C3 Double Bond Research Articles

Chemo‐ and Regioselective Dihydroxylation of Benzene to Hydroquinone Enabled by Engineered Cytochrome P450 Monooxygenase

AbstractHydroquinone (HQ) is produced commercially from benzene by multi‐step Hock‐type processes with equivalent amounts of acetone as side‐product. We describe an efficient biocatalytic alternative using the cytochrome P450‐BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi‐rational protein engineering strategy was developed. Highly active mutants were obtained which transform benzene in a one‐pot sequence first into phenol and then regioselectively into HQ without any overoxidation. A computational study shows that the chemoselective oxidation of phenol by the P450‐BM3 variant A82F/A328F leads to the regioselective formation of an epoxide intermediate at the C3=C4 double bond, which departs from the binding pocket and then undergoes fragmentation in aqueous medium with exclusive formation of HQ. As a practical application, an E. coli designer cell system was constructed, which enables the cascade transformation of benzene into the natural product arbutin, which has anti‐inflammatory and anti‐bacterial activities.

Chemo- and Regioselective Dihydroxylation of Benzene to Hydroquinone Enabled by Engineered Cytochrome P450 Monooxygenase.

Hydroquinone (HQ) is produced commercially from benzene by multi-step Hock-type processes with equivalent amounts of acetone as side-product. We describe an efficient biocatalytic alternative using the cytochrome P450-BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi-rational protein engineering strategy was developed. Highly active mutants were obtained which transform benzene in a one-pot sequence first into phenol and then regioselectively into HQ without any overoxidation. A computational study shows that the chemoselective oxidation of phenol by the P450-BM3 variant A82F/A328F leads to the regioselective formation of an epoxide intermediate at the C3=C4 double bond, which departs from the binding pocket and then undergoes fragmentation in aqueous medium with exclusive formation of HQ. As a practical application, an E. coli designer cell system was constructed, which enables the cascade transformation of benzene into the natural product arbutin, which has anti-inflammatory and anti-bacterial activities.

The influence of C2[dbnd]C3 double bond on the antiradical activity of flavonoid: Different mechanisms analysis

Flavonoids widely found in bee products are excellent antioxidants. The structural features are important in evaluating the antiradical activity of flavonoid. In this work, the density functional theory (DFT) methods were applied to investigate the influence of C2C3 double bond on the antiradical activity of flavonoid based on three prevalently accepted radical scavenging mechanisms from the thermodynamic aspect. It is found that the hydroxyl groups in different rings are affected variously by the C2C3 double bond and the 3OH group is most influenced. For the compounds that only differ with the C2C3 double bond, the antiradical activity of flavone or flavonol (possessing C2C3 double bond) is not always stronger than that of flavanone: in the weak polarity phases, only the antiradical activities of chrysin, galangin and morin are stronger than those of pinocembrin, pinobanksin and dihydro-morin, respectively. In polar phases, the C2C3 double bond would weaken the antiradical activity of flavonoid via enlarging the proton affinity and the antiradical activity of flavone or flavonol is weaker than that of flavanone.

Osmium Tag for Post-transcriptionally Modified RNA.

5-Methylcytidine (m5 C) and 5-methyluridine (m5 U) are highly abundant post-transcriptionally modified nucleotides that are observed in various natural RNAs. Such nucleotides were labeled through a chemical approach, as both underwent oxidation at the C5=C6 double bond, leading to the formation of osmium-bipyridine complexes, which could be identified by mass spectrometry. This osmium tag made it possible to distinguished m5 C and m5 U from their isomers, 2'-O-methylcytidine and 2'-O-methyluridine, respectively. Queuosine and 2-methylthio-N6 -isopentenyladenosine in tRNA were also tagged through complex formation.

Initial excited-state relaxation of locked retinal protonated schiff base chromophore. An insight from coupled cluster and multireference perturbation theory calculations.

The initial S1 excited-state relaxation of retinal protonated Schiff base (RPSB) analog with central C11C12 double bond locked by eight-membered ring (locked-11.8) was investigated by means of multireference perturbation theory methods (XMCQDPT2, XMS-CASPT2, MS-CASPT2) as well as single-reference coupled-cluster CC2 method. The analysis of XMCQDPT2-based geometries reveals rather weak coupling between in-plane and out-of-plane structural evolution and minor energetical relaxation of three locked-11.8 conformers. Therefore, a strong coupling between bonds length inversion and backbone out-of-plane deformation resulting in a very steep S1 energy profile predicted by CASSCF/CASPT2 calculations is in clear contradiction with the reference XMCQDPT2 results. Even though CC2 method predicts good quality ground-state structures, the excited-state structures display more advanced torsional deformation leading to ca. 0.2 eV exaggerated energy relaxation and significantly red shifted (0.4-0.7 eV) emission maxima. According to our findings, the initial photoisomerization process in locked-11.8, and possibly in other RPSB analogs, studied fully (both geometries and energies) by multireference perturbation theory may be somewhat slower than predicted by CASSCF/CASPT2 or CC2 methods. © 2018 Wiley Periodicals, Inc.

Deciphering the Mechanism of Human Carbonic Anhydrases Inhibition with Sulfocoumarins: Computational and Experimental Studies.

The reaction mechanism of the carbonic anhydrase-mediated hydrolysis of sulfocoumarins to sulfonic acids has been investigated on an enzyme cluster model using the B3LYP hybrid density functional theory (DFT) and the QST procedure for the Transition State (TS) search. A multistep process was highlighted, with the rate-determining step identified in the initial dual nucleophilic/acidic attack of the zinc-bound hydroxide ion to the sulfocoumarin sulfur atom and to the C3=C4 double bond. The reported multi-step process, combined to SAR analysis on a new set of derivatives, highlighted unprecedented mechanistic aspects of the CA-mediated prodrug activation, which in turn possess relevant consequences to the isoforms-selective inhibition profiles reported by such a class of compounds.

Hafnocene-based Bicyclo[2.1.1]hexene Germylenes - Formation, Reactivity, and Structural Flexibility.

2,5-Disilylsubstituted germole dianions 1 react with hafnocene dichloride to give hafnocene-based bicyclo[2.1.1]hexene germylenes 3. Their formation proceeds via hafnocene-germylene complexes 2 that were identified by NMR and UV spectroscopy. Germylenes 3 are stabilized by homoconjugation between the empty 4p(Ge) orbital and the π-bond of the innercyclic C2═C3 double bond. This interaction can be understood as σ2, π-coordination of the butadiene part to the dicoordinated germanium atom that leaves the 16e- hafnocene moiety electronically unsaturated. We demonstrate that this new class of germylenes might serve as ligand to a variety of low-valent transition-metal complexes. The structure of the germylene ligand in complexes with Fe(0), Ni(0), and Au(I) and in reaction products with N-heterocyclic carbenes showed an intriguing structural flexibility that allows to accommodate different electronic situations at the ligating germanium atom. The origin of this structural adaptability is the interplay between the topological flexible unsaturated germanium ring and the hafnocene group.

Stepwise Hydride Transfer in a Biological System: Insights into the Reaction Mechanism of the Light‐Dependent Protochlorophyllide Oxidoreductase

AbstractHydride transfer plays a crucial role in a wide range of biological systems. However, its mode of action (concerted or stepwise) is still under debate. Light‐dependent NADPH: protochlorophyllide oxidoreductase (POR) catalyzes the stereospecific trans addition of a hydride anion and a proton across the C17−C18 double bond of protochlorophyllide. Time‐resolved absorption and emission spectroscopy were used to investigate the hydride transfer mechanism in POR. Apart from excited states of protochlorophyllide, three discrete intermediates were resolved, consistent with a stepwise mechanism that involves an initial electron transfer from NADPH. A subsequent proton‐coupled electron transfer followed by a proton transfer yield distinct different intermediates for wild type and the C226S variant, that is, initial hydride attaches to either C17 or C18, but ends in the same chlorophyllide stereoisomer. This work provides the first evidence of a stepwise hydride transfer in a biological system.

Stepwise Hydride Transfer in a Biological System: Insights into the Reaction Mechanism of the Light-Dependent Protochlorophyllide Oxidoreductase.

Hydride transfer plays a crucial role in a wide range of biological systems. However, its mode of action (concerted or stepwise) is still under debate. Light‐dependent NADPH: protochlorophyllide oxidoreductase (POR) catalyzes the stereospecific trans addition of a hydride anion and a proton across the C17−C18 double bond of protochlorophyllide. Time‐resolved absorption and emission spectroscopy were used to investigate the hydride transfer mechanism in POR. Apart from excited states of protochlorophyllide, three discrete intermediates were resolved, consistent with a stepwise mechanism that involves an initial electron transfer from NADPH. A subsequent proton‐coupled electron transfer followed by a proton transfer yield distinct different intermediates for wild type and the C226S variant, that is, initial hydride attaches to either C17 or C18, but ends in the same chlorophyllide stereoisomer. This work provides the first evidence of a stepwise hydride transfer in a biological system.

Reaction Mechanism of Organocatalytic Michael Addition of Nitromethane to Cinnamaldehyde: A Case Study on Catalyst Regeneration and Solvent Effects.

The Michael addition of nitromethane to cinnamaldehyde has been computationally studied in the absence of a catalyst and the presence of a biotinylated secondary amine by a combined computational and experimental approach. The calculations were performed at the density functional theory (DFT) level with the M06-2X hybrid functional, and a polarizable continuum model has been employed to mimic the effect of two different solvents: dichloromethane (DCM) and water. Contrary to common assumption, the product-derived iminium intermediate was absent in both of the solvents tested. Instead, hydrating the C1–C2 double bond in the enamine intermediate directly yields the tetrahedral intermediate, which is key for forming the product and regenerating the catalyst. Enamine hydration is concerted and found to be rate-limiting in DCM but segregated into two non-rate-limiting steps when the solvent is replaced with water. However, further analysis revealed that the use of water as solvent also raises the energy barriers for other chemical steps, particularly the critical step of C–C bond formation between the iminium intermediate and nucleophile; this consequently lowers both the reaction yield and enantioselectivity of this LUMO-lowering reaction, as experimentally detected. These findings provide a logical explanation to why water often enhances organocatalysis when used as an additive but hampers the reaction progress when employed as a solvent.

Is Vitamin A an Antioxidant or a Pro-oxidant?

Antioxidant efficiency of all-trans-retinol has been studied on the basis of characteristic thermochemical properties using density functional theory. The influence of the solvent polarity has also been evaluated. It is found that retinol may act in parallel as an effective antioxidant via H atom donating as well as a pro-oxidant in yielding reactive hydroxyl radical. In fact, the lowest values of bond dissociation enthalpy were found at the C18-H and C18-OH positions. Retinol was also determined to be a good electron donor but bad acceptor in the single electron transfer (ET) reaction with hydroperoxyl (HOO•) radical. In addition, potential energy surfaces of H atom transfer (HAT) and radical adduct formation (RAF) reactions between retinol and HOO• radical were also investigated in the gas phase and in the solvent. The results demonstrated that the RAF mechanism was generally more predominant than ET and HAT ones. The most favored radical addition position was found at the C2═C3 double bond in the cyclohexenyl ring. Moreover, the radical scavenging reactivity via RAF reactions was strongly exergonic and thermodynamically feasible while the ET one was endergonic. Natural bond orbital analysis showed that the lone pairs of electrons on the oxygen atom of the HOO• radical were donated to the unoccupied antibonding orbital of transferred H atom in HAT reactions. In contrast, in the case of RAF reactions, strong interactions between 2p orbitals on oxygen atoms of the radical and the π orbital of the double bond on the retinol molecule were recognized. The results obtained in this work were in agreement with previous experimental observations.

Rapid C8-vinyl reduction of divinyl-chlorophyllide a by BciA from Rhodobacter capsulatus

Abstract Divinyl-chlorophyllide a (DV-Chlide a ) is a universal precursor for chlorophyll or bacteriochlorophyll biosynthesis in all photosynthetic organisms. Previous mutational analyses revealed that BciA works for reduction of the C8-vinyl group of DV-Chlide a. Chlorophyllide oxidoreductase (COR) reduces the C7=C8 double bond of Chlide a bearing the C8-ethyl group, but also potentially catalyzes the reduction of the C8-vinyl group of DV-Chlide a. In this study, we prepared a recombinant BciA protein from the purple photosynthetic bacterium Rhodobacter capsulatus and analyzed its C8-vinyl reduction activity towards DV-Chlide a . BciA formed a functional oligomeric complex consisting of at least three rigid dimers. BciA required NADPH as an electron donor for its C8-vinyl reduction activity. The enzymatic activity of BciA towards the substrate DV-Chlide a was much higher than that of COR towards Chlide a . Phylogenetic distribution and the enzymatic parameters of BciA and COR suggest that BciA is a more recently acquired auxiliary C8-vinyl reductase, attained to meet the high demand for bacteriochlorophylls used to produce large amounts of light-harvesting complexes.

Structures of Degradation Products and Degradation Pathways of Aflatoxin B1 by High-Voltage Atmospheric Cold Plasma (HVACP) Treatment.

High-voltage atmospheric cold plasma (HVACP) is a novel nonthermal decontamination technology that has potential for use in the food industry. In this study, HVACP was applied to treat pure aflatoxin B1 (AFB1) powder on a glass slide. AFB1 was degraded by 76% using a 5 min HVACP treatment in air having 40% relative humidity. The degradation products of AFB1 were separated, and their molecular formulas were elucidated using liquid-chromatography time-of-flight mass spectrometry (HPLC-TOF-MS). Six main degradation products were observed. The structures of the degradation products were further clarified via orbitrap mass spectrometry by means of fragmentation of the parental ions. Two degradation pathways were proposed on the basis of the structure of the degradation products. Among the six degradation products, two were ozonolysis products of AFB1. The appearance of the other four degradation products indicates that AFB1 was degraded by other reactive species besides ozone that were generated during HVACP treatment. Reactive oxygen gas species are suggested as the major agents for aflatoxin degradation during HVACP treatment. Two degradation pathways of AFB1 by HVACP treatment were proposed. One pathway involves reactions in which H•, OH•, CHO• radicals are added. The other involves epoxidation by HO2• radicals and oxidation of AFB1by the combined effects of the oxidative species OH•, H2O2, and O3. According to the structure-bioactivity relationship of AFB1, the bioactivity of the AFB1 samples subjected to HVACP treatment is significantly reduced because of the disappearance of the C8═C9 double bond in the furofuran ring in all of the major degradation products as well as the modification of the lactone ring, cyclopentanone, and the methoxyl group.

Mechanisms of the Diterpene Cyclases β-Pinacene Synthase from Dictyostelium discoideum and Hydropyrene Synthase from Streptomyces clavuligerus.

Two diterpene cyclases, one from the social amoeba Dictyostelium discoideum and the other from the bacterium Streptomyces clavuligerus, with products containing a Z-configured double bond between the original C2 and C3 of geranylgeranyl diphosphate, were extensively investigated for their mechanisms through isotopic labelling experiments. The participation of geranyllinalyl diphosphate, in analogy to the role of linalyl and nerolidyl diphosphate for mono- and sesquiterpene biosynthesis, as an intermediate towards diterpenes with a Z-configured C2=C3 double bond is discussed.

Molecular Structure-Affinity Relationship of Flavonoids in Lotus Leaf (Nelumbo nucifera Gaertn.) on Binding to Human Serum Albumin and Bovine Serum Albumin by Spectroscopic Method.

Lotus leaf has gained growing popularity as an ingredient in herbal formulations due to its various activities. As main functional components of lotus leaf, the difference in structure of flavonoids affected their binding properties and activities. In this paper, the existence of 11 flavonoids in lotus leaf extract was confirmed by High Performance Liquid Chromatography (HPLC) analysis and 11 flavonoids showed various contents in lotus leaf. The interactions between lotus leaf extract and two kinds of serum albumins (human serum albumin (HSA) and bovine serum albumin (BSA)) were investigated by spectroscopic methods. Based on the fluorescence quenching, the interactions between these flavonoids and serum albumins were further checked in detail. The relationship between the molecular properties of flavonoids and their affinities for serum albumins were analyzed and compared. The hydroxylation on 3 and 3’ position increased the affinities for serum albumins. Moreover, both of the methylation on 3’ position of quercetin and the C2=C3 double bond of apigenin and quercetin decreased the affinities for HSA and BSA. The glycosylation lowered the affinities for HSA and BSA depending on the type of sugar moiety. It revealed that the hydrogen bond force played an important role in binding flavonoids to HSA and BSA.

Characterization of the Old Yellow Enzyme Homolog from Bacillus subtilis (YqjM)

AbstractOld Yellow Enzymes (OYEs) have become practical catalysts for the stereoselective trans‐hydrogenation of conjugated C=C‐double bonds. Despite their importance, in‐depth characterization of their kinetic parameters and factors influencing their activity and stability have largely been neglected in the past years. In this study, we close this gap for one of the most prominent OYEs, i. e. the OYE from Bacillus subtilis (YqjM).

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged Gt activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates Gt in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.

The reactivity of the 5‐formylcytosine with hydroxyl radical: A theoretical perspective

AbstractHydroxyl radical (•OH) damages DNA/RNA by attacking pyrimidine nucleobases through the addition reaction and H‐atom abstraction. It may attack on the new cytosine derivative (5‐formylCytosine [5‐fCyt]) causing DNA oxidative damage, whereas the study of the related mechanism is still in its infancy. In the present work, 2 distinct mechanisms of •OH‐mediated 5‐fCyt at the complete basis set methods (CBS‐QB3) and CBS‐QB3/polarized continuum model approaches have firstly been explored, the addition reaction (paths R1 ~ R3) and the abstraction reaction (paths R4 ~ R6), respectively, and it shows that the addition of •OH to the C5═C6 double bond of 5‐fCyt is more favourable than other reactions, indicating that the •OH addition to the C5 and C6 atoms have relatively high probability to happen. The proportion of the C5 and C6 adducts is large and may be detectable experimentally, which is in agreement with the conclusions of •OH‐mediated cytosine reaction reported experimentally and theoretically. These hint that the new DNA base (5‐fCyt) is easily damaged when exposed the surrounding of •OH environment. Therefore, the reducing free radical production or the addition of some antioxidants should be taken in embryonic stem cells to resistance DNA damage. Our results provide some evidences between 5‐fCyt and tumor development for the experimental scientists.

Reactivity of 5-carboxycytosine toward addition and hydrogen abstraction by ·OH in acetonitrile: a computational study

Two distinct mechanisms of -mediated 5-carboxylcytosine (5-caCyt) at the CBS-QB3 approach with polarizable continuum model in acetonitrile have firstly been explored, the addition reaction (paths R1 and R2), the H-atom abstraction reaction (paths R3-R6), respectively. It indicates that the addition of to the C5 = C6 double bond of 5-caCyt remains more favourable than the H-atom abstraction, whether in the gas or aqueous phase. Meanwhile, there is a little difference in the free energy barrier between acetonitrile and water, showing that the solvent has small impact on the reactivity of -mediated 5-caCyt.

Formation of pyrimidine-pyrimidine type DNA intrastrand cross-links: a theoretical verification.

Pyrimidine-type radicals have been demonstrated to be able to attack their 3' or 5' neighboring purine nucleotides forming diverse DNA intrastrand cross-links, but whether or not these radicals can attack their surrounding pyrimidine nucleotides forming pyrimidine-pyrimidine type DNA intrastrand cross-links remains unclear. To resolve this question, probable additions of the uracil-5-methyl (˙UCH2) radical to the C5[double bond, length as m-dash]C6 double bond of its 3'/5' neighboring pyrimidine nucleotides in the four models, 5'-T(˙UCH2)-3', 5'-C(˙UCH2)-3', 5'-(˙UCH2)T-3', and 5'-(˙UCH2)C-3', are explored in the present work employing density functional theory (DFT) methods. The C6 site of its 5' neighboring thymidine is the preferred target for ˙UCH2 radical addition, while additions of the ˙UCH2 radical to the C6 and C5 sites of its 5' neighboring deoxycytidine are found to be competitive reactions. The ˙UCH2 radical can react with both the C6 and C5 sites of its 3' neighboring pyrimidine nucleotides, but the efficiencies of these reactions are predicted to be much lower than those of the corresponding addition reactions to its 5' neighboring pyrimidine nucleotides, indicating the existence of an obvious sequence effect. All the addition products could be finally transformed into closed-shell intrastrand cross-links, the molecular masses of which are found to be exactly the same as certain MS values determined in a recent study of an X-irradiated deoxygenated aqueous solution of calf thymus DNA. The present study thus not only definitely corroborates the fact that the reactive ˙UCH2 radical can attack its 3'/5' neighboring pyrimidine nucleotides forming several pyrimidine-pyrimidine type DNA intrastrand cross-links, but also provides a plausible explanation for the identities of these structurally unknown intrastrand cross-links.