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.

A Survey of Recently Discovered Naturally Occurring Organohalogen Compounds.

The discovery of naturally occurring organohalogen compounds has increased astronomically in the 55 years since they were first discovered─from fewer than 50 in 1968 to a combined 7,958 described examples in three comprehensive reviews. The present survey, which covers the period 2021-2023, brings the number of known natural organohalogens to approximately 8,400. The organization is according to species origin, and coverage includes marine and terrestrial plants, fungi, bacteria, marine sponges, corals, cyanobacteria, tunicates, and other marine organisms.

Substrate Profiling of Anion Methyltransferases for Promiscuous Synthesis of S-Adenosylmethionine Analogs from Haloalkanes.

Biocatalytic alkylation reactions can be performed with high chemo-, regio- and stereoselectivity using S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) and SAM analogs. Currently, however, this methodology is limited in application due to the rather laborious protocols to access SAM analogs. It has recently been shown that halide methyltransferases (HMTs) enable synthesis and recycling of SAM analogs with readily available haloalkanes as starting material. Here we expand this work by using substrate profiling of the anion MT enzyme family to explore promiscuous SAM analog synthesis. Our study shows that anion MTs are in general very promiscuous with respect to the alkyl chain as well as the halide leaving group. Substrate profiling further suggests that promiscuous anion MTs cluster in sequence space. Next to iodoalkanes, cheaper, less toxic, and more available bromoalkanes have been converted and several haloalkanes bearing short alkyl groups, alkyl rings, and functional groups such as alkene, alkyne and aromatic moieties are accepted as substrates. Further, we applied the SAM analogs as electrophiles in enzyme-catalyzed regioselective pyrazole allylation with 3-bromopropene as starting material.

Role of Voltage-Gated Sodium Channels in the Mechanism of Ether-Induced Unconsciousness.

Despite continuous clinical use for more than 170 years, the mechanism of general anesthetics has not been completely characterized. In this review, we focus on the role of voltage-gated sodium channels in the sedative-hypnotic actions of halogenated ethers, describing the history of anesthetic mechanisms research, the basic neurobiology and pharmacology of voltage-gated sodium channels, and the evidence for a mechanistic interaction between halogenated ethers and sodium channels in the induction of unconsciousness. We conclude with a more integrative perspective of how voltage-gated sodium channels might provide a critical link between molecular actions of the halogenated ethers and the more distributed network-level effects associated with the anesthetized state across species.

Monovalent Nickel-Mediated Radical Formation: A Concerted Halogen-Atom Dissociation Pathway Determined by Electroanalytical Studies.

The recent success of nickel catalysts in stereoconvergent cross-coupling and cross-electrophile coupling reactions partly stems from the ability of monovalent nickel species to activate C(sp3) electrophiles and generate radical intermediates. This electroanalytical study of the commonly applied (bpy)Ni catalyst elucidates the mechanism of this critical step. Data rule out outer-sphere electron transfer and two-electron oxidative addition pathways. The linear free energy relationship between rates and the bond-dissociation free energies, the electronic and steric effects of the nickel complexes and the electrophiles, and DFT calculations support a variant of the halogen-atom abstraction pathway, the inner-sphere electron transfer concerted with halogen-atom dissociation. This mechanism accounts for the observed reactivity of different electrophiles in cross-coupling reactions and provides a mechanistic rationale for the chemoselectivity obtained in cross-electrophile coupling over homocoupling.

Biocatalytic Cross-Coupling of Aryl Halides with a Genetically Engineered Photosensitizer Artificial Dehalogenase.

Devising artificial photoenzymes for abiological bond-forming reactions is of high synthetic value but also a tremendous challenge. Disclosed herein is the first photobiocatalytic cross-coupling of aryl halides enabled by a designer artificial dehalogenase, which features a genetically encoded benzophenone chromophore and site-specifically modified synthetic NiII(bpy) cofactor with tunable proximity to streamline the dual catalysis. Transient absorption studies suggest the likelihood of energy transfer activation in the elementary organometallic event. This design strategy is viable to significantly expand the catalytic repertoire of artificial photoenzymes for useful organic transformations.

Ground-State Electron Transfer as an Initiation Mechanism for Biocatalytic C-C Bond Forming Reactions.

The development of non-natural reaction mechanisms is an attractive strategy for expanding the synthetic capabilities of substrate promiscuous enzymes. Here, we report an "ene"-reductase catalyzed asymmetric hydroalkylation of olefins using α-bromoketones as radical precursors. Radical initiation occurs via ground-state electron transfer from the flavin cofactor located within the enzyme active site, an underrepresented mechanism in flavin biocatalysis. Four rounds of site saturation mutagenesis were used to access a variant of the "ene"-reductase nicotinamide-dependent cyclohexanone reductase (NCR) from Zymomonas mobiles capable of catalyzing a cyclization to furnish ß-chiral cyclopentanones with high levels of enantioselectivity. Additionally, wild-type NCR can catalyze intermolecular couplings with precise stereochemical control over the radical termination step. This report highlights the utility for ground-state electron transfers to enable non-natural biocatalytic C-C bond forming reactions.

Structural Dynamics of C2F4I2 in Cyclohexane Studied via Time-Resolved X-ray Liquidography.

The halogen elimination of 1,2-diiodoethane (C2H4I2) and 1,2-diiodotetrafluoroethane (C2F4I2) serves as a model reaction for investigating the influence of fluorination on reaction dynamics and solute-solvent interactions in solution-phase reactions. While the kinetics and reaction pathways of the halogen elimination reaction of C2H4I2 were reported to vary substantially depending on the solvent, the solvent effects on the photodissociation of C2F4I2 remain to be explored, as its reaction dynamics have only been studied in methanol. Here, to investigate the solvent dependence, we conducted a time-resolved X-ray liquidography (TRXL) experiment on C2F4I2 in cyclohexane. The data revealed that (ⅰ) the solvent dependence of the photoreaction of C2F4I2 is not as strong as that observed for C2H4I2, and (ⅱ) the nongeminate recombination leading to the formation of I2 is slower in cyclohexane than in methanol. We also show that the molecular structures of the relevant species determined from the structural analysis of TRXL data provide an excellent benchmark for DFT calculations, especially for investigating the relevance of exchange-correlation functionals used for the structural optimization of haloalkanes. This study demonstrates that TRXL is a powerful technique to study solvent dependence in the solution phase.

A Review: Halogenated Compounds from Marine Fungi.

Marine fungi produce many halogenated metabolites with a variety of structures, from acyclic entities with a simple linear chain to multifaceted polycyclic molecules. Over the past few decades, their pharmaceutical and medical application have been explored and still the door is kept open due to the need of new drugs from relatively underexplored sources. Biological properties of halogenated compounds such as anticancer, antiviral, antibacterial, anti-inflammatory, antifungal, antifouling, and insecticidal activity have been investigated. This review describes the chemical structures and biological activities of 217 halogenated compounds derived mainly from Penicillium and Aspergillus marine fungal strains reported from 1994 to 2019.

Anti-Pathogenic Properties of the Combination of a T3SS Inhibitory Halogenated Pyrrolidone with C-30 Furanone.

Antimicrobial resistance is one of the current public health challenges to be solved. The World Health Organization (WHO) has urgently called for the development of strategies to expand the increasingly limited antimicrobial arsenal. The development of anti-virulence therapies is a viable option to counteract bacterial infections with the possibility of reducing the generation of resistance. Here we report on the chemical structures of pyrrolidones DEXT 1-4 (previously identified as furan derivatives) and their anti-virulence activity on Pseudomonas aeruginosa strains. DEXT 1-4 were shown to inhibit biofilm formation, swarming motility, and secretion of ExoU and ExoT effector proteins. Also, the anti-pathogenic property of DEXT-3 alone or in combination with furanone C-30 (quorum sensing inhibitor) or MBX-1641 (type III secretion system inhibitor) was analyzed in a model of necrosis induced by P. aeruginosa PA14. All treatments reduced necrosis; however, only the combination of C-30 50 µM with DEXT-3 100 µM showed significant inhibition of bacterial growth in the inoculation area and systemic dispersion. In conclusion, pyrrolidones DEXT 1-4 are chemical structures capable of reducing the pathogenicity of P. aeruginosa and with the potential for the development of anti-virulence combination therapies.

A Borondifluoride-Complex-Based Photothermal Agent with an 80 % Photothermal Conversion Efficiency for Photothermal Therapy in the NIR-II Window.

Small organic photothermal agents (SOPTAs) that absorb in the second near-infrared (NIR-II, 1000-1700 nm) window are highly desirable in photothermal therapy for their good biocompatibility and deeper tissue penetration. However, the design of NIR-II absorbing SOPTAs remains a great challenge. Herein, we report that molecular engineering of BF2 complex via strengthening the donor-acceptor conjugation and increasing the intramolecular motions is an efficient strategy to achieve NIR-II absorbing SOPTAs with high photothermal performance. Based on this strategy, a BF2 complex, BAF4, was designed and synthesized. BAF4 exhibits an intense absorption maximum at 1000 nm and negligible fluorescence. Notably, the nanoparticles of BAF4 achieve a high photothermal conversion efficiency value of 80 % under 1064 nm laser irradiation (0.75 W cm-2 ). In vitro and in vivo studies reveal the great potential of BAF4 nanoparticles in photoacoustic imaging-guided photothermal therapy in the NIR-II window.

Convergent Catalytic Asymmetric Synthesis of Esters of Chiral Dialkyl Carbinols.

Because chiral dialkyl carbinols, as well as their derived esters, are significant as intermediates and end points in fields such as organic, pharmaceutical, and biological chemistry, the development of efficient approaches to their asymmetric synthesis is an important endeavor. In this report, we describe a method for the direct catalytic enantioselective synthesis of such esters, beginning with an alkyl halide (derived from an aldehyde and an acyl bromide), an olefin, and a hydrosilane, catalyzed by nickel, an earth-abundant metal. The method is versatile, tolerating substituents that vary in size and that bear a range of functional groups. We further describe a four-component variant of this process, wherein the alkyl halide is generated in situ, thus obviating the need to isolate either an alkyl electrophile or an alkylmetal, while still effecting an alkyl-alkyl coupling. Finally, we apply our convergent method to the efficient catalytic enantioselective synthesis of three esters that are bioactive themselves or that have been utilized in the synthesis of bioactive compounds.

The Impact of Halogenated Phenylalanine Derivatives on NFGAIL Amyloid Formation.

The hexapeptide hIAPP22-27 (NFGAIL) is known as a crucial amyloid core sequence of the human islet amyloid polypeptide (hIAPP) whose aggregates can be used to better understand the wild-type hIAPP's toxicity to ß-cell death. In amyloid research, the role of hydrophobic and aromatic-aromatic interactions as potential driving forces during the aggregation process is controversially discussed not only in case of NFGAIL, but also for amyloidogenic peptides in general. We have used halogenation of the aromatic residue as a strategy to modulate hydrophobic and aromatic-aromatic interactions and prepared a library of NFGAIL variants containing fluorinated and iodinated phenylalanine analogues. We used thioflavin T staining, transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) to study the impact of side-chain halogenation on NFGAIL amyloid formation kinetics. Our data revealed a synergy between aggregation behavior and hydrophobicity of the phenylalanine residue. This study introduces systematic fluorination as a toolbox to further investigate the nature of the amyloid self-assembly process.

Increased Lipophilicity of Halogenated Ruthenium(II) Polypyridyl Complexes Leads to Decreased Phototoxicity in†vitro when Used as Photosensitizers for Photodynamic Therapy.

In the fight against cancer, photodynamic therapy is generating great interest thanks to its ability to selectively kill cancer cells without harming healthy tissues. In this field, ruthenium(II) polypyridyl complexes, and more specifically, complexes with dipyrido[3,2-a:2',3'-c]phenazine (dppz) as a ligand are of particular interest due to their DNA-binding and photocleaving properties. However, ruthenium(II) polypyridyl complexes can sometimes suffer from low lipophilicity, which hampers cellular internalisation through passive diffusion. In this study, four new [Ru(dppz-X2 )3 ]2+ complexes (X=H, F, Cl, Br, I) were synthesized and their lipophilicity (logP), cytotoxicity and phototoxicity on cancerous and noncancerous cell lines were assessed. This study shows that, counterintuitively, the phototoxicity of these complexes decreases as their lipophilicity increases; this could be due solely to the atomic radius of the halogen substituents.

A competition between hydrophobic and electrostatic interactions in protein-ligand systems. Binding of heterogeneously halogenated benzotriazoles by the catalytic subunit of human protein kinase CK2.

A series of chlorine-substituted benzotriazole derivatives, representing all possible substitution patterns of halogen atoms attached to the benzotriazole benzene ring, were synthetized as potential inhibitors of human protein kinase CK2. Basic ADME parameters for the free solutes (hydrophobicity, electronic properties) together with their binding affinity to the catalytic subunit of protein kinase CK2 were determined with reverse-phase HPLC, spectrophotometric titration, and Thermal Shift Assay Method, respectively. The analysis of position-dependent thermodynamic contribution of a chlorine atom attached to the benzotriazole ring confirmed the previous observation for brominated benzotriazoles, in which substitution at positions 5 and 6 with bromine was found crucial for ligand binding. In all tested halogenated benzotriazoles the replacement of Br with Cl decreases the hydrophobicity, while the electronic properties remain virtually unaffected. Supramolecular architecture identified in the just resolved crystal structures of three of the four possible dichloro-benzotriazoles shows how substitution distant from the triazole ring affects the pattern of intermolecular interactions. Summarizing, the benzotriazole benzene ring substitution pattern has been identified as the main driver of ligand binding, predominating the non-specific hydrophobic effect.

One- and two-photon solvatochromism of the fluorescent dye Nile Red and its CF3, F and Br-substituted analogues.

The solvatochromic fluorophore Nile Red, 9-diethylamino-5H-benzo[a]phenoxazine-5-one, is one of the most commonly used stains to enhance contrast of lipid-rich areas of microscopic biosamples. Quite surprisingly, relatively little is known about the spectrally-resolved two-photon absorption (2PA) properties of this dye despite its promising features for two-photon microscopy of biological matter. For this reason, the two-photon solvatochromism of Nile Red still remains an uncharted territory as well. Also, no study has yet reported on how electron-withdrawing substituents attached to the Nile Red backbone affect its solvatochromic properties and two-photon brightness. In this paper, we demonstrate how solvent polarity influences the one- and two-photon absorption spectra of Nile Red as well as its fluorescence parameters, and we present new analogues that contain -CF3, -F and -Br substituents on its eastern side. Two-photon excited fluorescence experiments in a broad spectral range (780-1240 nm) and electronic structure calculations show that both the nature and location of the substituent have particular influence on the strength of 2PA, peaking in all cases at approx. 860 and 1050 nm. 2PA cross sections are higher at 1050 nm than at 860 nm, which suggests that Nile Red and its analogues are best suited for two-photon imaging employing excitation in the NIR-II optical transparency window of biological tissues.

1,2-cis-Selective glucosylation enabled by halogenated benzyl protecting groups.

We report on our initial results from a systematic effort to implement electron-withdrawing protecting groups and Lewis basic solvents/additives as an approach to 1,2-cis(α)-selective O-glucosylation. 1,2-cis-Selective O-glucosylations are reported with thioglucosides and glucosyl trichloroacetimidates and a range of acceptors. A correlation between electron-withdrawing effects and 1,2-cis selectivity has been established. This phenomenon may prove to be broadly applicable in the area of chemical O-glycosylation.

Synthesis of novel halogenated triarylpyrazoles as selective COX-2 inhibitors: Anti-inflammatory activity, histopatholgical profile and in-silico studies.

A novel series of halogenated triarylpyrazoles 12a-l was designed and synthesized. All target compounds showed good in vitro COX-2 inhibitory activity (IC50 = 0.043-0.17 µM) over COX-1 (IC50 = 7.8 - 15.4 µM) relative to celecoxib (COX-1/IC50 = 9.87, COX-2/IC50 = 0.055), with acceptable selectivity index values (SI = 50.6-253.1). Also, they displayed moderate to potent in vivo anti-inflammatory activity (% edema inhibition = 16.9-87.9) comparable to celecoxib (% edema inhibition = 46.6-72.1) as standard drug. Three fluorinated pyrazoles 12a, 12g and 12j, exhibited superior anti-inflammatory activity at all time intervals (% edema inhibition = 42.1-87.9) with better gastric profile (UI = 1.25-2.5) than the traditional NSAID; indomethacin (UI = 14) and were close to the selective COX-2 inhibitor; celecoxib (UI = 1.75). In-silico docking and ADME studies of 12a, 12g and 12j supported the obtained biological data and pointed out their potential use for the development of bio-available, safe and potent anti-inflammatory drugs.

Airborne brominated, chlorinated and organophosphate ester flame retardants inside the buildings of the Indian state of Bihar: Exploration of source and human exposure.

Since many household products used by individuals contain flame retardants (FRs), there is more chance that these chemicals may be present in the various exhibit of the indoor environment. Despite being one of the fastest-growing economies worldwide, the contamination level, sources, products, and pathways of FRs in India, is either not known or limited. This inspired us to investigate the level, profile, spatial distribution, and sources of different classes of FRs in the indoor air. For this purpose, 15 brominated, 2 chlorinated, and 8 organophosphate FRs (OPFRs) were investigated in indoor air samples from urban and suburban sites of an Indian state of Bihar. Additionally, inhalation health risk exposure to children and the adult was estimated to predict the risk of these chemicals. Overall, ∑8OPFRs (median 351 pg/m3) was the most prominent in air, followed by novel brominated FR (∑6NBFRs) (median 278 pg/m3), polybrominated diphenyl ether (∑9PBDE) (median 5.05 pg/m3), and dechlorane plus (∑2DPs) (median 2.52 pg/m3), and accounted for 55%, 44%, 0.8% and 0.4% of ∑FRs, respectively. Generally, ∑9PBDEs (median 6.29 pg/m3) and ∑8OPFRs (median 355 pg/m3) were measured high at sub-urban sites, while urban sites had the highest level of ∑2DPs (median 2.81 pg/m3) and ∑6NBFRs (median 740 pg/m3). BDE-209 was most abundant among ∑9PBDEs, while syn-DP dominated in ∑2DPs. Likewise, DBDPE was most prevalent in ∑6NBFRs, while TMPP topped among ∑8OPFRs. The principal component analysis revealed contribution from household items, food packaging and paints, hydraulic fluid, a gasoline additive, and de-bromination of BDE-209 as the primary sources of FRs. The estimated daily inhalation exposure (DIE) indicated a relatively high risk to children than the adult. The DIE of individual FR was several folds lower than their corresponding oral reference dose (RfDs), suggesting minimal risk. However, exposure risk, especially to children, may still need attention because other routes of intake may always be significant in the case of Bihar.

Trifluoroiodomethane (CF3I) (2019).

Trifluoroiodomethane (CF3I) is a colorless and odorless gas used primarily as a fire suppressant. CF3I has low acute inhalation toxicity. The no-observed adverse effect level (NOAEL) of CF3I for cardiac sensitization in dogs was 2000 ppm. The potential effects of 4-week inhalation exposure in both rats and mice have been examined. In rats, the NOAEL was 10,000 ppm, and in mice, the NOAEL was 10,000 ppm. In a subchronic inhalation study in rats, the lowest observed adverse effect level (LOAEL) was 20,000 ppm for thyroid-related effects; the study NOAEL (for non-thyroid-related effects) was 20,000 ppm. In a reproductive/developmental inhalation toxicity study in rats, 20,000 ppm CF3I produced minimal general toxicity and no indication of reproductive or developmental toxicity. The LOAEL for parental toxicity (based on thyroid hormone effects) was 2000 ppm; excluding thyroid effects, the parental NOAEL was 7000 ppm CF3I. The observed effects on the thyroid in rats were considered of less relevance to human risk assessment than the other observed systemic effects because of known species-specific differences in sensitivity to thyroid hormone perturbations. There are no chronic toxicity or carcinogenicity studies available. CF3I had mixed results in various in vitro and in vivo genotoxicity assays. The NOAEL of 7000 ppm from the reproductive/developmental inhalation study was used as the point of departure (POD) for workplace environmental exposure level (WEEL) value development. This POD was adjusted to account for interindividual variability, duration of exposure, and database limitations. The resulting 8-h time-weighted average WEEL value of 500 ppm is expected to provide a significant margin of safety against any potential adverse health effects in workers exposed to CF3I. A 15-min short-term exposure limit of 1500 ppm was also established to protect workers from potential cardiac effects produced by acute, high-dose inhalation of CF3I.