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Fluorine biocatalysis
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Fluorine biocatalysis Linrui Wu1,a, Fleurdeliz Maglangit1,2,a and Hai Deng1 Abstract
The introduction of fluorine atoms into organic molecules has received considerable attention as these organofluorines have often found widespread applications in bioorganic chemistry, medicinal chemistry and biomaterial science. Despite innovation of synthetic C–F forming methodologies, selective fluorination is still extremely challenging. Therefore, a biotransformation approach using fluorine biocatalysts is needed to selectively introduce fluorine into structurally diverse molecules. Yet, there are few ways that enable incorporation of fluorine into structurally complex bioactive molecules. One is to extend the substrate scope of the existing enzyme inventory. Another is to expand the biosynthetic pathways to accept fluorinated precursors for producing fluorinated bioactive molecules. Finally, an understanding of the physiological roles of fluorometabolites in the producing microorganisms will advance our ability to engineer a microorganism to produce novel fluorinated commodities. Here, we review the fluorinase biotechnology and fluorine biocatalysts that incorporate fluorine motifs to generate fluorinated molecules, and highlight areas for future developments. Addresses 1 Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen AB24 3UE, Scotland, UK 2 College of Science, University of the Philippines Cebu, Lahug, Cebu City, 6000, Philippines Corresponding author: Deng, Hai ([email protected]) These authors contribute equally.
a
Current Opinion in Chemical Biology 2020, 55:119–126 This review comes from a themed issue on Biocatalysis and biotransformation Edited by Jason Micklefield and Dominic J. Campopiano For a complete overview see the Issue and the Editorial
https://doi.org/10.1016/j.cbpa.2020.01.004 1367-5931/© 2020 Elsevier Ltd. All rights reserved.
Introduction Fluorine has emerged as a privileged element in medicinal chemistry, as well as in agrochemistry and materials science [1]. Fluorine substitutions are now considered a standard strategy for modulating the properties of chemical leads [2]. Although many chemical methods of selective synthesis of organofluorines have been www.sciencedirect.com
developed, it is attractive to consider biotransformation approaches to selectively access novel organofluorine chemicals, rather than using challenging chemical methods. This review focuses on two main approaches of fluorine biotransformation strategies: firstly to determine the substrate promiscuity of the fluorination enzymes, fluorinases, and secondly to modify biosynthetic pathways to accept fluorinated building blocks for precursordirected biosynthesis [3e6]. Particular emphasis is placed on exploring the existing enzyme inventory for new biofluorine chemistries. Furthermore, a good understanding of the physiological roles and metabolic fates of the fluorometabolites will certainly advance our abilities to engineer a suitable biotechnologically viable microbial host to generate novel organofluorines by fermentation strategies.
Extending the substrate scope of fluorination enzymes The hallmark of the fluorine biocatalysis research is the discovery of the first native fluorination enzyme, fluorinase, from Streptomyces cattleya, that catalyses CeF bond formation between fluoride ion and S-adenosyl0 0 L-methionine (SAM) 2 to generate 5 -fluoro-5 -deoxy0 adenosine (5 -FDA) 3 and L-methionine (L-Met) [7] (Figure 1a). Nature hardly finds a way for fluorine biochemistry, mainly due to the highest heat hydration of fluoride in water which makes fluoride a poor nucleophile. The fluorinase has evolved a unique desolvation strategy of the SN2 reaction [7e9]. During the turnover of the enzyme, fluoride binds first in the active site of the fluorinase by partial desolvation through hydrogen bonding exchanges [10e12]. SAM 2, which has 1000fold higher affinity than fluoride, then binds into the active site and during that progress drives full desolvation. The electrostatic interactions between the desolvated fluoride and the electropositive 50 C of the positively charged sulfonium species of 2 in the prereaction complex further maintain the fluoride as a good nucleophile, which finally progresses to a nucleophilic attack and CeF bond formation (Figure 1a) [10e12]. Since the seminal work on this fluorinase [3], a few fluorinase homologues from actinomycete organisms have been identified and biochemically characterised [13e15]. The fluorinases also catalyse the two-step transhalogenation reaction from 50 -chloro-50 -deoxyadenosine (50 -ClDA) 1 and L-Met to SAM 2, followed by the Current Opinion in Chemical Biology 2020, 55:119–126
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Figure 1
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De novo fluorination catalysed by fluorinase (a) The mechanism of fluorinase-mediated reaction with a representative of the fluoride/SAM 2 prereaction complex at the active site of the fluorinase together with selected hydrogen bonds among the key residues in the active site, fluoride and the ribose of 2. (b) Directed evolution provided two fluorinase variants with modest rate improvement in the first half of the two-step fluorinase-mediated transhalogenation reaction; (c) fluorinase-mediated biotransformation generated a series of 18F radiolabelling of cancer relevant targeting peptides and A2A adenosine receptor agonists for PET.
conversion of SAM 2 to 50 -FDA 3 in the presence of fluoride ion [16]. The substrate scope of this two-step transhalogenation reaction was further extended to the 2-deoxy analogue of 50 -ClDA 1 [16] and 50 -chloro-50 deoxy-2-ethynyladenosine (50 -ClDEA) 1a derivatives (Figure 1b) [17]. In an effort to explore the substrate flexibility and optimise the two-step transhalogenation, the fluorinase was subjected to directed evolution, allowing the identification of two fluorinase variants with the modest rate improvement for the first half reaction of the transhalogenation from 50 -ClDA 1 to SAM 2 [18,19]. Interestingly, modifications at F213 or A279 residues in fluorinase are also beneficial towards this transhalogenation even when using modified substrates, such as 50 -ClDEA (410-fold activity improvement) to produce 50 -fluorodeoxy-2-ethynyladenosine (50 -FDEA) [19]. During this transhalogenation, SAM synthesis is the ratelimiting step. To improve the overall transhalogenation Current Opinion in Chemical Biology 2020, 55:119–126
efficiency, a coupled chlorinaseefluorinase system was developed. In this system, the chlorinase is responsible for efficient conversion from 5-ClDA 1 to SAM 2, whereas the fluorinase is to convert SAM 2 to 50 -FDA 3 in the presence of fluoride ion [20]. More recently, the fluorinase was found to catalyse a one-step enzymatic Finkelstein reaction of 50 -bromodeoxyadenosine (50 BrDA) to 50 FDA 3 without the addition of SAM 2 or LMet (Figure 1b), offering a prospect of a simplified onestep fluorinase biotechnology for positron emission tomography (PET) [21].
Fluorinase method for use in biomedical applications PET is a highly sensitive, quantitative, noninvasive imaging technique. The 18F-radionuclide is often preferred for PET imaging because of its favourable half-life (109.8 min), high-resolution PET images. Traditional www.sciencedirect.com
Fluorine biocatalysis Wu et al.
labelling methods of the synthesis of 18F-labelled PET tracers usually demand multiple steps and/or harsh reaction conditions such as heating at high temperatures in strict anhydrous conditions because of the limited reactivity of the fluoride anion as a nucleophile in aqueous solution [22]. Fluorinase-mediated transhalogenation has emerged as a useful late-stage strategy for PET probe synthesis (Figure 1c). The fluorinase tolerates the acetylene functionality at the C-2 position of the adenine ring in the two-step transhalogenation reaction, leading to the conversion of 50 -ClDEA 1a to 50 -FDEA 3a [23]. Such an approach has been extended to generate a series of the 18 F-radiolabelling of cancer relevant targeting peptides [23e25] and A2A adenosine receptor agonists [26] for PET imaging applications, under experimentally mild conditions (Figure 1c).
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Biocatalysts that generate fluorinated building blocks There are few biocatalytic transformations for production of fluorine-containing molecules. Aldolases have emerged as a useful class of catalysts for controlling the formation of fluorine-containing stereocentres. Two types of aldolases have been explored for the synthesis of a range of chiral fluorinated building blocks (Figure 2). These aldolase-catalysed reactions often involve fluoropyruvate 5 as a nucleophile to lead stereoselective carbonecarbon bond formation enabling the synthesis of many a-fluoro-b-hydroxy molecules. Type I aldolases use a catalytic lysine residue to form a Schiff base with donor carbonyl-containing substrates enabling the controlled installation of fluorine-bearing stereocentres which has been reviewed recently [6]. Type II aldolases are metal dependent and have received much less attention in the research community in the past.
Figure 2
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(a) A type II aldolase-catalysed chemoenzymatic platform enabled the synthesis of more than twenty fluorinated acids, esters, amino acids and sugars involving fluoropyruvate 5 as a nucleophile. (b) A chemoenzymatic platform for synthesis of chiral a-trifluoromethylated organoborons 12 based on Rhodothermus marinus cytochrome c (Rma cyt c) mutant generated through directed evolution. www.sciencedirect.com
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Recently, type II 4-hydroxy-2-oxo-heptane-1,7-dioate aldolases (HpcH) have been explored as a chemoenzymatic platform to synthesise a series of a-fluoro-bhydroxy acids and esters with high stereopurity [27]. It was also demonstrated that the biotransformation platform displayed a good degree of flexibility further using these fluorinated acids and esters to generate a range of fluorinated analogues of amino acids, sugars and other valuable compounds by coupling with various transaminases and dehydrogenases (Figure 2a). Another new development of fluorine biocatalytic transformation is to introduce nonnatural chemistries into existing biocatalysts. Chiral a-trifluoromethylated (a-CF3) organoborons 12 are valuable synthetic building blocks that can be converted to a broad range of CF3containing products (Figure 2b). Cytochrome c (Rma cyt c), a P450 enzyme from Rhodothermus marinu, is a highly versatile platform for developing new chemistries of carbene transfer reactions [28,29]. Huang et al. [30]
further harnessed the versatility of this enzyme through directed evolution to develop a new enzymatic carbene transformation platform. The resulting variant, denoted as BOR-CF3, was able to accommodate structurally diverse trifluorodiazo alkanes 11 for highly enantioselective carbene BeH insertion reaction to provide chiral a- CF3 organoborons 12 with high total turnovers (Figure 2b).
Biotransformations that incorporate fluorinated building blocks into structurally complex organofluorines Polyketide metabolites contribute many of the important antibiotics used clinically, and these natural products are synthesised by multidomain polyketide synthase (PKS) complexes [31]. In an effort to engineer fluorinated building blocks into structurally complex molecules, an assembly module from the erythromycin polyketide pathway was used to
Figure 3
(a) The incorporation of fluorine atom using fluoromalonyl-CoA 14 as building blocks in mutated erythromycin polyketide assembly modules coupled with a trans-acting AT enzyme; (b) The in vivo biosynthesis of fluorinated building block, 2-fluoro-R-3-hydroxybutyrate (FHB) 20 and fluorinated poly(hydroxyalkanoate) polymers 21 from fluoromalonates 13.
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accommodate fluorinated extender units fluoromalonates 13 to introduce fluorine into the backbone of polyketides, albeit the low efficiency of chain extension with fluoromalonyl-CoA compared with the native substrate [32]. This is because the methylmalonyl-CoA 15 selective acyltransferase (AT) domain in the erythromycin PKS catalyses the hydrolysis of fluoromalonyl-CoA 14. It would be desirable to improve the efficient incorporation of fluoromalonyl-CoA 14 in the PKS assembly line. In this regard, replacement of a standalone trans-acting AT from the disorazole biosynthetic cluster to complement the deactivated cisAT domains of this erythromycin PKS module resulted in increased enzyme acylation with the fluoromalonyl CoA 14 extender unit in the presence of two other building blocks, 15 and propionyl-SNAC substrate mimic 16, thereby considerably improving the yield of monofluorinated or multifluorinated triketides 17e19 in vitro (Figure 3a) [33]. It is an exciting prospect to engineer the biosynthetic machinery of the cell to use simple fluorinated building blocks to produce new complex organofluorine molecules. More recently, the same group reported efficient production of the fluorinated diketide, 2-fluoro-3-hydroxybutyrate (FHB) 20 using fluoromalonate 13 as the feedstock in an engineered Escherichia coli (Figure 3b). Furthermore, the diketide was used as a monomer in vivo to generate fluorinated poly(hydroxyalkanoate) bioplastics 21 with fluorinated substitutions ranging from up to 15% (Figure 3b) [34].
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Understanding organofluorine physiology in fluorometabolisms It is attractive to consider engineering a microbial strain suitable for production of fluorinated products by fermentation. Such a strain would require a fluorinase gene, as well as its associated biosynthetic and regulatory genes. This strategy was partially achieved. Replacement of the chlorinase gene salL with flA gene in the biosynthetic gene cluster (BGC) of salinosporamide A, the chlorinated phase II anticancer metabolite, induced the production of fluorinated salinosporamide A when fluoride was added at the late stage of the fermentation [35]. However, problems emerged. The Salinispora tropica variant is extremely sensitive towards fluoride ion in culture, causing difficulty of scale-up fermentation [29]. Thus, there is considerable interest in understanding the toxic mitigation mechanisms of fluoride and fluorinated metabolites in the fluorometabolite producers [36,37]. It was found that S. cattleya evolves to manage the toxicity of fluoroacetate 24 and 4-fluorothreonine 25 by recruiting two specialised enzymes, fluoroacetyl-CoA hydrolase, FlK and a trans-acting fluorothreonyl-tRNA deacylase, FthB, to counteract the potential cellular toxicity of these two fluorometabolites, respectively. In the case of fluoroacetate 24, FlK displays high preference of hydrolysis towards fluoroacetyl-CoA 24a, the in vivo intermediate of fluoroacetate 24 as the lethal toxic to S. cattleya, due to the specific fluorine recognition with a hydrophobic residue in the binding site (Figure 4a) [38]. FthB, on the other hand, counteracts the misacylation of fluorothreonyl-
Figure 4
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(a) FlK, the specialised fluoroacetyl-CoA hydrolase, catalyses an entropy-driven hydrolysis of fluoroacetyl-CoA 24a, the toxic intermediate, to fluoroacetate 24 as part of toxicity mitigation strategy in vivo. (b) FthB, the specialised trans-acting fluorothreonyl-tRNA deacylase, counteracts the misacylation of fluorothreonyl-tRNA which otherwise would be misincorporated into protein in place of L-threonine. (c) Nucleocidin 26 consists of a unique tertiary fluorine at C-40 of the ribose ring. Recent investigations suggested that the BGC of 26 contains a new type of fluorination enzyme. Isotopic labelled moieties in glycerol highlighted in blue were demonstrated to be incorporated into nucleocidin 26.
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tRNA which otherwise would be misincorporated into protein in place of L-threonine (Figure 4b) [39]. These findings provide a deeper understanding of how microorganisms can evolve their own biosynthetic capacities by providing new chemistries to the BGCs. If a biotechnologically useful organism is to be engineered, such knowledge of fluorine toxic mitigation strategies will be useful.
Faculty, Reps and Staff Development Program (FRAS DP) PhD grant fellowship.
References Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Outlook/perspective Much is still needed to expand the scope of fluorine biocatalysts for organofluorine synthesis. It is exciting to fully use the existing enzyme inventory to accommodate new chemistries for bioactive organofluorines. As a complement to this, we should also turn our attention to nature for inspirations by discovering new fluorinated entities and new fluorometabolite pathways/enzymes. In this regard, studies of the various fluorometabolic pathways in the soil bacterium, Streptomyces sp MA37, could provide new strategies of organofluorine biotransformation because of its unusual capacity of producing a range of unknown fluorometabolites [40]. Another exciting development is to discover new CeF bond forming enzymes. Nucleocidin 26 is the only known fluorometabolite that possesses a tertiary fluorine at C-40 of the ribose ring, structurally different to other fluorometabolites containing a fluoromethyl group. Identification of the nucleocidin BGCs in the producing strains revealed that there is no known fluorinase encoded in microbial genomes [41,42]. Indeed, isotopic labelling experiments traced an oxidation occurring at C50 of nucleocidin after ribose ring assembly during the biosynthesis of 26 (Figure 4c) [43,44], significantly different to what was observed in the isotopic labelling studies of fluoroacetate 24 and 4-fluorothreonine 25 in S. ´ et al [43] idencattleya [45]. More recently, Bartholome tified two 40 -fluoro-30 -O-b-glucosylated metabolites, FMets I 27 and II 28, which were likely to be the last intermediates in the 26 biosynthesis (Figure 4c). It was also demonstrated that the UDP-glucose dependent glucosyl transferase (NucGT) and glucosidase (NucGS) encoded in the 26 BGC are responsible for attaching and removing glucose from the 30 -O-position of adenosine analogues (Figure 4c) [46]. Efforts directed towards greater understanding of the biosynthesis of nucleocidin 26 will certainly offer a promising opportunity of discovering a novel fluorination strategy.
Conflict of interest statement Nothing declared.
Acknowledgements HD is grateful for the financial supports through Biotechnology and Biological Sciences Research Council (BBSRC) grants (BB/P00380X/1 and BB/ R00479X/1) and Leverhulme Trust (RPG- 2014-418). HD and LW thank Industrial Biotechnology Innovation Centre (IBioIC) for the PhD studentship (LW). FM thanks the University of the Philippines for the Current Opinion in Chemical Biology 2020, 55:119–126
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46. Feng X, Bello D, Lowe P, Clark J, O’Hagan D: Two 3’-O-b glucosylated nucleoside fluorometabolites related to nucleocidin in Streptomyces calvus. Chem Sci 2019, 10:9501–9505. The authors disclosed the structures of two new 40 -fluoro-30 -O-bglucosylated fluorometabolites in the culture broth of Streptomyces calvus, the producer of nucleocidin and identified two key enzymes encoded in the nucleocidin BGC, UDP-glucose dependent glucosyl transferase and glucosidase, that have capacity to attach and remove glucose motif from the 30 -O-position of adenosin analogues, respectively.
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Fluorine biocatalysis Linrui Wu1,a, Fleurdeliz Maglangit1,2,a and Hai Deng1 Abstract
The introduction of fluorine atoms into organic molecules has received considerable attention as these organofluorines have often found widespread applications in bioorganic chemistry, medicinal chemistry and biomaterial science. Despite innovation of synthetic C–F forming methodologies, selective fluorination is still extremely challenging. Therefore, a biotransformation approach using fluorine biocatalysts is needed to selectively introduce fluorine into structurally diverse molecules. Yet, there are few ways that enable incorporation of fluorine into structurally complex bioactive molecules. One is to extend the substrate scope of the existing enzyme inventory. Another is to expand the biosynthetic pathways to accept fluorinated precursors for producing fluorinated bioactive molecules. Finally, an understanding of the physiological roles of fluorometabolites in the producing microorganisms will advance our ability to engineer a microorganism to produce novel fluorinated commodities. Here, we review the fluorinase biotechnology and fluorine biocatalysts that incorporate fluorine motifs to generate fluorinated molecules, and highlight areas for future developments. Addresses 1 Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen AB24 3UE, Scotland, UK 2 College of Science, University of the Philippines Cebu, Lahug, Cebu City, 6000, Philippines Corresponding author: Deng, Hai ([email protected]) These authors contribute equally.
a
Current Opinion in Chemical Biology 2020, 55:119–126 This review comes from a themed issue on Biocatalysis and biotransformation Edited by Jason Micklefield and Dominic J. Campopiano For a complete overview see the Issue and the Editorial
https://doi.org/10.1016/j.cbpa.2020.01.004 1367-5931/© 2020 Elsevier Ltd. All rights reserved.
Introduction Fluorine has emerged as a privileged element in medicinal chemistry, as well as in agrochemistry and materials science [1]. Fluorine substitutions are now considered a standard strategy for modulating the properties of chemical leads [2]. Although many chemical methods of selective synthesis of organofluorines have been www.sciencedirect.com
developed, it is attractive to consider biotransformation approaches to selectively access novel organofluorine chemicals, rather than using challenging chemical methods. This review focuses on two main approaches of fluorine biotransformation strategies: firstly to determine the substrate promiscuity of the fluorination enzymes, fluorinases, and secondly to modify biosynthetic pathways to accept fluorinated building blocks for precursordirected biosynthesis [3e6]. Particular emphasis is placed on exploring the existing enzyme inventory for new biofluorine chemistries. Furthermore, a good understanding of the physiological roles and metabolic fates of the fluorometabolites will certainly advance our abilities to engineer a suitable biotechnologically viable microbial host to generate novel organofluorines by fermentation strategies.
Extending the substrate scope of fluorination enzymes The hallmark of the fluorine biocatalysis research is the discovery of the first native fluorination enzyme, fluorinase, from Streptomyces cattleya, that catalyses CeF bond formation between fluoride ion and S-adenosyl0 0 L-methionine (SAM) 2 to generate 5 -fluoro-5 -deoxy0 adenosine (5 -FDA) 3 and L-methionine (L-Met) [7] (Figure 1a). Nature hardly finds a way for fluorine biochemistry, mainly due to the highest heat hydration of fluoride in water which makes fluoride a poor nucleophile. The fluorinase has evolved a unique desolvation strategy of the SN2 reaction [7e9]. During the turnover of the enzyme, fluoride binds first in the active site of the fluorinase by partial desolvation through hydrogen bonding exchanges [10e12]. SAM 2, which has 1000fold higher affinity than fluoride, then binds into the active site and during that progress drives full desolvation. The electrostatic interactions between the desolvated fluoride and the electropositive 50 C of the positively charged sulfonium species of 2 in the prereaction complex further maintain the fluoride as a good nucleophile, which finally progresses to a nucleophilic attack and CeF bond formation (Figure 1a) [10e12]. Since the seminal work on this fluorinase [3], a few fluorinase homologues from actinomycete organisms have been identified and biochemically characterised [13e15]. The fluorinases also catalyse the two-step transhalogenation reaction from 50 -chloro-50 -deoxyadenosine (50 -ClDA) 1 and L-Met to SAM 2, followed by the Current Opinion in Chemical Biology 2020, 55:119–126
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Figure 1
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De novo fluorination catalysed by fluorinase (a) The mechanism of fluorinase-mediated reaction with a representative of the fluoride/SAM 2 prereaction complex at the active site of the fluorinase together with selected hydrogen bonds among the key residues in the active site, fluoride and the ribose of 2. (b) Directed evolution provided two fluorinase variants with modest rate improvement in the first half of the two-step fluorinase-mediated transhalogenation reaction; (c) fluorinase-mediated biotransformation generated a series of 18F radiolabelling of cancer relevant targeting peptides and A2A adenosine receptor agonists for PET.
conversion of SAM 2 to 50 -FDA 3 in the presence of fluoride ion [16]. The substrate scope of this two-step transhalogenation reaction was further extended to the 2-deoxy analogue of 50 -ClDA 1 [16] and 50 -chloro-50 deoxy-2-ethynyladenosine (50 -ClDEA) 1a derivatives (Figure 1b) [17]. In an effort to explore the substrate flexibility and optimise the two-step transhalogenation, the fluorinase was subjected to directed evolution, allowing the identification of two fluorinase variants with the modest rate improvement for the first half reaction of the transhalogenation from 50 -ClDA 1 to SAM 2 [18,19]. Interestingly, modifications at F213 or A279 residues in fluorinase are also beneficial towards this transhalogenation even when using modified substrates, such as 50 -ClDEA (410-fold activity improvement) to produce 50 -fluorodeoxy-2-ethynyladenosine (50 -FDEA) [19]. During this transhalogenation, SAM synthesis is the ratelimiting step. To improve the overall transhalogenation Current Opinion in Chemical Biology 2020, 55:119–126
efficiency, a coupled chlorinaseefluorinase system was developed. In this system, the chlorinase is responsible for efficient conversion from 5-ClDA 1 to SAM 2, whereas the fluorinase is to convert SAM 2 to 50 -FDA 3 in the presence of fluoride ion [20]. More recently, the fluorinase was found to catalyse a one-step enzymatic Finkelstein reaction of 50 -bromodeoxyadenosine (50 BrDA) to 50 FDA 3 without the addition of SAM 2 or LMet (Figure 1b), offering a prospect of a simplified onestep fluorinase biotechnology for positron emission tomography (PET) [21].
Fluorinase method for use in biomedical applications PET is a highly sensitive, quantitative, noninvasive imaging technique. The 18F-radionuclide is often preferred for PET imaging because of its favourable half-life (109.8 min), high-resolution PET images. Traditional www.sciencedirect.com
Fluorine biocatalysis Wu et al.
labelling methods of the synthesis of 18F-labelled PET tracers usually demand multiple steps and/or harsh reaction conditions such as heating at high temperatures in strict anhydrous conditions because of the limited reactivity of the fluoride anion as a nucleophile in aqueous solution [22]. Fluorinase-mediated transhalogenation has emerged as a useful late-stage strategy for PET probe synthesis (Figure 1c). The fluorinase tolerates the acetylene functionality at the C-2 position of the adenine ring in the two-step transhalogenation reaction, leading to the conversion of 50 -ClDEA 1a to 50 -FDEA 3a [23]. Such an approach has been extended to generate a series of the 18 F-radiolabelling of cancer relevant targeting peptides [23e25] and A2A adenosine receptor agonists [26] for PET imaging applications, under experimentally mild conditions (Figure 1c).
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Biocatalysts that generate fluorinated building blocks There are few biocatalytic transformations for production of fluorine-containing molecules. Aldolases have emerged as a useful class of catalysts for controlling the formation of fluorine-containing stereocentres. Two types of aldolases have been explored for the synthesis of a range of chiral fluorinated building blocks (Figure 2). These aldolase-catalysed reactions often involve fluoropyruvate 5 as a nucleophile to lead stereoselective carbonecarbon bond formation enabling the synthesis of many a-fluoro-b-hydroxy molecules. Type I aldolases use a catalytic lysine residue to form a Schiff base with donor carbonyl-containing substrates enabling the controlled installation of fluorine-bearing stereocentres which has been reviewed recently [6]. Type II aldolases are metal dependent and have received much less attention in the research community in the past.
Figure 2
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(a) A type II aldolase-catalysed chemoenzymatic platform enabled the synthesis of more than twenty fluorinated acids, esters, amino acids and sugars involving fluoropyruvate 5 as a nucleophile. (b) A chemoenzymatic platform for synthesis of chiral a-trifluoromethylated organoborons 12 based on Rhodothermus marinus cytochrome c (Rma cyt c) mutant generated through directed evolution. www.sciencedirect.com
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Recently, type II 4-hydroxy-2-oxo-heptane-1,7-dioate aldolases (HpcH) have been explored as a chemoenzymatic platform to synthesise a series of a-fluoro-bhydroxy acids and esters with high stereopurity [27]. It was also demonstrated that the biotransformation platform displayed a good degree of flexibility further using these fluorinated acids and esters to generate a range of fluorinated analogues of amino acids, sugars and other valuable compounds by coupling with various transaminases and dehydrogenases (Figure 2a). Another new development of fluorine biocatalytic transformation is to introduce nonnatural chemistries into existing biocatalysts. Chiral a-trifluoromethylated (a-CF3) organoborons 12 are valuable synthetic building blocks that can be converted to a broad range of CF3containing products (Figure 2b). Cytochrome c (Rma cyt c), a P450 enzyme from Rhodothermus marinu, is a highly versatile platform for developing new chemistries of carbene transfer reactions [28,29]. Huang et al. [30]
further harnessed the versatility of this enzyme through directed evolution to develop a new enzymatic carbene transformation platform. The resulting variant, denoted as BOR-CF3, was able to accommodate structurally diverse trifluorodiazo alkanes 11 for highly enantioselective carbene BeH insertion reaction to provide chiral a- CF3 organoborons 12 with high total turnovers (Figure 2b).
Biotransformations that incorporate fluorinated building blocks into structurally complex organofluorines Polyketide metabolites contribute many of the important antibiotics used clinically, and these natural products are synthesised by multidomain polyketide synthase (PKS) complexes [31]. In an effort to engineer fluorinated building blocks into structurally complex molecules, an assembly module from the erythromycin polyketide pathway was used to
Figure 3
(a) The incorporation of fluorine atom using fluoromalonyl-CoA 14 as building blocks in mutated erythromycin polyketide assembly modules coupled with a trans-acting AT enzyme; (b) The in vivo biosynthesis of fluorinated building block, 2-fluoro-R-3-hydroxybutyrate (FHB) 20 and fluorinated poly(hydroxyalkanoate) polymers 21 from fluoromalonates 13.
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accommodate fluorinated extender units fluoromalonates 13 to introduce fluorine into the backbone of polyketides, albeit the low efficiency of chain extension with fluoromalonyl-CoA compared with the native substrate [32]. This is because the methylmalonyl-CoA 15 selective acyltransferase (AT) domain in the erythromycin PKS catalyses the hydrolysis of fluoromalonyl-CoA 14. It would be desirable to improve the efficient incorporation of fluoromalonyl-CoA 14 in the PKS assembly line. In this regard, replacement of a standalone trans-acting AT from the disorazole biosynthetic cluster to complement the deactivated cisAT domains of this erythromycin PKS module resulted in increased enzyme acylation with the fluoromalonyl CoA 14 extender unit in the presence of two other building blocks, 15 and propionyl-SNAC substrate mimic 16, thereby considerably improving the yield of monofluorinated or multifluorinated triketides 17e19 in vitro (Figure 3a) [33]. It is an exciting prospect to engineer the biosynthetic machinery of the cell to use simple fluorinated building blocks to produce new complex organofluorine molecules. More recently, the same group reported efficient production of the fluorinated diketide, 2-fluoro-3-hydroxybutyrate (FHB) 20 using fluoromalonate 13 as the feedstock in an engineered Escherichia coli (Figure 3b). Furthermore, the diketide was used as a monomer in vivo to generate fluorinated poly(hydroxyalkanoate) bioplastics 21 with fluorinated substitutions ranging from up to 15% (Figure 3b) [34].
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Understanding organofluorine physiology in fluorometabolisms It is attractive to consider engineering a microbial strain suitable for production of fluorinated products by fermentation. Such a strain would require a fluorinase gene, as well as its associated biosynthetic and regulatory genes. This strategy was partially achieved. Replacement of the chlorinase gene salL with flA gene in the biosynthetic gene cluster (BGC) of salinosporamide A, the chlorinated phase II anticancer metabolite, induced the production of fluorinated salinosporamide A when fluoride was added at the late stage of the fermentation [35]. However, problems emerged. The Salinispora tropica variant is extremely sensitive towards fluoride ion in culture, causing difficulty of scale-up fermentation [29]. Thus, there is considerable interest in understanding the toxic mitigation mechanisms of fluoride and fluorinated metabolites in the fluorometabolite producers [36,37]. It was found that S. cattleya evolves to manage the toxicity of fluoroacetate 24 and 4-fluorothreonine 25 by recruiting two specialised enzymes, fluoroacetyl-CoA hydrolase, FlK and a trans-acting fluorothreonyl-tRNA deacylase, FthB, to counteract the potential cellular toxicity of these two fluorometabolites, respectively. In the case of fluoroacetate 24, FlK displays high preference of hydrolysis towards fluoroacetyl-CoA 24a, the in vivo intermediate of fluoroacetate 24 as the lethal toxic to S. cattleya, due to the specific fluorine recognition with a hydrophobic residue in the binding site (Figure 4a) [38]. FthB, on the other hand, counteracts the misacylation of fluorothreonyl-
Figure 4
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(a) FlK, the specialised fluoroacetyl-CoA hydrolase, catalyses an entropy-driven hydrolysis of fluoroacetyl-CoA 24a, the toxic intermediate, to fluoroacetate 24 as part of toxicity mitigation strategy in vivo. (b) FthB, the specialised trans-acting fluorothreonyl-tRNA deacylase, counteracts the misacylation of fluorothreonyl-tRNA which otherwise would be misincorporated into protein in place of L-threonine. (c) Nucleocidin 26 consists of a unique tertiary fluorine at C-40 of the ribose ring. Recent investigations suggested that the BGC of 26 contains a new type of fluorination enzyme. Isotopic labelled moieties in glycerol highlighted in blue were demonstrated to be incorporated into nucleocidin 26.
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tRNA which otherwise would be misincorporated into protein in place of L-threonine (Figure 4b) [39]. These findings provide a deeper understanding of how microorganisms can evolve their own biosynthetic capacities by providing new chemistries to the BGCs. If a biotechnologically useful organism is to be engineered, such knowledge of fluorine toxic mitigation strategies will be useful.
Faculty, Reps and Staff Development Program (FRAS DP) PhD grant fellowship.
References Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Outlook/perspective Much is still needed to expand the scope of fluorine biocatalysts for organofluorine synthesis. It is exciting to fully use the existing enzyme inventory to accommodate new chemistries for bioactive organofluorines. As a complement to this, we should also turn our attention to nature for inspirations by discovering new fluorinated entities and new fluorometabolite pathways/enzymes. In this regard, studies of the various fluorometabolic pathways in the soil bacterium, Streptomyces sp MA37, could provide new strategies of organofluorine biotransformation because of its unusual capacity of producing a range of unknown fluorometabolites [40]. Another exciting development is to discover new CeF bond forming enzymes. Nucleocidin 26 is the only known fluorometabolite that possesses a tertiary fluorine at C-40 of the ribose ring, structurally different to other fluorometabolites containing a fluoromethyl group. Identification of the nucleocidin BGCs in the producing strains revealed that there is no known fluorinase encoded in microbial genomes [41,42]. Indeed, isotopic labelling experiments traced an oxidation occurring at C50 of nucleocidin after ribose ring assembly during the biosynthesis of 26 (Figure 4c) [43,44], significantly different to what was observed in the isotopic labelling studies of fluoroacetate 24 and 4-fluorothreonine 25 in S. ´ et al [43] idencattleya [45]. More recently, Bartholome tified two 40 -fluoro-30 -O-b-glucosylated metabolites, FMets I 27 and II 28, which were likely to be the last intermediates in the 26 biosynthesis (Figure 4c). It was also demonstrated that the UDP-glucose dependent glucosyl transferase (NucGT) and glucosidase (NucGS) encoded in the 26 BGC are responsible for attaching and removing glucose from the 30 -O-position of adenosine analogues (Figure 4c) [46]. Efforts directed towards greater understanding of the biosynthesis of nucleocidin 26 will certainly offer a promising opportunity of discovering a novel fluorination strategy.
Conflict of interest statement Nothing declared.
Acknowledgements HD is grateful for the financial supports through Biotechnology and Biological Sciences Research Council (BBSRC) grants (BB/P00380X/1 and BB/ R00479X/1) and Leverhulme Trust (RPG- 2014-418). HD and LW thank Industrial Biotechnology Innovation Centre (IBioIC) for the PhD studentship (LW). FM thanks the University of the Philippines for the Current Opinion in Chemical Biology 2020, 55:119–126
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46. Feng X, Bello D, Lowe P, Clark J, O’Hagan D: Two 3’-O-b glucosylated nucleoside fluorometabolites related to nucleocidin in Streptomyces calvus. Chem Sci 2019, 10:9501–9505. The authors disclosed the structures of two new 40 -fluoro-30 -O-bglucosylated fluorometabolites in the culture broth of Streptomyces calvus, the producer of nucleocidin and identified two key enzymes encoded in the nucleocidin BGC, UDP-glucose dependent glucosyl transferase and glucosidase, that have capacity to attach and remove glucose motif from the 30 -O-position of adenosin analogues, respectively.
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