Investigation of Reactive Oxygen Species production in Human Hepatocytes

1. Aim/Background: Reactive oxygen species (ROS) have been identified as compounds responsible for producing cellular damage. The purpose of this research is to examine if there is production of reactive oxygen species through free radical intermediates within human hepatocytes treated with morphine, bilirubin, or furosemide. The investigation examines the early stages of biotransformation by measuring the levels of reactive oxygen species produced inside of the treated hepatocytes within the first and second hours of treatment. The experiment was designed upon a case of a jaundiced (elevated bilirubin) infant who received morphine and furosemide and later died through unknown mechanisms. The experiment looks to examine if these drug compounds could contribute to cellular damage. This can help to further understand the potential interactions and complications of free radical intermediates produced during the phases of biotransformation. 2. Method: Previously cultured human hepatocytes were washed by centrifugation and re-suspended in 1x supplemental buffer to a concentration of 1x106 cells/mL and seeded in a dark clear bottom 96-well microplate at 100,000 stained cells/well. The cells were treated with either furosemide, morphine, bilirubin, a Tert-Butyl hydro peroxide (TBHP) positive control, or left as a background. Reactive oxygen generated in the presence of these agents were quantified by fluorescence excitation/emission measurement at 495nm/529nm. Fluorescence was measured at one and two hours. ROS generated convert 2',7'-dichlorodihydrofluorescein diacetate to 2',7'-dichlorodihydrofluorescein within the cells, which fluoresces. The fluorescence intensity detected is equivalent to the level of ROS generated. Wells that were untreated were used as blanks and subtracted from background and TBPH. 3. Results: Furosemide and Morphine did not produce statistically significant levels of ROS (p >0.05) above the background in both hours 1 and 2 of biotransformation and ROS measurement (Figure 1). Although Bilirubin did not produce statistically significant (p >0.05) levels of ROS above the background (Figure 2) during the first hour, it did produce statistically significant levels in the second hour of biotransformation. Each compound’s level of ROS was reduced during the second hour, signaling the removal of intermediate ROS metabolites (Figure 2). The production of ROS in each compound signifies that there is biotransformation to an intermediate that produces ROS. 4. Conclusion: The production of ROS above the background by each of the compounds shows there is an intermediate free radical compound that is produced during the biotransformation of each compound [21]. In this study, although furosemide and morphine did not produce statistically significant levels of ROS in both hours of biotransformation, bilirubin did produce significant levels of ROS in the second hour of biotransformation. This finding is in line with previous studies that shows morphine to offer protective effects against ROS production [16, 17]; and bilirubin demonstrating deleterious production of ROS at high doses [18]. Further work must be done to examine the correlation between the levels of ROS and extent of hepatocellular damage.

Photoinitiated Multicomponent Anti-Markovnikov Alkoxylation over Graphene Oxide

The development of graphene oxide–based heterogeneous materials with an economical and environmentally–friendly manner has the potential to facilitate many important organic transformations but proves to have few relevant reported reactions. Herein, we explore the synergistic role of catalytic systems driven by graphene oxide and visible light that form nucleophilic alkoxyl radical intermediates, which enable an anti-Markovnikov addition exclusively to the terminal alkenes, and then the produced benzyl radicals are subsequently added with N–methylquinoxalones. This photoinduced cascade radical difunctionalization of olefins offers a concise and applicable protocol for constructing alkoxyl–substituted N–methylquinoxalones.

Visible-Light-Induced Transition-Metal and Photosensitizer Free Decarbonylative Addition of Amino-Arylaldehydes to Ketones

Decarbonylative-coupling reaction is generally promoted by transition-metal (via organometallic complexes) or peroxide (via radical intermediates), often at high temperature to facilitate the CO release. Herein, a visible-light-induced, transition-metal and external...

Using Enzymes to Tame Nitrogen-Centered Radicals for Enantioselective Hydroamination

The construction of C–N bonds is essential for the preparation of numerous molecules critical to modern society1,2. Nature has evolved enzymes to facilitate these transformations using nucleophilic and nitrene transfer mechanisms3,4. However, neither natural nor engineered enzymes are known to generate and control nitrogen-centered radicals, which serve as valuable species for C–N bond formation. Herein, we describe a platform for generating nitrogen-centered radicals within protein active sites, thus enabling asymmetric hydroamination reactions. Using flavin- dependent ‘ene’-reductases with an exogenous photoredox catalyst, amidyl radicals are generated selectively within the protein active site. Empowered by directed evolution, these enzymes are engineered to catalyze 5-exo, 6-endo, 7-endo, 8-endo, and intermolecular hydroamination reactions with high levels of enantioselectivity. Mechanistic studies suggest that radical initiation occurs via an enzyme-gated mechanism, where the protein thermodynamically activates the substrate for reduction by the photocatalyst. Molecular dynamics studies suggest that the enzymes bind substrates using non-canonical binding interactions, which may serve as a handle to further manipulate reactivity. This approach demonstrates the versatility of these enzymes for controlling the reactivity of high-energy radical intermediates and highlight the opportunity for synergistic catalyst strategies to unlock new enzymatic functions.

Tropane and Related Alkaloid Skeletons via a Radical [3+3]-Annulation Process

A mild and simple protocol for the synthesis of 8-azabicyclo[3.2.1]octane and 9-azabicyclo[3.3.1]nonane derivatives is described. It provides these valuable bicyclic alkaloid skeletons in good yields and high levels of diastereoselectivity from simple and readily available starting materials using visible-light photoredox catalysis. This unprecedented annulation process takes advantage of the unique reactivity of ethyl 2-(acetoxymethyl)acrylate as a 1,3-bis radical acceptor and of cyclic N,N-dialkylanilines as radical 1,3-bis radical donors. The success of this process relies on efficient electron transfer processes and highly selective deprotonation of aminium radical cations leading to the key α-amino radical intermediates.

Tropane and Related Alkaloid Skeletons via a Radical [3+3]-Annulation Process

A mild and simple protocol for the synthesis of 8-azabicyclo[3.2.1]octane and 9-azabicyclo[3.3.1]nonane derivatives is described. It provides these valuable bicyclic alkaloid skeletons in good yields and high levels of diastereoselectivity from simple and readily available starting materials using visible-light photoredox catalysis. This unprecedented annulation process takes advantage of the unique reactivity of ethyl 2-(acetoxymethyl)acrylate as a 1,3-bis radical acceptor and of cyclic N,N-dialkylanilines as radical 1,3-bis radical donors. The success of this process relies on efficient electron transfer processes and highly selective deprotonation of aminium radical cations leading to the key α-amino radical intermediates.

Switchable radical carbonylation by polarity-regulation

Carbonylation reactions involving CO as readily available C1 synthons have become one of the most important tools for construction of carbonyl compounds from feedstock chemicals in modern chemical synthesis. Whereas numerous catalytic methods for carbonylation reactions proceeding via ionic or radical pathways have been reported, an inherent limitation to these methods is the need to control switchable single and double carbonylative formation of value-added products from the same and simple starting materials. Here we describe a new strategy that exploits simple visible-light-driven photoredox catalysis to regulate the polarity of coupling partners to drive switchable radical carbonylation reactions. Controlled trap of various alkyl radicals by single or double CO thereby proceed smoothly with excellent selectivity in the presence of various amine nucleophiles at room temperature, generating valuable amides and α-ketoamides in a versatile and controlled fashion. Combined experimental and DFT computational studies suggest that trap of the initially formed acyl radical by the second molecule of CO to form α-ketoacyl radical is a facile but reversible process; and photoredox-catalyzed SET oxidation of natural nucleophilic amines into relatively electrophilic nitrogen radical cations is responsible for switchable coupling with such two radical intermediates.