difficult to transfer between species and exhibit limited mechanistic diversity. Here we demonstrate how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules. Our novel riboswitch-ligand pairings are shown to regulate physiologically important genes required for bacterial motility in Escherichia coli and cell morphology in Bacillus subtilis. These findings are relevant for future gene function studies and antimicrobial target validation, while providing new modular and orthogonal regulatory components for deployment in synthetic biology regimes.

Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro.

The fluorinase enzyme represents the only biological mechanism capable of forming stable C-F bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (KM ) for S-adenosyl-l-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l-methionine affinity points to a long-range effect of hexamerization on substrate binding - likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.

A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida.

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.

Two 3'-O-ß-glucosylated nucleoside fluorometabolites related to nucleocidin in Streptomyces calvus.

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-ß-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.