Unlocking carbene reactivity by metallaphotoredox α-elimination.

The ability to tame high-energy intermediates is important for synthetic chemistry, enabling the construction of complex molecules and propelling advances in the field of synthesis. Along these lines, carbenes and carbenoid intermediates are particularly attractive, but often unknown, high-energy intermediates1,2. Classical methods to access metal carbene intermediates exploit two-electron chemistry to form the carbon-metal bond. However, these methods are usually prohibitive because of reagent safety concerns, limiting their broad implementation in synthesis3-6. Mechanistically, an alternative approach to carbene intermediates that could circumvent these pitfalls would involve two single-electron steps: radical addition to metal to forge the initial carbon-metal bond followed by redox-promoted α-elimination to yield the desired metal carbene intermediate. Here we realize this strategy through a metallaphotoredox platform that exploits iron carbene reactivity using readily available chemical feedstocks as radical sources and α-elimination from six classes of previously underexploited leaving groups. These discoveries permit cyclopropanation and σ-bond insertion into N-H, S-H and P-H bonds from abundant and bench-stable carboxylic acids, amino acids and alcohols, thereby providing a general solution to the challenge of carbene-mediated chemical diversification.

Interactive features of proteins composing eukaryotic circadian clocks.

Research into the molecular mechanisms of eukaryotic circadian clocks has proceeded at an electrifying pace. In this review, we discuss advances in our understanding of the structures of central molecular players in the timing oscillators of fungi, insects, and mammals. A series of clock protein structures demonstrate that the PAS (Per/Arnt/Sim) domain has been used with great variation to formulate the transcriptional activators and repressors of the clock. We discuss how posttranslational modifications and external cues, such as light, affect the conformation and function of core clock components. Recent breakthroughs have also revealed novel interactions among clock proteins and new partners that couple the clock to metabolic and developmental pathways. Overall, a picture of clock function has emerged wherein conserved motifs and structural platforms have been elaborated into a highly dynamic collection of interacting molecules that undergo orchestrated changes in chemical structure, conformational state, and partners.

Enzyme inhibitor discovery by activity-based protein profiling.

Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.

Targeted Photoconvertible BODIPYs Based on Directed Photooxidation-Induced Conversion for Applications in Photoconversion and Live Super-Resolution Imaging.

Photomodulable fluorescent probes are drawing increasing attention due to their applications in advanced bioimaging. Whereas photoconvertible probes can be advantageously used in tracking, photoswitchable probes constitute key tools for single-molecule localization microscopy to perform super-resolution imaging. Herein, we shed light on a red and far-red BODIPY, namely, BDP-576 and BDP-650, which possess both properties of conversion and switching. Our study demonstrates that these pyrrolyl-BODIPYs convert into typical green- and red-emitting BODIPYs that are perfectly adapted to microscopy. We also showed that this pyrrolyl-BODIPYs undergo Directed Photooxidation Induced Conversion, a photoconversion mechanism that we recently introduced, where the pyrrole moiety plays a central role. These unique features were used to develop targeted photoconvertible probes toward different organelles or subcellular units (plasma membrane, mitochondria, nucleus, actin, Golgi apparatus, etc.) using chemical targeting moieties and a Halo tag. We notably showed that BDP-650 could be used to track intracellular vesicles over more than 20 min in two-color imagings with laser scanning confocal microscopy, demonstrating its robustness. The switching properties of these photoconverters were studied at the single-molecule level and were then successfully used in live single-molecule localization microscopy in epithelial cells and neurons. Both membrane- and mitochondria- targeted probes could be used to decipher membrane 3D architecture and mitochondrial dynamics at the nanoscale. This study builds a bridge between the photoconversion and photoswitching properties of probes undergoing directed photooxidation and shows the versatility and efficacy of this mechanism in advanced live imaging.

Crystal structure reveals the binding mode and selectivity of a photoswitchable ligand for the adenosine A2A receptor.

Rational synthetic expansion of photoresponsive ligands is important for photopharmacological studies. Adenosine A2A receptor (A2AR) is stimulated by adenosine and related in Parkinson's disease and other diseases. Here, we report the crystal structure of the A2AR in complex with the novel photoresponsive ligand photoNECA (blue) at 3.34 Å resolution. PhotoNECA (blue) was designed for this structural study and the cell-based assay showed a photoresponsive and receptor selective characteristics of photoNECA (blue) for A2AR. The crystal structure explains the binding mode, photoresponsive mechanism and receptor selectivity of photoNECA (blue). Our study would promote not only the rational design of photoresponsive ligands but also dynamic structural studies of A2AR.

Enhancing the potential of enantioselective organocatalysis with light.

Organocatalysis-catalysis mediated by small chiral organic molecules-is a powerful technology for enantioselective synthesis, and has extensive applications in traditional ionic, two-electron-pair reactivity domains. Recently, organocatalysis has been successfully combined with photochemical reactivity to unlock previously inaccessible reaction pathways, thereby creating new synthetic opportunities. Here we describe the historical context, scientific reasoning and landmark discoveries that were essential in expanding the functions of organocatalysis to include one-electron-mediated chemistry and excited-state reactivity.

Cooperative asymmetric reactions combining photocatalysis and enzymatic catalysis.

Living organisms rely on simultaneous reactions catalysed by mutually compatible and selective enzymes to synthesize complex natural products and other metabolites. To combine the advantages of these biological systems with the reactivity of artificial chemical catalysts, chemists have devised sequential, concurrent, and cooperative chemoenzymatic reactions that combine enzymatic and artificial catalysts1-9. Cooperative chemoenzymatic reactions consist of interconnected processes that generate products in yields and selectivities that cannot be obtained when the two reactions are carried out sequentially with their respective substrates2,7. However, such reactions are difficult to develop because chemical and enzymatic catalysts generally operate in different media at different temperatures and can deactivate each other1-9. Owing to these constraints, the vast majority of cooperative chemoenzymatic processes that have been reported over the past 30 years can be divided into just two categories: chemoenzymatic dynamic kinetic resolutions of racemic alcohols and amines, and enzymatic reactions requiring the simultaneous regeneration of a cofactor2,4,5. New approaches to the development of chemoenzymatic reactions are needed to enable valuable chemical transformations beyond this scope. Here we report a class of cooperative chemoenzymatic reaction that combines photocatalysts that isomerize alkenes with ene-reductases that reduce carbon-carbon double bonds to generate valuable enantioenriched products. This method enables the stereoconvergent reduction of E/Z mixtures of alkenes or reduction of the unreactive stereoisomers of alkenes in yields and enantiomeric excesses that match those obtained from the reduction of the pure, more reactive isomers. The system affords a range of enantioenriched precursors to biologically active compounds. More generally, these results show that the compatibility between photocatalysts and enzymes enables chemoenzymatic processes beyond cofactor regeneration and provides a general strategy for converting stereoselective enzymatic reactions into stereoconvergent ones.

Laser-induced CdS/TiO2/graphene dual photoanodes for ratiometric self-powered photoelectrochemical sensor: an innovative approach for aflatoxin B1 detection.

A ratiometric self-powered photoelectrochemical sensor based on laser direct writing technology was constructed to address the problem that the conventional single-signal detection mode was susceptible to the influence of instrumentation and environmental factors, which interfered with the detection results. Laser-induced CdS/TiO2/Graphene was prepared as dual photoanodes (PA1 and PA2), which were controlled by multiplexed switches to form a photocatalytic fuel cell with Pt cathode. By modifying the aptamer of aflatoxin B1 (AFB1) on the photoanode surface, the target was specifically captured to the electrode surface to form a biological complex, which increased the steric hindrance and affected the electron transfer, thus reducing the output signal of the sensor. Targets with different concentrations were incubated on the surface of PA1, and targets with fixed concentrations were incubated on the surface of PA2. Under the control of the multiplex switch, the output signals of the two photoanodes were recorded, and the ratio of these two signals was used as the basis for the quantitative detection of AFB1. The sensor output was linearly increasing with the logarithm of AFB1 concentration from 1.0 to 150 ng mL-1 and the detection limit was 0.0974 ng mL-1. Additionally, this method had good stability, fast response, and good selectivity to real samples, providing an effective method for food safety monitoring.

Photo-Uncaging by C(sp3)-C(sp3) Bond Cleavage Restores ß-Lapachone Activity.

ß-Lapachone is an ortho-naphthoquinone natural product with significant antiproliferative activity but suffers from adverse systemic toxicity. The use of photoremovable protecting groups to covalently inactivate a substrate and then enable controllable release with light in a spatiotemporal manner is an attractive prodrug strategy to limit toxicity. However, visible light-activatable photocages are nearly exclusively enabled by linkages to nucleophilic functional sites such as alcohols, amines, thiols, phosphates, and sulfonates. Herein, we report covalent inactivation of the electrophilic quinone moiety of ß-lapachone via a C(sp3)-C(sp3) bond to a coumarin photocage. In contrast to ß-lapachone, the designed prodrug remained intact in human whole blood and did not induce methemoglobinemia in the dark. Under light activation, the C-C bond cleaves to release the active quinone, recovering its biological activity when evaluated against the enzyme NQO1 and human cancer cells. Investigations into this report of a C(sp3)-C(sp3) photoinduced bond cleavage suggest a nontraditional, radical-based mechanism of release beginning with an initial charge-transfer excited state. Additionally, caging and release of the isomeric para-quinone, α-lapachone, are demonstrated. As such, we describe a photocaging strategy for the pair of quinones and report a unique light-induced cleavage of a C-C bond. We envision that this photocage strategy can be extended to quinones beyond ß- and α-lapachone, thus expanding the chemical toolbox of photocaged compounds.

Optical Control of Protein Functions via Genetically Encoded Photocaged Aspartic Acids.

Site-specific protein decaging by light has become an effective approach for in situ manipulation of protein activities in a gain-of-function fashion. Although successful decaging of amino acid side chains of Lys, Tyr, Cys, and Glu has been demonstrated, this strategy has not been extended to aspartic acid (Asp), an essential amino acid residue with a range of protein functions and protein-protein interactions. We herein reported a genetically encoded photocaged Asp and applied it to the photocontrolled manipulation of a panel of proteins including firefly luciferase, kinases (e.g., BRAF), and GTPase (e.g., KRAS) as well as mimicking the in situ phosphorylation event on kinases. As a new member of the increasingly expanded amino acid-decaging toolbox, photocaged Asp may find broad applications for gain-of-function study of diverse proteins as well as biological processes in living cells.

Fluorous Liquids for Magnetic Resonance-Based Thermometry with Enhanced Responsiveness and Environmental Degradation.

Accurate temperature measurement via magnetic resonance is valuable for both in vitro and in vivo analysis of local tissue for evaluating disease pathology and medical interventions. 1H MRI-based thermometry is used clinically but is susceptible to error from magnetic field drift and low sensitivity in fatty tissue and requires a reference for absolute temperature determination. As an alternative, perfluorotributylamine (PFTBA), a perfluorocarbon liquid for 19F MRI thermometry, is based on chemical shift responsiveness and approaches the sensitivity of 1H MRI thermometry agents; however, environmental persistence, greenhouse gas concerns, and multiple resonances which can lead to MRI artifacts indicate a need for alternative sensors. Using a 19F NMR-based structure-property study of synthetic organofluorine molecules, this research develops new organofluorine liquids with improved temperature responsiveness, high signal, and reduced nonmagnetically equivalent fluorine resonances. Environmental degradation analysis using reverse-phase HPLC and quantitative 19F NMR demonstrates a rapid degradation profile mediated via the aryl fluorine core of temperature sensors. Our findings show that our lead liquid temperature sensor, DD-1, can be made in high yield in a single step and possesses an improved responsiveness over our prior work and an 83% increase in aqueous thermal responsiveness over PFTBA. Degradation studies indicate robust degradation with half-lives of less than two hours under photolysis conditions for the parent compound and formation of other fluorinated products. The improved performance of DD-1 and its susceptibility to environmental degradation highlight a new lead fluorous liquid for thermometry applications.

Selective Photodynamic Activity of Tetrakis(4-aminophenyl)porphyrins with and without Acetyl Protecting Groups on Cancer and Normal Cells.

Tetrakis(4-aminophenyl)porphyrin (1) and tetrakis(4-acetamidophenyl)porphyrin (2) were dissolved in water with the incorporation of a polysaccharide (λ-carrageenan (CGN)) as a water-solubilizing agent. Although the photodynamic activity of the CGN-2 complex was considerably lower than that of the CGN-1 complex, the selectivity index (SI; IC50 in a normal cell/IC50 in a cancer cell) of the CGN-2 complex was considerably higher than that of the CGN-1 complex. This is because the photodynamic activity of the CGN-2 complex was significantly affected by the intracellular uptakes by the normal and cancer cells. During in vivo experiments, the CGN-2 complex inhibited tumor growth under light irradiation with high blood retention compared with the CGN-1 complex and Photofrin, which exhibited lower blood retention. This study showed that the photodynamic activity and SI are influenced by substituent groups of arene in the meso-positions of porphyrin analogs.

Chemical synthesis of on demand-activated SUMO-based probe by a photocaged glycine-assisted strategy.

The transiently-activated SUMO probes are conducive to understand the dynamic control of SENPs activity. Here, we developed a photocaged glycine-assisted strategy for the construction of on demand-activated SUMO-ABPs. The light-sensitive groups installed at G92 and G64 backbone of SUMO-2 can temporarily block probes activity and hamper aspartimide formation, respectively, which enabled the efficient synthesis of inert SUMO-2 propargylamide (PA). The probe could be activated to capture SENPs upon photo-irradiation not only in vitro but also in intact cells, providing opportunities to further perform intracellular time-resolved proteome-wide profiling of SUMO-related enzymes.

Photoclick Chemistry: A Bright Idea.

At its basic conceptualization, photoclick chemistry embodies a collection of click reactions that are performed via the application of light. The emergence of this concept has had diverse impact over a broad range of chemical and biological research due to the spatiotemporal control, high selectivity, and excellent product yields afforded by the combination of light and click chemistry. While the reactions designated as "photoclick" have many important features in common, each has its own particular combination of advantages and shortcomings. A more extensive realization of the potential of this chemistry requires a broader understanding of the physical and chemical characteristics of the specific reactions. This review discusses the features of the most frequently employed photoclick reactions reported in the literature: photomediated azide-alkyne cycloadditions, other 1,3-dipolarcycloadditions, Diels-Alder and inverse electron demand Diels-Alder additions, radical alternating addition chain transfer additions, and nucleophilic additions. Applications of these reactions in a variety of chemical syntheses, materials chemistry, and biological contexts are surveyed, with particular attention paid to the respective strengths and limitations of each reaction and how that reaction benefits from its combination with light. Finally, challenges to broader employment of these reactions are discussed, along with strategies and opportunities to mitigate such obstacles.

Light-Triggered Click Chemistry.

The merging of click chemistry with discrete photochemical processes has led to the creation of a new class of click reactions, collectively known as photoclick chemistry. These light-triggered click reactions allow the synthesis of diverse organic structures in a rapid and precise manner under mild conditions. Because light offers unparalleled spatiotemporal control over the generation of the reactive intermediates, photoclick chemistry has become an indispensable tool for a wide range of spatially addressable applications including surface functionalization, polymer conjugation and cross-linking, and biomolecular labeling in the native cellular environment. Over the past decade, a growing number of photoclick reactions have been developed, especially those based on the 1,3-dipolar cycloadditions and Diels-Alder reactions owing to their excellent reaction kinetics, selectivity, and biocompatibility. This review summarizes the recent advances in the development of photoclick reactions and their applications in chemical biology and materials science. A particular emphasis is placed on the historical contexts and mechanistic insights into each of the selected reactions. The in-depth discussion presented here should stimulate further development of the field, including the design of new photoactivation modalities, the continuous expansion of λ-orthogonal tandem photoclick chemistry, and the innovative use of these unique tools in bioconjugation and nanomaterial synthesis.

Light-driven carbon-carbon bond formation via CO2 reduction catalyzed by complexes of CdS nanorods and a 2-oxoacid oxidoreductase.

Redox enzymes are capable of catalyzing a vast array of useful reactions, but they require redox partners that donate or accept electrons. Semiconductor nanocrystals provide a mechanism to convert absorbed photon energy into redox equivalents for enzyme catalysis. Here, we describe a system for photochemical carbon-carbon bond formation to make 2-oxoglutarate by coupling CO2 with a succinyl group. Photoexcited electrons from cadmium sulfide nanorods (CdS NRs) transfer to 2-oxoglutarate:ferredoxin oxidoreductase from Magnetococcus marinus MC-1 (MmOGOR), which catalyzes a carbon-carbon bond formation reaction. We thereby decouple MmOGOR from its native role in the reductive tricarboxylic acid cycle and drive it directly with light. We examine the dependence of 2-oxoglutarate formation on a variety of factors and, using ultrafast transient absorption spectroscopy, elucidate the critical role of electron transfer (ET) from CdS NRs to MmOGOR. We find that the efficiency of this ET depends strongly on whether the succinyl CoA (SCoA) cosubstrate is bound at the MmOGOR active site. We hypothesize that the conformational changes due to SCoA binding impact the CdS NR-MmOGOR interaction in a manner that decreases ET efficiency compared to the enzyme with no cosubstrate bound. Our work reveals structural considerations for the nano-bio interfaces involved in light-driven enzyme catalysis and points to the competing factors of enzyme catalysis and ET efficiency that may arise when complex enzyme reactions are driven by artificial light absorbers.

Reverse Engineering Caged Compounds: Design Principles for their Application in Biology.

Light passes through biological tissue, and so it is used for imaging biological processes in situ. Such observation is part of the very essence of science, but mechanistic understanding requires intervention. For more than 50 years a "second function" for light has emerged; namely, that of photochemical control. Caged compounds are biologically inert signaling molecules that are activated by light. These optical probes enable external instruction of biological processes by stimulation of an individual element in complex signaling cascades in its native environment. Cause and effect are linked directly in spatial, temporal, and frequency domains in a quantitative manner by their use. I provide a guide to the basic properties required to make effective caged compounds for the biological sciences.

Engineering Photomechanical Molecular Crystals to Achieve Extraordinary Expansion Based on Solid-State [2 + 2] Photocycloaddition.

Photomechanical molecular crystals are promising candidates for photoactuators and can potentially be implemented as smart materials in various fields. Here, we synthesized a new molecular crystal, (E)-3-(naphthalen-1-yl)acrylaldehyde malononitrile ((E)-NAAM), that can undergo a solid-state [2 + 2] photocycloaddition reaction under visible light (≥400 nm) illumination. (E)-NAAM microcrystals containing symmetric twinned sealed cavities were prepared using a surfactant-mediated crystal seeded growth method. When exposed to light, the hollow microcrystals exhibited robust photomechanical motions, including bending and dramatic directional expansion of up to 43.1% elongation of the original crystal length before fragmentation due to the photosalient effect. The sealed cavities inside the microcrystals could store different aqueous dye solutions for approximately one month and release the solutions instantly upon light irradiation. A unique slow-fast-slow crystal elongation kinematic process was observed, suggesting significant molecular rearrangements during the illumination period, leading to an average anisotropic crystal elongation of 37.0% (±3.8%). The significant molecular structure and geometry changes accompanying the photocycloaddition reaction, which propels photochemistry to nearly 100% completion, also facilitate photomechanical crystal expansion. Our results provide a possible way to rationally design molecular structures and engineer crystal morphologies to promote more interesting photomechanical behaviors.

THE MAKING OF A FOOTPRINT IN PROTEIN FOOTPRINTING: A REVIEW IN HONOR OF MICHAEL L. GROSS.

Within the past decade protein footprinting in conjunction with mass spectrometry has become a powerful and versatile means to unravel the higher order structure of proteins. Footprinting-based approaches has demonstrated the capacity to inform on interaction sites and dynamic regions that participate in conformational changes. These findings when set in a biological perspective inform on protein folding/unfolding, protein-protein interactions, and protein-ligand interactions. In this review, we will look at the contribution of Dr. Michael L. Gross to protein footprinting approaches such as hydrogen deuterium exchange mass spectrometry and hydroxyl radical protein footprinting. This review details the development of novel footprinting methods as well as their applications to study higher order protein structure. © 2020 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd. Mass Spec Rev.

Wide range of metabolic adaptations to the acquisition of the Calvin cycle revealed by comparison of microbial genomes.

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO2 as a potential substrate for biorefining. The most common CO2 fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO2 concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indicate yet unknown regulatory mechanisms in Calvin cycle-utilizing microbes. Our gene ranking provides targets for experiments seeking to improve CO2 fixation, or engineer novel CO2-fixing organisms.