A light-driven enzymatic enantioselective radical acylation.

Enzymes are recognized as exceptional catalysts for achieving high stereoselectivities1-3, but their ability to control the reactivity and stereoinduction of free radicals lags behind that of chemical catalysts4. Thiamine diphosphate (ThDP)-dependent enzymes5 are well-characterized systems that inspired the development of N-heterocyclic carbenes (NHCs)6-8 but have not yet been proved viable in asymmetric radical transformations. There is a lack of a biocompatible and general radical-generation mechanism, as nature prefers to avoid radicals that may be harmful to biological systems9. Here we repurpose a ThDP-dependent lyase as a stereoselective radical acyl transferase (RAT) through protein engineering and combination with organophotoredox catalysis10. Enzyme-bound ThDP-derived ketyl radicals are selectively generated through single-electron oxidation by a photoexcited organic dye and then cross-coupled with prochiral alkyl radicals with high enantioselectivity. Diverse chiral ketones are prepared from aldehydes and redox-active esters (35 examples, up to 97% enantiomeric excess (e.e.)) by this method. Mechanistic studies reveal that this previously elusive dual-enzyme catalysis/photocatalysis directs radicals with the unique ThDP cofactor and evolvable active site. This work not only expands the repertoire of biocatalysis but also provides a unique strategy for controlling radicals with enzymes, complementing existing chemical tools.

Direct radical functionalization of native sugars.

Naturally occurring (native) sugars and carbohydrates contain numerous hydroxyl groups of similar reactivity1,2. Chemists, therefore, rely typically on laborious, multi-step protecting-group strategies3 to convert these renewable feedstocks into reagents (glycosyl donors) to make glycans. The direct transformation of native sugars to complex saccharides remains a notable challenge. Here we describe a photoinduced approach to achieve site- and stereoselective chemical glycosylation from widely available native sugar building blocks, which through homolytic (one-electron) chemistry bypasses unnecessary hydroxyl group masking and manipulation. This process is reminiscent of nature in its regiocontrolled generation of a transient glycosyl donor, followed by radical-based cross-coupling with electrophiles on activation with light. Through selective anomeric functionalization of mono- and oligosaccharides, this protecting-group-free 'cap and glycosylate' approach offers straightforward access to a wide array of metabolically robust glycosyl compounds. Owing to its biocompatibility, the method was extended to the direct post-translational glycosylation of proteins.

Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling.

Photobiocatalysis-where light is used to expand the reactivity of an enzyme-has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2-9. Here we report photobiocatalytic asymmetric sp3-sp3 oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5'-phosphate-dependent enzymes, threonine aldolases10-12, for the α-C-H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids 13-15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox-pyridoxal biocatalysis provides a platform for sp3-sp3 oxidative coupling16, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology.

From Ground-State to Excited-State Activation Modes: Flavin-Dependent "Ene"-Reductases Catalyzed Non-natural Radical Reactions.

Enzymes are desired catalysts for chemical synthesis, because they can be engineered to provide unparalleled levels of efficiency and selectivity. Yet, despite the astonishing array of reactions catalyzed by natural enzymes, many reactivity patterns found in small molecule catalysts have no counterpart in the living world. With a detailed understanding of the mechanisms utilized by small molecule catalysts, we can identify existing enzymes with the potential to catalyze reactions that are currently unknown in nature. Over the past eight years, our group has demonstrated that flavin-dependent "ene"-reductases (EREDs) can catalyze various radical-mediated reactions with unparalleled levels of selectivity, solving long-standing challenges in asymmetric synthesis.This Account presents our development of EREDs as general catalysts for asymmetric radical reactions. While we have developed multiple mechanisms for generating radicals within protein active sites, this account will focus on examples where flavin mononucleotide hydroquinone (FMNhq) serves as an electron transfer radical initiator. While our initial mechanistic hypotheses were rooted in electron-transfer-based radical initiation mechanisms commonly used by synthetic organic chemists, we ultimately uncovered emergent mechanisms of radical initiation that are unique to the protein active site. We will begin by covering intramolecular reactions and discussing how the protein activates the substrate for reduction by altering the redox-potential of alkyl halides and templating the charge transfer complex between the substrate and flavin-cofactor. Protein engineering has been used to modify the fundamental photophysics of these reactions, highlighting the opportunity to tune these systems further by using directed evolution. This section highlights the range of coupling partners and radical termination mechanisms available to intramolecular reactions.The next section will focus on intermolecular reactions and the role of enzyme-templated ternary charge transfer complexes among the cofactor, alkyl halide, and coupling partner in gating electron transfer to ensure that it only occurs when both substrates are bound within the protein active site. We will highlight the synthetic applications available to this activation mode, including olefin hydroalkylation, carbohydroxylation, arene functionalization, and nitronate alkylation. This section also discusses how the protein can favor mechanistic steps that are elusive in solution for the asymmetric reductive coupling of alkyl halides and nitroalkanes. We are aware of several recent EREDs-catalyzed photoenzymatic transformations from other groups. We will discuss results from these papers in the context of understanding the nuances of radical initiation with various substrates.These biocatalytic asymmetric radical reactions often complement the state-of-the-art small-molecule-catalyzed reactions, making EREDs a valuable addition to a chemist's synthetic toolbox. Moreover, the underlying principles studied with these systems are potentially operative with other cofactor-dependent proteins, opening the door to different types of enzyme-catalyzed radical reactions. We anticipate that this Account will serve as a guide and inspire broad interest in repurposing existing enzymes to access new transformations.

Harnessing in vivo synthesis of bioactive multiarylmethanes in Escherichia coli via oxygen-mediated free radical reaction induced by simple phenols.

BACKGROUND: Xanthenes and multi-aryl carbon core containing compounds represent different types of complex and condensed architectures that have impressive wide range of pharmacological, industrial and synthetic applications. Moreover, indoles as building blocks were only found in naturally occurring metabolites with di-aryl carbon cores and in chemically synthesized tri-aryl carbon core containing compounds. Up to date, rare xanthenes with indole bearing multicaryl carbon core have been reported in natural or synthetic products. The underlying mechanism of fluorescein-like arthrocolins with tetra-arylmethyl core were synthesized in an engineered Escherichia coli fed with toluquinol remained unclear. RESULTS: In this study, the Keio collection of single gene knockout strains of 3901 mutants of E. coli BW25113, together with 14 distinct E. coli strains, was applied to explore the origins of endogenous building blocks and the biogenesis for arthrocolin assemblage. Deficiency in bacterial respiratory and aromatic compound degradation genes ubiX, cydB, sucA and ssuE inhibited the mutant growth fed with toluquinol. Metabolomics of the cultures of 3897 mutants revealed that only disruption of tnaA involving in transforming tryptophan to indole, resulted in absence of arthrocolins. Further media optimization, thermal cell killing and cell free analysis indicated that a non-enzyme reaction was involved in the arthrocolin biosynthesis in E. coli. Evaluation of redox potentials and free radicals suggested that an oxygen-mediated free radical reaction was responsible for arthrocolins formation in E. coli. Regulation of oxygen combined with distinct phenol derivatives as inducer, 31 arylmethyl core containing metabolites including 13 new and 8 biological active, were isolated and characterized. Among them, novel arthrocolins with p-hydroxylbenzene ring from tyrosine were achieved through large scale of aerobic fermentation and elucidated x-ray diffraction analysis. Moreover, most of the known compounds in this study were for the first time synthesized in a microbe instead of chemical synthesis. Through feeding the rat with toluquinol after colonizing the intestines of rat with E. coli, arthrocolins also appeared in the rat blood. CONCLUSION: Our findings provide a mechanistic insight into in vivo synthesis of complex and condensed arthrocolins induced by simple phenols and exploits a quinol based method to generate endogenous aromatic building blocks, as well as a methylidene unit, for the bacteria-facilitated synthesis of multiarylmethanes.

Cysteine Radical and Glutamate Collaboratively Enable C-H Bond Activation and C-N Bond Cleavage in a Glycyl Radical Enzyme HplG.

Hydroxyprolines are abundant in nature and widely utilized by many living organisms. Isomerization of trans-4-hydroxy-d-proline (t4D-HP) to generate 2-amino-4-ketopentanoate has been found to need a glycyl radical enzyme HplG, which catalyzes the cleavage of the C-N bond, while dehydration of trans-4-hydroxy-l-proline involves a homologous enzyme of HplG. Herein, molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM) calculations are employed to understand the reaction mechanism of HplG. Two possible reaction pathways of HplG have been explored to decipher the origin of its chemoselectivity. The QM/MM calculations reveal that the isomerization proceeds via an initial hydrogen shift from the Cγ site of t4D-HP to a catalytic cysteine radical, followed by cleavage of the Cδ-N bond in t4D-HP to form a radical intermediate that captures a hydrogen atom from the cysteine. Activation of the Cδ-H bond in t4D-HP to bring about dehydration of t4D-HP possesses an extremely high energy barrier, thus rendering the dehydration pathway implausible in HplG. On the basis of the current calculations, conserved residue Glu429 plays a pivotal role in the isomerization pathway: the hydrogen bonding between it and t4D-HP weakens the hydroxyalkyl Cγ-Hγ bond, and it acts as a proton acceptor to trigger the cleavage of the C-N bond in t4D-HP. Our current QM/MM calculations rationalize the origin of the experimentally observed chemoselectivity of HplG and propose an H-bond-assisted bond activation strategy in radical-containing enzymes. These findings have general implications on radical-mediated enzymatic catalysis and expand our understanding of how nature wisely and selectively activates the C-H bond to modulate catalytic selectivity.

INDIVIDUAL ARTICLE: Sugar Sag: What Is Skin Glycation and How Do You Combat It?

Skin aging is influenced by various exogenous and endogenous factors, ranging from ultraviolet (UV) light exposure and environmental toxins to biological sources, such as those that arise from normal metabolic processes (eg, free radicals). Glycation is the normal process by which glucose and other reducing sugars react with proteins to form an array of heterogeneous biomolecular structures known as advanced glycation end-products (AGEs) over time. However, AGEs are toxic to human cells and are implicated in the acceleration of inflammatory and oxidative processes, with their accumulation in the skin being associated with increased skin dulling and yellowing, fine lines, wrinkles, and skin laxity. Clinicians should become cognizant of how AGEs develop, what their biological consequences are, and familiarize themselves with available strategies to mitigate their formation. J Drugs Dermatol.  2024;23:4(Suppl 1):s5-10.

Free Radical-Associated Gene Signature Predicts Survival in Sepsis Patients.

Sepsis continues to overwhelm hospital systems with its high mortality rate and prevalence. A strategy to reduce the strain of sepsis on hospital systems is to develop a diagnostic/prognostic measure that identifies patients who are more susceptible to septic death. Current biomarkers fail to achieve this outcome, as they only have moderate diagnostic power and limited prognostic capabilities. Sepsis disrupts a multitude of pathways in many different organ systems, making the identification of a single powerful biomarker difficult to achieve. However, a common feature of many of these perturbed pathways is the increased generation of reactive oxygen species (ROS), which can alter gene expression, changes in which may precede the clinical manifestation of severe sepsis. Therefore, the aim of this study was to evaluate whether ROS-related circulating molecular signature can be used as a tool to predict sepsis survival. Here we created a ROS-related gene signature and used two Gene Expression Omnibus datasets from whole blood samples of septic patients to generate a 37-gene molecular signature that can predict survival of sepsis patients. Our results indicate that peripheral blood gene expression data can be used to predict the survival of sepsis patients by assessing the gene expression pattern of free radical-associated -related genes in patients, warranting further exploration.

Oxidative Stress and Bio-Regulation.

Reactive oxygen species (ROS) and free radicals work to maintain homeostasis in the body, but their excessive production causes damage to the organism. The human body is composed of a variety of cells totaling over 60 trillion cells. Each cell performs different functions and has a unique lifespan. The lifespan of cells is preprogrammed in their genes, and the death of cells that have reached the end of their lifespan is called apoptosis. This is contrary to necrosis, which is the premature death of cells brought about by physical or scientific forces. Each species has its own unique lifespan, which in humans is estimated to be up to 120 years. Elucidating the mechanism of the death of a single cell will lead to a better understanding of human death, and, conversely, the death of a single cell will lead to exploring the mechanisms of life. In this sense, research on active oxygen and free radicals, which are implicated in biological disorders and homeostasis, requires an understanding of both the physicochemical as well as the biochemical aspects. Based on the discussion above, it is clear to see that active oxygen and free radicals have dual functions of both injuring and facilitating homeostasis in living organisms.

Biphasic Fermentation of Trapa bispinosa Shells by Ganoderma sinense and Characterization of Its Polysaccharides and Alcoholic Extract and Analysis of Their Bioactivity.

BACKGROUND: Trapa bispinosa shells (TBs) and its flesh (TBf) have been recognized for their medicinal properties, including antioxidant, antitumor, and immunomodulatory effects. Despite these benefits, TBs are often discarded as waste material, and their applications remain to be further explored. METHODS: In this study, we optimized the solid-state fermentation process of Ganoderma sinense (GS) with TBs using a response surface experiment methodology to obtain the fermented production with the highest water extract rate and DPPH free radical scavenging activity. We prepared and characterized pre-fermentation purified polysaccharides (P1) and post-fermentation purified polysaccharides (P2). Alcoholic extracts before (AE1) and after (AE2) fermentation were analyzed for active components such as polyphenols and flavonoids using UPLC-QTOF-MS/MS (ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry). Mouse macrophages (RAW 264.7) were employed to compare the immune-stimulating ability of polysaccharides and the antioxidant activity of AE1 and AE2. RESULTS: Optimal fermentation conditions comprised a duration of 2 days, a temperature of 14 °C, and a humidity of 77%. The peak water extract yield and DPPH free radical scavenging rate of the water extract from TBs fermented by GS were observed under these conditions. The enhanced activity may be attributed to changes in the polysaccharide structure and the components of the alcoholic extract. The P2 treatment group indicated more secretion of RAW 264.7 cells of NO, iNOS, IL-2, IL-10, and TNF-α than P1, which shows that the polysaccharides demonstrated increased immune-stimulating ability, with their effect linked to the NF-кB pathway. Moreover, the results of the AE2 treatment group indicated that secretion of RAW 264.7 cells of T-AOC and T-SOD increased and MDA decreased, which shows that the alcoholic extract demonstrated enhanced antioxidant activity, with its effect linked to the Nrf2/Keap1-ARE pathway. CONCLUSIONS: Biphasic fermentation of Trapa bispinosa shells by Ganoderma sinense could change the composition and structure of the polysaccharides and the composition of the alcoholic extract, which could increase the products' immunomodulatory and antioxidant activity.

Carbon Free Radical (R⋠) Inactivates NF-κB for Radical Capping Therapy.

Inactivating hyperactivated transcription factors can overcome tumor therapy resistance, but their undruggable features limit the development of conventional inhibitors. Here, we report that carbon-centered free radicals (R⋅) can inactivate NF-κB transcription by capping the active sites in both NF-κB and DNA. We construct a type of thermosensitive R⋅ initiator loaded amphiphilic nano-micelles to facilitate intracellular delivery of R⋅. At a temperature of 43 °C, the generated R⋅ engage in electrophilic radical addition towards double bonds in nucleotide bases, and simultaneously cap the sulfhydryl residues in NF-κB through radical chain reaction. As a result, both NF-κB nuclear translocation and NF-κB-DNA binding are suppressed, leading to a remarkable NF-κB inhibition of up to 94.1 %. We have further applied R⋅ micelles in a clinical radiofrequency ablation tumor therapy model, showing remarkable NF-κB inactivation and consequently tumor metastasis inhibition. Radical capping strategy not only provides a method to solve the heat-sink effect in clinic tumor hyperthermia, but also suggests a new perspective for controllable modification of biomacromolecules in cancer therapy.

THE IMPACT OF DIAMIDE DERIVATIVES OF OXALIC ACID ON FREE RADICAL LIPID OXIDATION IN WHITE RAT BRAIN AND LIVER.

Antioxidants are widely used in medicine due to their ability to bind free radicals - active biomolecules that destroy the genetic apparatus of cells and the structure of their membranes, which makes it possible to reduce the intensity of oxidative processes in the body. In a living organism, free radicals are involved in various processes, but their activity is controlled by antioxidants. The purpose of this work was to conduct a series of studies to identify the antioxidant activity of new synthesized compounds of a series of oxalic acid diamides in the brain and liver tissue of white rats in vivo and in vitro experiments, as well as to determine their potential pharmacological properties. The studies were conducted on outbred white male rats, weighing 180-200 g, kept on a normal diet. After autopsy, the brain and liver were isolated, washed with saline, cleared of blood vessels, and homogenized in Tris-HCl buffer (pH-7.4) (in vitro). The research results showed significant antioxidant activity (AOA) of all compounds with varying effectiveness. The most pronounced activity was demonstrated by compound SV-425 in both brain and liver tissues. Compound SV-427 demonstrated the least activity, with levels in brain tissue and liver tissue. In addition, all physicochemical descriptors of the studied compounds comply with Lipinski's rule of five to identify new molecules for the treatment of oxidative stress. From the data obtained, it can be concluded that the studied compounds have antioxidant properties, helping to protect cells from oxidative stress. This is important for the prevention and treatment of diseases associated with increased levels of free radicals.

[Is Biological Aging a Treatable Disease? A Consideration Based on Proposed Mechanisms of Aging].

In view of the current claim by many researchers that biological aging is a treatable disease, the possibility is discussed whether the claim is realistic, based on several proposed mechanisms of aging. The definition of biological aging is stated referring to physiological aging and pathological aging, since biological aging must be defined for the discussion of whether it can be cured. Aging in animal model is compared with that in humans in terms of common age-associated phenotypes. Major proposed mechanisms of aging are next examined including Genome Instability Theory of aging, Free Radical or Oxidative Stress Theory of Aging, Mitochondrial Theory of Aging, Error Catastrophe Theory of Aging/Translational Error Theory of Aging, Altered Protein Theory of Aging/Proteostasis Theory of Aging, and Epigenetic Theory of Aging. Finally, we discuss whether treatment of aging as a disease is realistic in comparison with possible lifespan extension by retardation of biological aging.

Quantum Sensing of Free Radical Generation in Mitochondria of Single Heart Muscle Cells during Hypoxia and Reoxygenation.

Cells are damaged during hypoxia (blood supply deprivation) and reoxygenation (oxygen return). This damage occurs in conditions such as cardiovascular diseases, cancer, and organ transplantation, potentially harming the tissue and organs. The role of free radicals in cellular metabolic reprogramming under hypoxia is under debate, but their measurement is challenging due to their short lifespan and limited diffusion range. In this study, we employed a quantum sensing technique to measure the real-time production of free radicals at the subcellular level. We utilize fluorescent nanodiamonds (FNDs) that exhibit changes in their optical properties based on the surrounding magnetic noise. This way, we were able to detect the presence of free radicals. To specifically monitor radical generation near mitochondria, we coated the FNDs with an antibody targeting voltage-dependent anion channel 2 (anti-VDAC2), which is located in the outer membrane of mitochondria. We observed a significant increase in the radical load on the mitochondrial membrane when cells were exposed to hypoxia. Subsequently, during reoxygenation, the levels of radicals gradually decreased back to the normoxia state. Overall, by applying a quantum sensing technique, the connections among hypoxia, free radicals, and the cellular redox status has been revealed.

Hybrid mechanism of microplastics degradation via biological and chemical process during composting.

Little is known about the synergistic effects of abiotic aging and biodegradation on microplastics (MPs) transformation in the environment. Herein, a hybrid process of MPs degradation was proposed by analyzing the effect of microorganisms and abiotic aging on aging MPs and non-aging MPs degradation during composting. The results showed that composting facilitated the oxidation and depolymerization of aging MPs, and its degradation efficiency was about three times that of non-aging MPs. Further investigation revealed that aging MPs contained higher abundance of plastic-degrading bacteria and enzyme activity than non-aging MPs. In addition, free radicals also influenced the degradation of MPs. However, path model and shielding experiments confirmed that free radicals mainly facilitated the non-aging MPs degradation (contribution was 68.8 %), while aging MPs was easily degraded by microorganisms (contribution was 72.6 %). This study provides promising strategies for scaling up plastic treatment in bioreactors through a hybrid collaboration of biological and abiotic processes.

[What is the Initiating Reaction for the Lipid Radical Chain Reaction System That Can Induce Ferroptotic Cell Death at the Lower Oxygen Content?]

The neural cell death in cerebral infarction is suggested to be ferroptosis-like cell death, involving the participation of 15-lipoxygenase (15-LOx). Ferroptosis is induced by lipid radical species generated through the one-electron reduction of lipid hydroperoxides, and it has been shown to propagate intracellularly and intercellularly. At lower oxygen concentration, it appeared that both regiospecificity and stereospecificity of conjugated diene moiety in lipoxygenase-catalysed lipid hydroperoxidation are drastically lost. As a result, in the reaction with linoleic acid, the linoleate 9-peroxyl radical-ferrous lipoxygenase complex dissolves into the linoleate 9-peroxyl radical and ferrous 15-lipoxygenase. Subsequently, the ferrous 15-lipoxygenase then undergoes one-electron reduction of 13-hydroperoxy octadecadienoic acid, generating an alkoxyl radical (pseudoperoxidase reaction). A part of the produced lipid alkoxyl radicals undergoes cleavage of C-C bonds, liberating small molecular hydrocarbon radicals. Particularly, in ω-3 polyunsaturated fatty acids, which are abundant in the vascular and nervous systems, the liberation of small molecular hydrocarbon radicals was more pronounced compared to ω-6 polyunsaturated fatty acids. The involvement of these small molecular hydrocarbon radicals in the propagation of membrane lipid damage is suggested.

Enhancing lipid peroxidation via radical chain transfer reaction for MRI guided and effective cancer therapy in mice.

Lipid peroxidation (LPO), the process of membrane lipid oxidation, is a potential new form of cell death for cancer treatment. However, the radical chain reaction involved in LPO is comprised of the initiation, propagation (the slowest step), and termination stages, limiting its effectiveness in vivo. To address this limitation, we introduce the radical chain transfer reaction into the LPO process to target the propagation step and overcome the sluggish rate of lipid peroxidation, thereby promoting endogenous lipid peroxidation and enhancing therapeutic outcomes. Firstly, radical chain transfer agent (CTA-1)/Fe nanoparticles (CTA-Fe NPs-1) was synthesized. Notably, CTA-1 convert low activity peroxyl radicals (ROO·) into high activity alkoxyl radicals (RO·), creating the cycle of free radical oxidation and increasing the propagation of lipid peroxidation. Additionally, CTA-1/Fe ions enhance reactive oxygen species (ROS) generation, consume glutathione (GSH), and thereby inactivate GPX-4, promoting the initiation stage and reducing termination of free radical reaction. CTA-Fe NPs-1 induce a higher level of peroxidation of polyunsaturated fatty acids in lipid membranes, leading to highly effective treatment in cancer cells. In addition, CTA-Fe NPs-1 could be enriched in tumors inducing potent tumor inhibition and exhibit activatable T1-MRI contrast of magnetic resonance imaging (MRI). In summary, CTA-Fe NPs-1 can enhance intracellular lipid peroxidation by accelerating initiation, propagation, and inhibiting termination step, promoting the cycle of free radical reaction, resulting in effective anticancer outcomes in tumor-bearing mice.

Modeling the Initiation Phase of the Catalytic Cycle in the Glycyl-Radical Enzyme Benzylsuccinate Synthase.

The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.

Stereodivergent photobiocatalytic radical cyclization through the repurposing and directed evolution of fatty acid photodecarboxylases.

Despite their intriguing photophysical and photochemical activities, naturally occurring photoenzymes have not yet been repurposed for new-to-nature activities. Here we engineered fatty acid photodecarboxylases to catalyse unnatural photoredox radical C-C bond formation by leveraging the strongly oxidizing excited-state flavoquinone cofactor. Through genome mining, rational engineering and directed evolution, we developed a panel of radical photocyclases to facilitate decarboxylative radical cyclization with excellent chemo-, enantio- and diastereoselectivities. Our high-throughput experimental workflow allowed for the directed evolution of fatty acid photodecarboxylases. An orthogonal set of radical photocyclases was engineered to access all four possible stereoisomers of the stereochemical dyad, affording fully diastereo- and enantiodivergent biotransformations in asymmetric radical biocatalysis. Molecular dynamics simulations show that our evolved radical photocyclases allow near-attack conformations to be easily accessed, enabling chemoselective radical cyclization. The development of stereoselective radical photocyclases provides unnatural C-C-bond-forming activities in natural photoenzyme families, which can be used to tame the stereochemistry of free-radical-mediated reactions.

Association of Free Radical Product and Polycystic Ovary Syndrome: A Systematic Review and Meta-analysis.

PURPOSE: Polycystic ovary syndrome (PCOS) is an endocrine disorder that primarily affects women of reproductive age. It is recognized as the leading cause of infertility due to anovulation. This research aims to evaluate the diagnostic potential of oxidative stress biomarkers, including advanced oxidation protein products (AOPP), malondialdehyde (MDA), uric acid (UA), and nitric oxide (NO), in identifying PCOS. METHODS: A literature search was conducted in the EMBASE, PubMed, Cochrane Library, and Scopus databases. The standardized mean difference (SMD) and 95% confidence interval (CI) were employed to assess the correlation between free radical product and PCOS. Moreover, the presence of heterogeneity among the studies was assessed utilizing the I2 statistic and Cochran Q test. The methodological rigor of the incorporated studies was assessed through the application of the Newcastle-Ottawa Scale. Furthermore, the presence of publication bias was determined via Begg and Egger tests. RESULTS: This meta-analysis reviewed 38 observational studies, including 17,845 women. The results revealed a significant association between PCOS in women and alterations in free radical levels. The study revealed that the PCOS group had significantly higher levels of AOPP (SMD = 3.193; 95% CI, 2.86 to 3.25), UA (SMD = 0.68; 95% CI, 0.24 to 1.13), and MDA (SMD = 1.16; 95% CI, 0.77 to 1.56) compared to the healthy control group. Furthermore, the analysis found a significantly lower level of NO (SMD = (- 0.59); 95% CI, - 1.15 to - 0.03) in the PCOS patient. CONCLUSION: Screening of specific biomarkers associated with free radical products could provide valuable benefits in the prognosis and diagnosis of PCOS.