Engineering and Characterization of Cytochrome P450 Enzymes for Nitrogen-Atom Transfer Reactions

The creation of novel enzyme activity is a great challenge to protein engineers, but nature has done so repeatedly throughout the process of natural selection. I begin by outlining the multitude of distinct reactions catalyzed by a single enzyme class, cytochrome P450 monooxygenases. I discuss the ability of cytochrome P450 to generate reactive intermediates capable of diverse reactivity, suggesting this enzyme can also be used to generate novel reactive intermediates in the form of metal-carbenoid and nitrenoid species. I then show that cytochrome P450 from Bacillus megaterium (P450_BM3) and its isolated cofactor can catalyze metal-nitrenoid transfer in the form of intramolecular C–H bond amination. Mutations to the protein sequence can enhance the reactivity and selectivity of this transformation significantly beyond that of the free cofactor. Next, I demonstrate an intermolecular nitrene transfer reaction catalyzed by P450_BM3 in the form of sulfide imidation. Understanding that sulfur heteroatoms are strong nucleophiles, I show that increasing the sulfide nucleophilicity through substituents on the aryl sulfide ring can dramatically increase reaction productivity. To explore engineering nitrenoid transfer in P450BM3, active site mutagenesis is employed to tune the regioselectivity intramolecular C–H amination catalysts. The solution of the crystal structure of a highly selective variant demonstrates that hydrophobic residues in the active site strongly modulate reactivity and regioselectivity. Finally, I use a similar strategy to develop P450-based catalysts for intermolecular olefin aziridination, demonstrating that active site mutagenesis can greatly enhance this nitrene transfer reaction. The resulting variant can catalyze intermolecular aziridination with more than 1000 total turnovers and enantioselectivity of up to 99% ee.

Bonding quartz wafers by the atom transfer radical polymerization of the glycidyl methacrylate at mild temperature

In this paper, we presented a novel covalent bonding process between two quartz wafers at 300 degrees C. High-quality wafer bonding was formed by the hydroxylization, aminosilylation and atom transfer radical polymerization (ATRP) of glycidyl methacrylate (GMA), respectively, on quartz wafer surfaces, followed by close contact of the GMA functional wafer and the aminosilylation wafer, the epoxy group opening ring reaction was catalyzed by the amino and solidified to form the covalent bonding of the quartz wafers. The shear force between two wafers in all bonding samples was higher than 1.5 MPa. Microfluidic chips bonded by the above procedures had high transparency and the present procedure avoided the adhesive to block or flow into the channel.

The study of synthesis of poly(ethylene oxide)-b-poly(N,N-dimethylacrylamide) by atom transfer radical polymerization and self-assembly in selective solvents

Poly( ethylene oxide)-b-poly(N, N-dimethylacrylamide) (PEO-b-PDMA) was synthesized by successive atom transfer radical polymerization (ATRP) of N, N-dimethylacrylamide (DMA) monomer using PEO-Br macro initiators as initiator, CuBr and 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazamacrocyclotetra decane (Me-6[14] aneN(4)) as catalyst and ligand. PEO-Br macroinitiator was synthesized by esterification of PEO with 2-bromoisobutyryl bromide. GPC and H-1 NMR studies show that the plot of ln([DMA](0)/[ DMA]) against the reaction time is linear, and the molecular weight of the resulting PDMA increased linearly with the conversion. Within 3 h, the polymerization can reach almost 60% of conversion. PEO-b-PDMA copolymer with low polydispersity index (M-w/M-n approximate to 1.1) is obtained. Self-assembly of PEO-b-PDMA in selective solvents is also studied. It could self-assemble into micelles in methanol/acetone (1/10, v/v) solution. TEM analyses of the PEO-b-PDMA micelles with narrow size distribution revealed that their size and shape depend much on the copolymer composition.

Oxygen Atom Transfer Reactions from Mimoun Complexes to Sulfides and Sulfoxides. A Bonding Evolution Theory Analysis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Synthesis of Macrocyclic Polymers Formed via Intramolecular Radical Trap-Assisted Atom Transfer Radical Coupling

The synthesis of cyclic polystyrene (Pst) with an alkoxyamine functionality has been accomplished by intramolecular radical coupling in the presence of a nitroso radical trap Linear alpha,omega-dibrominated polystyrene, produced by the atom transfer radical polymerization (ATRP) of styrene using a dibrominated initiator, was subjected to chain-end activation via the atom transfer radical coupling (ATRC) process under pseudodilute conditions in the presence of 2-methyl-2-nitrosopropane (MNP). This radical trap-assisted, intramolecular ATRC (RTA-ATRC) produced cyclic polymers in greater than 90% yields possessing < G > values in the 0.8-0.9 range as determined by gel permeation chromatography (GPC). Thermal-induced opening of the cycles, made possible by the incorporated alkoxyamine, resulted in a return to the original apparent molecular weight, further supporting the formation of cyclic polymers in the RTA-ATRC reaction. Liquid chromatography-mass spectrometry (LC-MS) provided direct confirmation of the cyclic architecture and the incorporation of the nitroso group into the macrocycle RTA-ATRC cyclizations carried out with faster rates of polymer addition into the redox active solution and/or in the presence of a much larger excess of MNP (up to a 250:1 ratio of MNP:C-Br chain end) still yielded cyclic polymers that contained alkoxyamine functionality.

One Pot, Two Step Sequence Converting Atom Transfer Radical Polymerization Directly to Radical Trap-Assisted Atom Transfer Radical Coupling

Monobrominated polystyrene (PStBr) chains were prepared using standard atom transfer radical polymerization (ATRP) procedures at 80 °C in THF, with monomer conversions allowed to proceed to approximately 40%. At this time, additional copper catalyst, reducing agent, and ligand were added to the unpurified reaction mixture, and the reaction was allowed to proceed at 50 °C in an atom transfer radical coupling (ATRC) phase. During this phase, polymerization continued to occur as well as coupling; expected due to the substantial amount of residual monomer remaining. This was confirmed using gel permeation chromatography (GPC), which showed increases in molecular weight not matching a simple doubling of the PStBr formed during ATRP, and an increase in monomer conversion after the second phase. When the radical trap 2-methyl-2-nitrosopropane (MNP) was added to the ATRC phase, no further monomer conversion occurred and the resulting product showed a doubling of peak molecular weight (Mp), consistent with a radical trap-assisted ATRC (RTA-ATRC) reaction.

One Pot, Two Step Sequence Converting Atom Transfer Radical Polymerization Directly to Radical Trap-Assisted Atom Transfer Radical Coupling

Monobrominated polystyrene (PStBr) chains were prepared using standard atom transfer radical polymerization (ATRP) procedures at 80 degrees C in THF, with monomer conversions allowed to proceed to approximately 40%. At this time, additional copper catalyst, reducing agent, and ligand were added to the unpurified reaction mixture, and the reaction was allowed to proceed at 50 degrees C in an atom transfer radical coupling (ATRC) phase. During this phase, polymerization continued to occur as well as coupling; expected due to the substantial amount of residual monomer remaining. This was confirmed using gel permeation chromatography (GPC), which showed increases in molecular weight not matching a simple doubling of the PStBr formed during ATRP, and an increase in monomer conversion after the second phase. When the radical trap 2-methyl-2-nitrosopropane (MNP) was added to the ATRC phase, no further monomer conversion occurred and the resulting product showed a doubling of peak molecular weight (M-p), consistent with a radical trap-assisted ATRC (RTA-ATRC) reaction. (C) 2013 Elsevier Ltd. All rights reserved.

The Synthesis of Macrocyclic Polystyrene by Sequential Atom Transfer Radical Reactions

A method for the production of macrocyclic polystyrene via ring closing of a linear !,"-dibrominated polystyrene by an Atom Transfer Radical Coupling (ATRC) reaction is described. The dibrominated polystyrene chain was produced from two simultaneous atom transfer radical polymerizations (ATRPs) originating from a dibrominated benzal bromide initiator. To ensure the retention of the halogen end groups polymerization was allowed to proceed to less than 50% conversion. Using this precursor in an intramolecular ATRC (ring closing) reaction was found to yield in excess of 90% cyclic product based on refractive index-gel permeation chromatography (GPC) analysis. The cyclic architecture of the polymer was verified by GPC, Nuclear Magnetic Resonance (NMR), and mass spectrometry analysis. The utility of this method has been expanded by the addition of 2-methyl-2-nitrosopropane to the coupling reaction, which allows for the coupling to proceed at a faster rate and to yield macrocycles with incorporated alkoxyamine functionality. The alkoxyamine functionality allows for degradation of the cycles at high temperatures (>125° C) and we hypothesize that it may allow the macrocycles to act as a macroinitiator for a ring expansion polymerization in future studies.

Dimerization of Poly(methyl methacrylate) Chains Using Radical Trap-Assisted Atom Transfer Radical Coupling

End-brominated poly(methyl methacrylate) (PMMABr) was prepared by atom transfer radical polymerization (ATRP) and employed in a series of atom transfer radical coupling (ATRC) and radical trap-assisted ATRC (RTA-ATRG) reactions. When coupling reactions were performed in the absence of a nitroso radical trap-traditional ATRC condition-very little coupling of the PMMA chains was observed, consistent with disproportionation as the major termination pathway for two PMMA chain-end radicals in our reactions. When 2-methyl-2-nitrosopropane (MNP) was used as the radical trap, coupling of the PMMA chains in this attempted RTA-ATRC reaction was again unsuccessful, owing to capping of the PMMA chains with a bulky nitroxide and preventing further coupling. Analogous reactions performed using nitrosobenzene (NBz) as the radical trap showed significant dimerization, as observed by gel permeation chromatography (GPC) by a shift in the apparent molecular weight compared to the PMMABr precursors. The extent of coupling was found to depend on the concentrion of NBz compared to the PMMABr chain ends, as well as the temperature and time of the coupling reaction. To a lesser extent, the concentrations of copper(I) bromide (CuBr), nitrogen ligand (N,N,N',N',N"-pentamethyldiethylenetriamine = PMDETA), and elemental copper (Cu) were also found to play a role in the success of the RTA-ATRC reaction. The highest levels of dimerization were observed when the coupling reaction was carried out at 80 degrees C for 0.5h, with ratio of 1:4:2.5:8:1 equiv of NBz: CuBr:Cu:PMDETA:PMMABr.

On the Methyl-Transfer Reaction in Crystalline Methyl 2-(Methylthio)benzenesulfonate: a Thermally Induced Non-Topochemical Solid-State Reaction

The rearrangement of methyl 2-(methylthio)benzenesulfonate (1) to the zwitterionic 2-(dimethyl-sulfonium)benzenesulfonate (2) is known to proceed in solution by intermolecular Me transfers. The same rearrangement has been observed to occur in crystalline 1, but the crystal structure shows that the molecular packing is not conducive to intermolecular Me transfer. The reaction has been carried out with mixed crystals composed of 1 and deuteriomethylated (D6)-l. By fast-atom-bombardment mass spectroscopy, it has been shown that the product consists of a 1:2:1 mixture of the non-, tri-, and hexadeuterated species, the mixture expected, if the solid-state reaction proceeds by intermolecular Me transfers. From this result, together with the slower rates of conversion in the single crystal compared with the melt, it can be concluded that the reaction must occur not topochemically but rather at defects such as microcavities, surfaces, and other irregularities in the ordered crystal arrangement.

Atom transfer radical polymerisation - towards the synthesis of a fully-functional photorefractive polymer

Atom transfer radical polymerisation (ATRP) of styrene in xylene solution initiated with 1-phenylethyl bromide and mediated by CuBr/N-propyl-2- pyridinemethanimine catalyst complex was studied. The polymerisation was ill-controlled, yielding polymers with broad molecular weight distributions and values of number average molecular weight considerably higher than the theoretical values calculated from 100% initiator efficiency. The degree of control afforded over the polymerisation was enhanced by use of a more soluble catalyst complex, CuBr/N-octyl-2-pyridinemethanimine. Furthermore, the use of a more polar solvent, diglyme, generated a homogeneous catalyst complex that facilitated the production of polymers having narrow molecular weight distributions (1.10 < PDi < 1.20). The kinetics of the atom transfer radical polymerisation of methyl methacrylate at 90°C in diglyme solution initiated with ethyl-2-bromoisobutyrate and mediated by CuBr/N-octyl-2-pyridinemethanimine was studied and the orders of the reaction were established. The effect on the rate of polymerisation of the ratio of CuBr:N-octyl-2-pyridinemethanimine was also determined. The temperature dependencies of the rate of polymerisation of methyl methacrylate in diglyme solution and xylene solution were studied, and were found to be non-linear and dependent upon the polarity of the solvent. The use of highly polar aprotic solvents, such as N,N-dimethylformamide and dimethylsulphoxide, was found to be detrimental to the degree of control afforded over the polymerisation of methyl methacrylate. This was circumvented by use of a 5-fold excess, over that conventionally used, of catalyst complex. The atom transfer radical polymerisation of (4-nitrophenyl)-[3-[N-[2- (methacryloyloxy)ethyl]carbazolyl]]diazene in dimethyl sulphoxide solution was studied. Although homopolymerisation yielded only oligomers, copolymerisation of this monomer with methyl methacrylate was found to be readily achievable. Keywords: ATRP, Styrene; Methyl methacrylate; Polar solvents; Fully-functional photorefractive polymer. 2

Influence of Solvent on Photoinduced Electron-Transfer Reaction: Time-Resolved Resonance Raman Study

Time-resolved resonance Raman spectroscopy (TR3) has been used to study the effect of solvent polarity on the mechanism and nature of intermediates formed in photoinduced electron-transfer reaction between triplet flouranil ((FL)-F-3) and tetramethylbenzene (TMB). Comparison of the TR3 spectra in polar, nonpolar, and medium polar media suggests that formation of radical anion due to electron-transfer reaction between (FL)-F-3 and TMB is favored in more polar solvents, whereas ketyl radical formation is more favored in less polar media. Compared to ketyl radical, the extent of radical anion formation is negligible in nonpolar solvents. Therefore, it is inferred that in nonpolar media ketyl radical is mainly generated by hydrogen-transfer reaction in the encounter complex between (FL)-F-3 and TMB. In solvents of medium polarity, the ion-pair decay leads to the formation of both ketyl radical and ketyl radical formed from the encounter between triplet state and the donor. Thus, competition between the formation of ketyl radical and ion pair is influenced by the solvent polarity. The nature of the ion pair in different solvent polarity has been investigated from the changes observed in the vibrational frequency of (fluoranil) FL part of the complex.

Disjunct eddy covariance measurements of volatile organic compound fluxes using proton transfer reaction mass spectrometry

Volatile organic compounds (VOCs) are emitted into the atmosphere from natural and anthropogenic sources, vegetation being the dominant source on a global scale. Some of these reactive compounds are deemed major contributors or inhibitors to aerosol particle formation and growth, thus making VOC measurements essential for current climate change research. This thesis discusses ecosystem scale VOC fluxes measured above a boreal Scots pine dominated forest in southern Finland. The flux measurements were performed using the micrometeorological disjunct eddy covariance (DEC) method combined with proton transfer reaction mass spectrometry (PTR-MS), which is an online technique for measuring VOC concentrations. The measurement, calibration, and calculation procedures developed in this work proved to be well suited to long-term VOC concentration and flux measurements with PTR-MS. A new averaging approach based on running averaged covariance functions improved the determination of the lag time between wind and concentration measurements, which is a common challenge in DEC when measuring fluxes near the detection limit. The ecosystem scale emissions of methanol, acetaldehyde, and acetone were substantial. These three oxygenated VOCs made up about half of the total emissions, with the rest comprised of monoterpenes. Contrary to the traditional assumption that monoterpene emissions from Scots pine originate mainly as evaporation from specialized storage pools, the DEC measurements indicated a significant contribution from de novo biosynthesis to the ecosystem scale monoterpene emissions. This thesis offers practical guidelines for long-term DEC measurements with PTR-MS. In particular, the new averaging approach to the lag time determination seems useful in the automation of DEC flux calculations. Seasonal variation in the monoterpene biosynthesis and the detailed structure of a revised hybrid algorithm, describing both de novo and pool emissions, should be determined in further studies to improve biological realism in the modelling of monoterpene emissions from Scots pine forests. The increasing number of DEC measurements of oxygenated VOCs will probably enable better estimates of the role of these compounds in plant physiology and tropospheric chemistry. Keywords: disjunct eddy covariance, lag time determination, long-term flux measurements, proton transfer reaction mass spectrometry, Scots pine forests, volatile organic compounds