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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.
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.
Abstract
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.
The antibiotic nucleocidin is a product of the soil bacterium
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
the fluorinase. It seems to have a unique enzymatic fluorination process. We disclose here the structures of two 4'-fluoro-3'-
-β-glucosylated metabolites (F-Mets I and II) which appear and then disappear before nucleocidin production in batch cultures of
. Full genome sequencing of
T-3018 and an analysis of the putative biosynthetic gene cluster for nucleocidin identified UDP-glucose dependent glucosyl transferase (
) and glucosidase (n
) genes within the cluster. We demonstrate that these genes express enzymes that have the capacity to attach and remove glucose from the 3'-
-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
abolished nucelocidin production.
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.
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.
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.
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.
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