Chemistry

To predict the branching between energetically allowed product channels, chemists often rely on statistical transition state theories or exact quantum scattering calculations on a single adiabatic potential energy surface. The potential energy surface gives the energetic barriers to each chemical reaction and allows prediction of the reaction rates. Yet, chemical reactions evolve on a single potential energy surface only if, in simple terms, the electronic wavefunction can evolve from the reactant electronic configuration to the product electronic configuration on a time scale that is fast compared to the nuclear dynamics through the transition state. The experiments reviewed here investigate how the breakdown of the Born-Oppenheimer approximation at a barrier along an adiabatic reaction coordinate can alter the dynamics of and the expected branching between molecular dissociation pathways. The work reviewed focuses on three questions that have come to the forefront with recent theory and experiments: Which classes of chemical reactions evidence dramatic nonadiabatic behavior that influences the branching between energetically allowed reaction pathways? How do the intramolecular distance and orientation between the electronic orbitals involved influence the nonadiabaticity in the reaction? How can the detailed nuclear dynamics mediate the effective nonadiabatic coupling encountered in a chemical reaction?

Advances in the study of photodissociation dynamics over the past 30 years are reviewed. An overview of experimental techniques that have been developed to extract photofragment energy and angular distributions is presented, followed by a discussion on several current topics of interest in the field of photodissociation.

This work investigates the predissociative C (1 1 B 2 ) state of SO 2 by cooling the SO 2 in a pulsed molecular beam and dispersing the emission upon resonant excitation into several different vibronic absorption features in the C state between 197 and 212 nm. Unlike at the lower excitation energies, the dispersed emission spectra at the higher excitation energies are dominated by progressions with odd quanta in the antisymmetric stretch mode 3 and combination bands with up to six quanta in the bending mode 2 . The formidable intensity for emission into vibrational states with odd quanta in the antisymmetric stretch of the jet-cooled molecule suggests that the intermediate state at high energies in the excited state is of mixed electronic character at nonsymmetric geometries, so the operative components of the transition moment for excitation and emission may be different. We discuss our results by considering the avoided crossing seam and coupling between the C (1 1 B 2 ) state and two dissociative states potentially involved in the electronic predissociation, the 3 1 A 1 and the 1 3 A 1 states.

The photodissociation dynamics of allyl chloride at 235 nm producing atomic Cl( 2 P J ;J ϭ1/2,3/2) fragments is investigated using a two-dimensional photofragment velocity ion imaging technique. Detection of the Cl( 2 P 1/2 ) and Cl( 2 P 3/2 ) products by ͓2ϩ1͔ resonance enhanced multiphoton ionization shows that primary C-Cl bond fission of allyl chloride generates 66.8% Cl( 2 P 3/2 ) and 33.2% Cl( 2 P 1/2 ). The Cl( 2 P 3/2 ) fragments evidenced a bimodal translational energy distribution with a relative weight of low kinetic energy Cl( 2 P 3/2 )/high kinetic energy Cl( 2 P 3/2 ) of 0.097/0.903. The minor dissociation channel for C-Cl bond fission, producing low kinetic energy chlorine atoms, formed only chlorine atoms in the Cl( 2 P 3/2 ) spin-orbit state. The dominant C-Cl bond fission channel, attributed to an electronic predissociation that results in high kinetic energy Cl atoms, produced both Cl( 2 P 1/2 ) and Cl( 2 P 3/2 ) atomic fragments. The relative branching for this dissociation channel is Cl( 2 P 1/2 )/͓Cl( 2 P 1/2 )ϩCl( 2 P 3/2 )͔ϭ35.5%. The average fraction of available energy imparted into product recoil for the high kinetic energy products was found to be 59%, in qualitative agreement with that predicted by a rigid radical impulsive model. Both the spin-orbit ground and excited chlorine atom angular distributions were close to isotropic. We compare the observed Cl( 2 P 1/2 )/͓Cl( 2 P 1/2 )ϩCl( 2 P 3/2 )͔ ratio produced in the electronic predissociation channel of allyl chloride with a prior study of the chlorine atom spin-orbit states produced from HCl photodissociation, concluding that angular momentum recoupling in the exit channel at long interatomic distance determines the chlorine atom spin-orbit branching.

The emission spectroscopy of acrolein ͑C 3 H 4 O͒, acrylic acid ͑C 2 H 3 COOH͒, and acryloyl chloride ͑C 2 H 3 COCl͒ excited at 199 nm elucidates the dominant electronic character of the excited state reached by the optical transition at this wavelength. Progressions in the CvC and CvO stretching overtones and various combination bands suggest the antibonding orbital has mixed *͑CvC͒/*͑CvO͒ character. We interpret the results in conjunction with ab initio calculations at the configuration interaction singles level to identify the influence of resonance in the excited state of these conjugated molecules. The results on acrylic acid are of particular interest as excitation in this absorption band produces the HOCO intermediate of the OHϩCO→HϩCO 2 reaction that is important in combustion.

This work investigates the predissociative C (1 1 B 2 ) state of SO 2 by cooling the SO 2 in a pulsed molecular beam and dispersing the emission upon resonant excitation into several different vibronic absorption features in the C state between 197 and 212 nm. Unlike at the lower excitation energies, the dispersed emission spectra at the higher excitation energies are dominated by progressions with odd quanta in the antisymmetric stretch mode 3 and combination bands with up to six quanta in the bending mode 2 . The formidable intensity for emission into vibrational states with odd quanta in the antisymmetric stretch of the jet-cooled molecule suggests that the intermediate state at high energies in the excited state is of mixed electronic character at nonsymmetric geometries, so the operative components of the transition moment for excitation and emission may be different. We discuss our results by considering the avoided crossing seam and coupling between the C (1 1 B 2 ) state and two dissociative states potentially involved in the electronic predissociation, the 3 1 A 1 and the 1 3 A 1 states.

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. ...

Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkynesubstituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor-acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

Supramolecular catalysis involves the design and characterization of synthetic macromolecules that catalyze chemical reactions. While enzymes are often cited as the inspiration for such catalysts, enzymes can also serve as hosts for non-native catalytic components. Protein-based hosts can be readily produced in E. coli and rapidly evolved for particular applications. Moreover, inherent properties of these systems, including their conformational dynamics, can be exploited for non-native transformations that occur within their interior. Studies on the peptidase activity of a prolyl oligopeptidase from Pyrococcus furiosus (Pfu POP) suggest that its unique two-domain architecture regulates substrate access and specificity. We have established that Pfu POP also serves as an efficient host for asymmetric cyclopropanation upon active-site modification with a dirhodium cofactor. To understand how Pfu POP controls both peptidase and dirhodium catalysis, we determined the crystal structures o...

Artificial metalloenzymes (ArMs) have the potential to merge key benefits of transition metal catalysts, particularly their ability to catalyze a wide range of challenging transformations, with those of enzymes, including their evolvability and capacity for molecular (i.e., substrate) recognition [1]. These topics and more are discussed in detail elsewhere in this volume, but their pursuit requires robust methods for ArM formation. Such methods are in and of themselves quite challenging to develop. Site-specific metal incorporation is required to ensure that single-site catalysts can be obtained. Compatibility with a wide range of metals and scaffolds is desirable to maximize the range of chemistries that can be explored. Compatibility with aqueous, ideally aerobic, reaction conditions and a wide range of functional groups, including those found in cellular milieu, are also important. An additional synthetic challenge is faced for ArMs generated from preformed catalysts, since these...

A bicyclo[6,1,0]nonyne-substituted 9-mesityl-10-methyl-acridinium cofactor was prepared and covalently linked to a prolyl oligopeptidase scaffold containing a genetically encoded 4-azido-Lphenylalanine residue in its active site. The resulting artificial enzyme catalyzed sulfoxidation when irradiated with visible light in the presence of air. This reaction proceeds via initial electron abstraction from the sulfide within the enzyme active site, and the protein scaffold extended the fluorescence lifetime of the acridium cofactor. The mode of sulfide activation and placement of 5 in POP-ZA 4-5 make this artificial enzyme a promising platform for developing selective photocatalytic transformations. Keywords artificial enzyme; photocatalyst; oxygenase; prolyl oligopeptidase; unnatural amino acid Chemists have long sought to design catalysts that mimic the ability of natural photosynthetic systems to harvest visible light photons and use the energy from photon absorption to drive chemical reactions. [1] The protein complexes that evolved to accomplish these tasks, photosystems II and I, catalyze light-driven water oxidation and transmembrane electron transport, respectively. [2] Photosynthetic organisms use the electrons transported by photosystem I (PSI) to reduce NADP + to NADPH, but a number of biohybrid systems capable of harnessing these electrons to reduce protons to H 2 for solar fuels applications have been developed by linking PSI to synthetic catalysts. [3] These systems show that the unique photophysical properties imparted to natural chromophores embedded within the PSI reaction center protein scaffolds [4] can be exploited for non-natural chemical reactions. Recently, an alternative biohybrid catalyst for light-driven proton reduction was created via bioconjugation of a hydrogen-evolving diiron complex to a protein scaffold lacking any bound chromaphores. [5] The photosensitizer [Ru(bpy) 3 ] 2+ was added to a solution of the biohybrid to enable intermolecular electron transfer to the diiron complex upon irradiation with visible light and catalytic proton reduction in the presence of ascorbate. Prior to this work, Gray [6] and Cheruzel [7] had demonstrated that electrons supplied to a cytochrome P450 BM3 variant by a covalently linked [Ru(bpy) 3 ] 2+ derivative upon irradiation with

New catalysts for non-directed hydrocarbon functionalization have great potential in organic synthesis. We hypothesized that incorporating a Mn-terpyridine cofactor into a protein scaffold would lead to artificial metalloenzymes (ArMs) in which the selectivity of the Mn cofactor could be controlled by the protein scaffold. We designed and synthesized a maleimide-substituted Mn-terpyridine cofactor and demonstrated that this cofactor could be incorporated into two different scaffold proteins to generate the desired ArMs. The structure and reactivity of one of these ArMs was explored, and the broad oxygenation capability of the Mn-terpyridine catalyst was maintained, providing a robust platform for optimization of ArMs for selective hydrocarbon functionalization.

Iron is an essential nutrient that is actively acquired by bacterial pathogens during infections. Clinically important Staphylococcus aureus obtains iron by extracting heme from hemoglobin (Hb) using the closely related IsdB and IsdH surface receptors. In IsdH, extraction is mediated by a conserved tridomain unit that contains its second (N2) and third (N3) NEAT domains joined by a helical linker, called IsdH N2N3. Leveraging the crystal structure of the IsdH N2N3 :Hb complex, we have probed the mechanism of heme capture using NMR, stopped-flow transfer kinetics measurements, and molecular dynamics (MD) simulations. NMR studies of the 220 kDa IsdH N2N3 :Hb complex reveal that it is dynamic, with persistent interdomain motions enabling the linker and N3 domains in the receptor to transiently engage Hb to remove its heme. An alanine mutagenesis analysis reveals that two receptor subsites positioned ~20 Å apart trigger heme release by contacting Hb's F-helix. These subsites are located within the N3 and linker domains and appear to play distinct roles in stabilizing the heme transfer transition state. Linker domain

Overexpression of rhIFN-α2b was obtained by synthesizing a codon optimized gene for IFN-α2b and expressing it in the form of inclusion bodies (IBs) in Escherichia coli. The recombinant plasmid pRSET-IFNα, which had the IFN-α2b gene under the T7 promoter, was coexpressed with plasmid pGP1-2, which carried the gene for T7 RNA polymerase under the heat inducible λPL promoter. This two plasmid expression system was optimized with respect to heat shock time, media, and time of induction in shake flask cultures. This was then scaled up into a bioreactor to get a maximum volumetric product yield of 5.2 g/L at a final OD600 of 67. At this point, the IBs represented ∼40% of the total cellular protein. This high specific product yields eased the further downstream processing steps and improved product recoveries. The IBs were isolated and purified through ion exchange followed by step refolding to give a final product yield of ∼3 g/L, which is maximum reported in the literature. The bioassay of the refolded protein gave a specific activity of ∼3 × 109 IU/mg protein.