Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis.

Flavin-dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes have been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications.

Electrocatalytic reductive deuteration of arenes and heteroarenes.

The incorporation of deuterium in organic molecules has widespread applications in medicinal chemistry and materials science1,2. For example, the deuterated drugs austedo3, donafenib4 and sotyktu5 have been recently approved. There are various methods for the synthesis of deuterated compounds with high deuterium incorporation6. However, the reductive deuteration of aromatic hydrocarbons-ubiquitous chemical feedstocks-to saturated cyclic compounds has rarely been achieved. Here we describe a scalable and general electrocatalytic method for the reductive deuteration and deuterodefluorination of (hetero)arenes using a prepared nitrogen-doped electrode and deuterium oxide (D2O), giving perdeuterated and saturated deuterocarbon products. This protocol has been successfully applied to the synthesis of 13 highly deuterated drug molecules. Mechanistic investigations suggest that the ruthenium-deuterium species, generated by electrolysis of D2O in the presence of a nitrogen-doped ruthenium electrode, are key intermediates that directly reduce aromatic compounds. This quick and cost-effective methodology for the preparation of highly deuterium-labelled saturated (hetero)cyclic compounds could be applied in drug development and metabolism studies.

Catalytic asymmetric synthesis of cannabinoids and menthol from neral.

The selective conversion of natural or synthetic neral to (1R,6S)-trans-isopiperitenol would enable and expedite sustainable routes to menthol1,2 and cannabinoids3-5. However, this reaction has been considered impossible because its product is more reactive to the required acid catalysts than its starting material, resulting in several side products6-9. We now show that an unsymmetric, strong and confined chiral acid, a highly fluorinated imino-imidodiphosphate, catalyses this process with excellent efficiency and selectivity. Expanding the method to other α,ß-unsaturated aldehydes could enable access to new cannabinoids and menthol derivatives not readily accessible previously. Mechanistic studies suggest that the confined catalyst accomplishes this reaction by binding the product in an unreactive conformation, thereby preventing its decomposition. We also show how (1R,6S)-trans-isopiperitenol can be readily converted to pharmaceutically useful cannabinoids and menthol, each in the shortest and most atom-economic routes so far.

Bayesian reaction optimization as a tool for chemical synthesis.

Reaction optimization is fundamental to synthetic chemistry, from optimizing the yield of industrial processes to selecting conditions for the preparation of medicinal candidates1. Likewise, parameter optimization is omnipresent in artificial intelligence, from tuning virtual personal assistants to training social media and product recommendation systems2. Owing to the high cost associated with carrying out experiments, scientists in both areas set numerous (hyper)parameter values by evaluating only a small subset of the possible configurations. Bayesian optimization, an iterative response surface-based global optimization algorithm, has demonstrated exceptional performance in the tuning of machine learning models3. Bayesian optimization has also been recently applied in chemistry4-9; however, its application and assessment for reaction optimization in synthetic chemistry has not been investigated. Here we report the development of a framework for Bayesian reaction optimization and an open-source software tool that allows chemists to easily integrate state-of-the-art optimization algorithms into their everyday laboratory practices. We collect a large benchmark dataset for a palladium-catalysed direct arylation reaction, perform a systematic study of Bayesian optimization compared to human decision-making in reaction optimization, and apply Bayesian optimization to two real-world optimization efforts (Mitsunobu and deoxyfluorination reactions). Benchmarking is accomplished via an online game that links the decisions made by expert chemists and engineers to real experiments run in the laboratory. Our findings demonstrate that Bayesian optimization outperforms human decisionmaking in both average optimization efficiency (number of experiments) and consistency (variance of outcome against initially available data). Overall, our studies suggest that adopting Bayesian optimization methods into everyday laboratory practices could facilitate more efficient synthesis of functional chemicals by enabling better-informed, data-driven decisions about which experiments to run.

Synergistic material-microbe interface toward deeper anaerobic defluorination.

Per- and polyfluoroalkyl substances (PFAS), particularly the perfluorinated ones, are recalcitrant to biodegradation. By integrating an enrichment culture of reductive defluorination with biocompatible electrodes for the electrochemical process, a deeper defluorination of a C6-perfluorinated unsaturated PFAS was achieved compared to the biological or electrochemical system alone. Two synergies in the bioelectrochemical system were identified: i) The in-series microbial-electrochemical defluorination and ii) the electrochemically enabled microbial defluorination of intermediates. These synergies at the material-microbe interfaces surpassed the limitation of microbial defluorination and further turned the biotransformation end products into less fluorinated products, which could be less toxic and more biodegradable in the environment. This material-microbe hybrid system brings opportunities in the bioremediation of PFAS driven by renewable electricity and warrants future research on mechanistic understanding of defluorinating and electroactive microorganisms at the material-microbe interface for system optimizations.

Fluorinated macromolecular amphiphiles as prototypic molecular drones.

The advent of drones has revolutionized various aspects of our lives, and in the realm of biological systems, molecular drones hold immense promise as "magic bullets" for major diseases. Herein, we introduce a unique class of fluorinated macromolecular amphiphiles, designed in the shape of jellyfish, serving as exemplary molecular drones for fluorine-19 MRI (19F MRI) and fluorescence imaging (FLI)-guided drug delivery, status reporting, and targeted cancer therapy. Functioning akin to their mechanical counterparts, these biocompatible molecular drones autonomously assemble with hydrophobic drugs to form uniform nanoparticles, facilitating efficient drug delivery into cells. The status of drug delivery can be tracked through aggregation-induced emission (AIE) of FLI and 19F MRI. Furthermore, when loaded with a heptamethine cyanine fluorescent dye IR-780, these molecular drones enable near-infrared (NIR) FL detection of tumors and precise delivery of the photosensitizer. Similarly, when loaded with doxorubicin (DOX), they enable targeted chemotherapy with fluorescence resonance energy transfer (FRET) FL for real-time status updates, resulting in enhanced therapeutic efficacy. Compared to conventional drug delivery systems, molecular drones stand out for their simplicity, precise structure, versatility, and ability to provide instantaneous status updates. This study presents prototype molecular drones capable of executing fundamental drone functions, laying the groundwork for the development of more sophisticated molecular machines with significant biomedical implications.

Desymmetrization of difluoromethylene groups by C-F bond activation.

Tertiary stereogenic centres containing one fluorine atom are valuable for medicinal chemistry because they mimic common tertiary stereogenic centres containing one hydrogen atom, but they possess distinct charge distribution, lipophilicity, conformation and metabolic stability1-3. Although tertiary stereogenic centres containing one hydrogen atom are often set by enantioselective desymmetrization reactions at one of the two carbon-hydrogen (C-H) bonds of a methylene group, tertiary stereocentres containing fluorine have not yet been constructed by the analogous desymmetrization reaction at one of the two carbon-fluorine (C-F) bonds of a difluoromethylene group3. Fluorine atoms are similar in size to hydrogen atoms but have distinct electronic properties, causing C-F bonds to be exceptionally strong and geminal C-F bonds to strengthen one another4. Thus, exhaustive defluorination typically dominates over the selective replacement of a single C-F bond, hindering the development of the enantioselective substitution of one fluorine atom to form a stereogenic centre5,6. Here we report the catalytic, enantioselective activation of a single C-F bond in an allylic difluoromethylene group to provide a broad range of products containing a monofluorinated tertiary stereogenic centre. By combining a tailored chiral iridium phosphoramidite catalyst, which controls regioselectivity, chemoselectivity and enantioselectivity, with a fluorophilic activator, which assists the oxidative addition of the C-F bond, these reactions occur in high yield and selectivity. The design principles proposed in this work extend to palladium-catalysed benzylic substitution, demonstrating the generality of the approach.

CnoX Is a Chaperedoxin: A Holdase that Protects Its Substrates from Irreversible Oxidation.

Bleach (HOCl) is a powerful oxidant that kills bacteria in part by causing protein aggregation. It inactivates ATP-dependent chaperones, rendering cellular proteins mostly dependent on holdases. Here we identified Escherichia coli CnoX (YbbN) as a folding factor that, when activated by bleach via chlorination, functions as an efficient holdase, protecting the substrates of the major folding systems GroEL/ES and DnaK/J/GrpE. Remarkably, CnoX uniquely combines this function with the ability to prevent the irreversible oxidation of its substrates. This dual activity makes CnoX the founding member of a family of proteins, the "chaperedoxins." Because CnoX displays a thioredoxin fold and a tetratricopeptide (TPR) domain, two structural motifs conserved in all organisms, this investigation sets the stage for the discovery of additional chaperedoxins in bacteria and eukaryotes that could cooperate with proteins from both the Hsp60 and Hsp70 families.

Formation of left-handed helices by C2'-fluorinated nucleic acids under physiological salt conditions.

Recent findings in cell biology have rekindled interest in Z-DNA, the left-handed helical form of DNA. We report here that two minimally modified nucleosides, 2'F-araC and 2'F-riboG, induce the formation of the Z-form under low ionic strength. We show that oligomers entirely made of these two nucleosides exclusively produce left-handed duplexes that bind to the Zα domain of ADAR1. The effect of the two nucleotides is so dramatic that Z-form duplexes are the only species observed in 10 mM sodium phosphate buffer and neutral pH, and no B-form is observed at any temperature. Hence, in contrast to other studies reporting formation of Z/B-form equilibria by a preference for purine glycosidic angles in syn, our NMR and computational work revealed that sequential 2'F…H2N and intramolecular 3'H…N3' interactions stabilize the left-handed helix. The equilibrium between B- and Z- forms is slow in the 19F NMR time scale (≥ms), and each conformation exhibited unprecedented chemical shift differences in the 19F signals. This observation led to a reliable estimation of the relative population of B and Z species and enabled us to monitor B-Z transitions under different conditions. The unique features of 2'F-modified DNA should thus be a valuable addition to existing techniques for specific detection of new Z-binding proteins and ligands.

Biocatalytic control of site-selectivity and chain length-selectivity in radical amino acid halogenases.

Biocatalytic C-H activation has the potential to merge enzymatic and synthetic strategies for bond formation. FeII/αKG-dependent halogenases are particularly distinguished for their ability both to control selective C-H activation as well as to direct group transfer of a bound anion along a reaction axis separate from oxygen rebound, enabling the development of new transformations. In this context, we elucidate the basis for the selectivity of enzymes that perform selective halogenation to yield 4-Cl-lysine (BesD), 5-Cl-lysine (HalB), and 4-Cl-ornithine (HalD), allowing us to probe how site-selectivity and chain length selectivity are achieved. We now report the crystal structure of the HalB and HalD, revealing the key role of the substrate-binding lid in positioning the substrate for C4 vs C5 chlorination and recognition of lysine vs ornithine. Targeted engineering of the substrate-binding lid further demonstrates that these selectivities can be altered or switched, showcasing the potential to develop halogenases for biocatalytic applications.

Halogenated Antimicrobial Agents to Combat Drug-Resistant Pathogens.

Antimicrobial resistance presents us with a potential global crisis as it undermines the abilities of conventional antibiotics to combat pathogenic microbes. The history of antimicrobial agents is replete with examples of scaffolds containing halogens. In this review, we discuss the impacts of halogen atoms in various antibiotic types and antimicrobial scaffolds and their modes of action, structure-activity relationships, and the contributions of halogen atoms in antimicrobial activity and drug resistance. Other halogenated molecules, including carbohydrates, peptides, lipids, and polymeric complexes, are also reviewed, and the effects of halogenated scaffolds on pharmacokinetics, pharmacodynamics, and factors affecting antimicrobial and antivirulence activities are presented. Furthermore, the potential of halogenation to circumvent antimicrobial resistance and rejuvenate impotent antibiotics is addressed. This review provides an overview of the significance of halogenation, the abilities of halogens to interact in biomolecular settings and enhance pharmacological properties, and their potential therapeutic usages in preventing a postantibiotic era. SIGNIFICANCE STATEMENT: Antimicrobial resistance and the increasing impotence of antibiotics are critical threats to global health. The roles and importance of halogen atoms in antimicrobial drug scaffolds have been established, but comparatively little is known of their pharmacological impacts on drug resistance and antivirulence activities. This review is the first to extensively evaluate the roles of halogen atoms in various antibiotic classes and pharmacological scaffolds and to provide an overview of their ability to overcome antimicrobial resistance.

4S-fluorination of ProB29 in insulin lispro slows fibril formation.

Recombinant insulin is a life-saving therapeutic for millions of patients affected by diabetes mellitus. Standard mutagenesis has led to insulin variants with improved control of blood glucose; for instance, the fast-acting insulin lispro contains two point mutations that suppress dimer formation and expedite absorption. However, insulins undergo irreversible denaturation, a process accelerated for the insulin monomer. Here we replace ProB29 of insulin lispro with 4R-fluoroproline, 4S-fluoroproline, and 4,4-difluoroproline. All three fluorinated lispro variants reduce blood glucose in diabetic mice, exhibit similar secondary structure as measured by CD, and rapidly dissociate from the zinc- and resorcinol-bound hexamer upon dilution. Notably, however, we find that 4S-fluorination of ProB29 delays the formation of undesired insulin fibrils that can accumulate at the injection site in vivo and can complicate insulin production and storage. These results demonstrate how subtle molecular changes achieved through non-canonical amino acid mutagenesis can improve the stability of protein therapeutics.

Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates. Oxidized flavin needs to be reduced for regeneration of the enzyme, which can be performed in vitro by a photoreduction with blue light. Here, we employed this photoreduction to study characteristic structural changes associated with the transition from oxidized to fully reduced flavin in PyrH from Streptomyces rugosporus as a model for tryptophan-5-halogenases. The effect of the presence of bromide and chloride or the absence of any halides on the UV-vis spectrum of the enzyme demonstrated a halide-dependent structure of the flavin-binding pocket. Light-induced FTIR difference spectroscopy was applied and the signals assigned by selective isotope labeling of the protein moiety. The identified structural changes in α-helix and ß-sheet elements were strongly dependent on the presence of bromide, chloride, the substrate tryptophan, and the product 5-chloro-tryptophan, respectively. We identified a clear allosteric coupling in solution at ambient conditions between cofactor-binding site and substrate-binding site that is active in both directions, despite their separation by a tunnel. We suggest that this coupling constitutes a fine-tuned mechanism for the promotion of the enzymatic reaction of flavin-dependent halogenases in dependence of halide and substrate availability.

Identifying and Engineering Flavin Dependent Halogenases for Selective Biocatalysis.

ConspectusOrganohalogen compounds are extensively used as building blocks, intermediates, pharmaceuticals, and agrochemicals due to their unique chemical and biological properties. Installing halogen substituents, however, frequently requires functionalized starting materials and multistep functional group interconversion. Several classes of halogenases evolved in nature to enable halogenation of a different classes of substrates; for example, site-selective halogenation of electron rich aromatic compounds is catalyzed by flavin-dependent halogenases (FDHs). Mechanistic studies have shown that these enzymes use FADH2 to reduce O2 to water with concomitant oxidation of X- to HOX (X = Cl, Br, I). This species travels through a tunnel within the enzyme to access the FDH active site. Here, it is believed to interact with an active site lysine proximal to bound substrate, enabling electrophilic halogenation with selectivity imparted via molecular recognition, rather than directing groups or strong electronic activation.The unique selectivity of FDHs led to several early biocatalysis efforts, preparative halogenation was rare, and the hallmark catalyst-controlled selectivity of FDHs did not translate to non-native substrates. FDH engineering was limited to site-directed mutagenesis, which resulted in modest changes in site-selectivity or substrate preference. To address these limitations, we optimized expression conditions for the FDH RebH and its cognate flavin reductase (FRed), RebF. We then showed that RebH could be used for preparative halogenation of non-native substrates with catalyst-controlled selectivity. We reported the first examples in which the stability, substrate scope, and site selectivity of a FDH were improved to synthetically useful levels via directed evolution. X-ray crystal structures of evolved FDHs and reversion mutations showed that random mutations throughout the RebH structure were critical to achieving high levels of activity and selectivity on diverse aromatic substrates, and these data were used in combination with molecular dynamics simulations to develop predictive model for FDH selectivity. Finally, we used family wide genome mining to identify a diverse set of FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds.The diversity of our evolved and mined FDHs allowed us to pursue synthetic applications beyond simple aromatic halogenation. For example, we established that FDHs catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. These results highlight the ability of FDH active sites to tolerate different substrate topologies. This utility was further expanded by our recent studies on the single component FDH/FRed, AetF. While we were initially drawn to AetF because it does not require a separate FRed, we found that it halogenates substrates that are not halogenated efficiently or at all by other FDHs and provides high enantioselectivity for reactions that could only be achieved using RebH variants after extensive mutagenesis. Perhaps most notably, AetF catalyzes site-selective aromatic iodination and enantioselective iodoetherification. Together, these studies highlight the origins of FDH engineering, the utility and limitations of the enzymes developed to date, and the promise of FDHs for an ever-expanding range of biocatalytic halogenation reactions.

The digitization of organic synthesis.

Organic chemistry has largely been conducted in an ad hoc manner by academic laboratories that are funded by grants directed towards the investigation of specific goals or hypotheses. Although modern synthetic methods can provide access to molecules of considerable complexity, predicting the outcome of a single chemical reaction remains a major challenge. Improvements in the prediction of 'above-the-arrow' reaction conditions are needed to enable intelligent decision making to select an optimal synthetic sequence that is guided by metrics including efficiency, quality and yield. Methods for the communication and the sharing of data will need to evolve from traditional tools to machine-readable formats and open collaborative frameworks. This will accelerate innovation and require the creation of a chemistry commons with standardized data handling, curation and metrics.

Discovery of a pathway for terminal-alkyne amino acid biosynthesis.

Living systems can generate an enormous range of cellular functions, from mechanical infrastructure and signalling networks to enzymatic catalysis and information storage, using a notably limited set of chemical functional groups. This observation is especially notable when compared to the breadth of functional groups used as the basis for similar functions in synthetically derived small molecules and materials. The relatively small cross-section between biological and synthetic reactivity space forms the foundation for the development of bioorthogonal chemistry, in which the absence of a pair of reactive functional groups within the cell allows for a selective in situ reaction1-4. However, biologically 'rare' functional groups, such as the fluoro5, chloro6,7, bromo7,8, phosphonate9, enediyne10,11, cyano12, diazo13, alkene14 and alkyne15-17 groups, continue to be discovered in natural products made by plants, fungi and microorganisms, which offers a potential route to genetically encode the endogenous biosynthesis of bioorthogonal reagents within living organisms. In particular, the terminal alkyne has found broad utility via the Cu(I)-catalysed azide-alkyne cycloaddition 'click' reaction18. Here we report the discovery and characterization of a unique pathway to produce a terminal alkyne-containing amino acid in the bacterium Streptomyces cattleya. We found that L-lysine undergoes an unexpected reaction sequence that includes halogenation, oxidative C-C bond cleavage and triple bond formation through a putative allene intermediate. This pathway offers the potential for de novo cellular production of halo-, alkene- and alkyne-labelled proteins and natural products from glucose for a variety of downstream applications.

Chlorination of arenes via the degradation of toxic chlorophenols.

Aryl chlorides are among the most versatile synthetic precursors, and yet inexpensive and benign chlorination techniques to produce them are underdeveloped. We propose a process to generate aryl chlorides by chloro-group transfer from chlorophenol pollutants to arenes during their mineralization, catalyzed by Cu(NO3)2/NaNO3 under aerobic conditions. A wide range of arene substrates have been chlorinated using this process. Mechanistic studies show that the Cu catalyst acts in cooperation with NOx species generated from the decomposition of NaNO3 to regulate the formation of chlorine radicals that mediate the chlorination of arenes together with the mineralization of chlorophenol. The selective formation of aryl chlorides with the concomitant degradation of toxic chlorophenol pollutants represents a new approach in environmental pollutant detoxication. A reduction in the use of traditional chlorination reagents provides another (indirect) benefit of this procedure.

Polar Interactions between Substrate and Flavin Control Iodotyrosine Deiodinase Function.

Flavin cofactors offer a wide range of chemical mechanisms to support a great diversity in catalytic function. As a corollary, such diversity necessitates careful control within each flavoprotein to limit its function to an appropriate subset of possible reactions and substrates. This task falls to the protein environment surrounding the flavin in most enzymes. For iodotyrosine deiodinase that catalyzes a reductive dehalogenation of halotyrosines, substrates can dictate the chemistry available to the flavin. Their ability to stabilize the necessary one-electron reduced semiquinone form of flavin strictly depends on a direct coordination between the flavin and α-ammonium and carboxylate groups of its substrates. While perturbations to the carboxylate group do not significantly affect binding to the resting oxidized form of the deiodinase, dehalogenation (kcat/Km) is suppressed by over 2000-fold. Lack of the α-ammonium group abolishes detectable binding and dehalogenation. Substitution of the ammonium group with a hydroxyl group does not restore measurable binding but does support dehalogenation with an efficiency greater than those of the carboxylate derivatives. Consistent with these observations, the flavin semiquinone does not accumulate during redox titration in the presence of inert substrate analogues lacking either the α-ammonium or carboxylate groups. As a complement, a nitroreductase activity based on hydride transfer is revealed for the appropriate substrates with perturbations to their zwitterion.

Enzyme Catalyzed Formation of CoA Adducts of Fluorinated Hexanoic Acid Analogues using a Long-Chain acyl-CoA Synthetase from Gordonia sp. Strain NB4-1Y.

Per and polyfluoroalkyl substances (PFAS) are a large family of anthropogenic fluorinated chemicals of increasing environmental concern. Over recent years, numerous microbial communities have been found to be capable of metabolizing some polyfluoroalkyl substances, generating a range of low-molecular-weight PFAS metabolites. One proposed pathway for the microbial breakdown of fluorinated carboxylates includes ß-oxidation, this pathway is initiated by the formation of a CoA adduct. However, until recently no PFAS-CoA adducts had been reported. In a previous study, we were able to use a bacterial medium-chain acyl-CoA synthetase (mACS) to form CoA adducts of fluorinated adducts of propanoic acid and pentanoic acid but were not able to detect any products of fluorinated hexanoic acid analogues. Herein, we expressed and purified a long-chain acyl-CoA synthetase (lACS) and a A461K variant of mACS from the soil bacterium Gordonia sp. strain NB4-1Y and performed an analysis of substrate scope and enzyme kinetics using fluorinated and nonfluorinated carboxylates. We determined that lACS can catalyze the formation of CoA adducts of 1:5 fluorotelomer carboxylic acid (FTCA), 2:4 FTCA and 3:3 FTCA, albeit with generally low turnover rates (<0.02 s-1) compared with the nonfluorinated hexanoic acid (5.39 s-1). In addition, the A461K variant was found to have an 8-fold increase in selectivity toward hexanoic acid compared with wild-type mACS, suggesting that Ala-461 has a mechanistic role in selectivity toward substrate chain length. This provides further evidence to validate the proposed activation step involving the formation of CoA adducts in the enzymatic breakdown of PFAS.

α,ß-Desaturation and Formal ß-C(sp3)-H Fluorination of N-Substituted Amines: A Late-Stage Functionalization Strategy Enabled by Electrochemistry.

Incorporation of C(sp3)-F bonds in biologically active compounds is a common strategy employed in medicinal and agricultural chemistry to tune pharmacokinetic and pharmacodynamic properties. Due to the limited number of robust strategies for C(sp3)-H fluorination of complex molecules, time-consuming de novo syntheses of such fluorinated analogs are typically required, representing a major bottleneck in the drug discovery process. In this work, we present a general and operationally simple strategy for site-specific ß-C(sp3)-H fluorination of amine derivatives including carbamates, amides, and sulfonamides, which is compatible with a wide range of functional groups including N-heteroarenes. In this approach, an improved electrochemical Shono oxidation is used to set the site of functionalization via net α,ß-desaturation to access enamine derivatives. We further developed a series of new transformations of these enamine intermediates to synthesize a variety of ß-fluoro-α-functionalized structures, allowing efficient access to pertinent targets to accelerate drug discovery campaigns.