In-situ TEM and Spectroscopy of Structures and Processes in Graphene Sandwiches and Graphene Liquid Cells

Recent developments in microfabrication technology have resulted a surge of interest in in-situ transmission electron microscopy (TEM). This dissertation will focus on in-situ imaging and spectroscopy of liquids and materials suspended in liquids using aberration-corrected scanning transmission electron microscopy. I have developed a novel approach to in-situ microscopy that allows the encapsulation of liquid-containing samples using monolayers of graphene. Transmission electron microscopy and spectroscopy is utilized to characterize several beam sensitive materials and processes in a liquid environment at atomic resolution, obtaining information including structures, elemental distribution, bonding information, even phase change and valence state transition in physical and biochemical activities. Radiolysis modeling is performed to assist liquid cell design, as well as control of electron microscope parameters, allowing liquid chemistry modulation by electronic signal. This also allows implementation of graphene liquid cells as nano-scale chemical reactors which enable the precise control of radial and ionic concentration for reaction kinetics modulation as a function of space and time. These approaches can be combined to solve problems in a liquid phase with unprecedented resolution

Machine Learning Approach to Enable Spectral Imaging Analysis for Particularly Complex Nanomaterial Systems

Scanning transmission electron microscopy-based electron energy loss spectroscopy spectral imaging (STEM-EELS-SI) has been widely used in material research to capture a wealth of information, including elemental, electron density, and bonding state distributions. However, its exploitation still faces many challenges due to the difficulty of extracting information from noisy and overlapping edges in the convoluted spatial and spectroscopic data set. A traditional EELS spectral imaging analysis lacks the capability to isolate noise and deconvolute such overlapping edges, which either limits the resolution or the signal-to-noise ratio of the maps generated by EELS-SI. Existing machine learning (ML) algorithms can achieve denoising and deconvolution to a certain extent, but the extracted spectra lack physical meaning. To address these challenges, we have developed a ML method tailored to a spectral imaging analysis system and based on a non-negative robust principal component analysis. This approach offers an effective way to analyze EELS spectral images with improved space-time resolution, signal-to-noise ratio, and the capability to separate subtle differences in the spectrum. We apply this algorithm to 13 nanomaterial systems to show that ML can greatly improve image quality compared to a traditional approach, especially for more challenging systems. This will expand the type of nanomaterial systems that can be characterized by EELS-SI, and aid the analysis of structural, chemical, and electronic properties that are otherwise difficult to obtain

Molecular Dynamics and Density Functional Studies of Substrate Binding and Catalysis of Arginine Deiminase

The active-site dynamics of arginine deiminase (ADI) complexed with the arginine substrate are investigated with ns molecular dynamics for the wildtype ADI and several mutants. It is shown that the substrate is held in the active site by an extensive hydrogen bond network, which may be weakened by substitution of active-site residues. In addition, the initial step of the catalysis is explored in several truncated active-site models with density functional theory. Evidence is presented in support of the hypothesis that the nucleophilic attack of the ADI Cys thiol at the guanidino carbon of the substrate is initiated by substrate-mediated proton transfer to a His residue in the catalytic triad (Cys-His-Glu). In addition, the active-site residues are found to strongly influence the reaction profile, consistent with their important role in catalysis

Photocyclization Reactions of Cyclohexa- and Cyclopenta-Fused Pyridinium Salts. Factors Governing Regioselectivity

The results of studies described in this report show that irradiation of 1,2-cyclopenta-fused pyridinium perchlorate in aqueous base promotes a remarkably regioselective photocyclization reaction that results in exclusive formation of a single tricyclic allylic alcohol. Moreover, transformation of this photoproduct to a spirocyclic amido diester followed by enzymatic desymmetrization produces an enantiomerically pure monoalcohol. This chemistry comprises a highly concise sequence for the preparation of what should become a useful synthon in synthetic organic chemistry

Photocyclization Reactions of Cyclohexa- and Cyclopenta-Fused Pyridinium Salts. Factors Governing Regioselectivity

The results of studies described in this report show that irradiation of 1,2-cyclopenta-fused pyridinium perchlorate in aqueous base promotes a remarkably regioselective photocyclization reaction that results in exclusive formation of a single tricyclic allylic alcohol. Moreover, transformation of this photoproduct to a spirocyclic amido diester followed by enzymatic desymmetrization produces an enantiomerically pure monoalcohol. This chemistry comprises a highly concise sequence for the preparation of what should become a useful synthon in synthetic organic chemistry

Synthesis of Enantiopure <i>tert</i>-Butanesulfinamide from <i>tert</i>-Butanesulfinyloxazolidinone

A three-step procedure for the preparation of enantiopure tert-butanesulfinamide 6 in 51% overall yield is described starting from (1R,2S)-N-Cbz-1,2-diphenylaminoethanol. The key step is the reaction of tert-butylmagnesium chloride with N-Cbz-4,5-diphenyl-1,2,3-oxathiazolidine-2-oxide 2 to afford the optical pure tert-butylsulfinyl-4,5-diphenyl-1,3-oxazolidinone 5 via an 1,5-alkoxy anion rearrangement, which is then subject to ammonia hydrolysis with LiNH2 in liquid ammonia to give (R)-tert-butanesulfinamide 6

Photocyclization Reactions of Cyclohexa- and Cyclopenta-Fused Pyridinium Salts. Factors Governing Regioselectivity

The results of studies described in this report show that irradiation of 1,2-cyclopenta-fused pyridinium perchlorate in aqueous base promotes a remarkably regioselective photocyclization reaction that results in exclusive formation of a single tricyclic allylic alcohol. Moreover, transformation of this photoproduct to a spirocyclic amido diester followed by enzymatic desymmetrization produces an enantiomerically pure monoalcohol. This chemistry comprises a highly concise sequence for the preparation of what should become a useful synthon in synthetic organic chemistry

Contributions of Long-Range Electrostatic Interactions to 4-Chlorobenzoyl-CoA Dehalogenase Catalysis: A Combined Theoretical and Experimental Study^†

It is well established that electrostatic interactions play a vital role in enzyme catalysis. In this work, we report theory-guided mutation experiments that identified strong electrostatic contributions of a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase catalysis. The Glu232Asp mutant was found to bind the substrate analogue 4-methylbenzoyl-CoA more tightly than does the wild-type dehalogenase. In contrast, the kcat for 4-chlorobenzoyl-CoA conversion to product was reduced 10000-fold in the mutant. UV difference spectra measured for the respective enzyme−ligand complexes revealed an ∼3-fold shift in the equilibrium of the two active site conformers away from that inducing strong π-electron polarization in the ligand benzoyl ring. Increased substrate binding, decreased ring polarization, and decreased catalytic efficiency indicated that the repositioning of the point charge in the Glu232Asp mutant might affect the orientation of the Asp145 carboxylate with respect to the substrate aromatic ring. The time course for formation and reaction of the arylated enzyme intermediate during a single turnover was measured for wild-type and Glu232Asp mutant dehalogenases. The accumulation of arylated enzyme in the wild-type dehalogenase was not observed in the mutant. This indicates that the reduced turnover rate in the mutant is the result of a slow arylation of Asp145, owing to decreased efficiency in substrate nucleophilic attack by Asp145. To rationalize the experimental observations, a theoretical model is proposed, which computes the potential of mean force for the nucleophilic aromatic substitution step using a hybrid quantum mechanical/molecular mechanical method. To this end, the removal or reorientation of the side chain charge of residue 232, modeled respectively by the Glu232Gln and Glu232Asp mutants, is shown to increase the rate-limiting energy barrier. The calculated 23.1 kcal/mol free energy barrier for formation of the Meisenheimer intermediate in the Glu232Asp mutant represents an increase of 6 kcal/mol relative to that of the wild-type enzyme, consistent with the 5.6 kcal/mol increase calculated from the difference in experimentally determined rate constants. On the basis of the combination of the experimental and theoretical evidence, we hypothesize that the Glu232(B) residue contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile

The Electrostatic Driving Force for Nucleophilic Catalysis in l-Arginine Deiminase: A Combined Experimental and Theoretical Study

l-Arginine deiminase (ADI) catalyzes the hydrolysis of l-arginine to form l-citrulline and ammonia via two partial reactions. A working model of the ADI catalytic mechanism assumes nucleophilic catalysis by a stringently conserved active site Cys and general acid−general base catalysis by a stringently conserved active site His. Accordingly, in the first partial reaction, the Cys attacks the substrate guanidino Cζ atom to form a tetrahedral covalent adduct, which is protonated by the His at the departing ammonia group to facilitate the formation of the Cys-S-alkylthiouronium intermediate. In the second partial reaction, the His activates a water molecule for nucleophilic addition at the thiouronium Cζ atom to form the second tetrahedral intermediate, which eliminates the Cys in formation of the l-citrulline product. The absence of a basic residue near the Cys thiol suggested that the electrostatic environment of the Cys thiol, in the enzyme–substrate complex, stabilizes the Cys thiolate anion. The studies described in this paper explore the mechanism of stabilization of the Cys thiolate. First, the log(kcat/Km) and log kcat pH rate profiles were measured for several structurally divergent ADIs to establish the pH range for ADI catalysis. All ADIs were optimally active at pH 5, which suggested that the Cys pKa is strongly perturbed by the prevailing electrostatics of the ADI active site. The pKa of the Bacillus cereus ADI (BcADI) was determined by UV−pH titration to be 9.6. In contrast, the pKa determined by iodoacetamide Cys alkylation is 6.9. These results suggest that the negative electrostatic field from the two opposing Asp carboxylates perturbs the Cys pKa upward in the apoenzyme and that the binding of the iodoacetamide (a truncated analogue of the citrulline product) between the Cys thiol and the two Asp carboxylates shields the Cys thiol, thereby reducing its pKa. It is hypothesized that the bound positively charged guanidinium group of the l-arginine substrate further stabilizes the Cys thiolate. The so-called “substrate-assisted” Cys ionization, first reported by Fast and co-workers to operate in the related enzyme dimethylarginine dimethylaminohydrolase [Stone, E. M., Costello, A. L., Tierney, D. L., and Fast, W. (2006) Biochemistry 45, 5618–5630], was further explored computationally in ADI by using an ab initio quantum mechanics/molecular mechanics method. The energy profiles for formation of the tetrahedral intermediate in the first partial reaction were calculated for three different reaction scenarios. From these results, we conclude that catalytic turnover commences from the active configuration of the ADI(l-arginine) complex which consists of the Cys thiolate (nucleophile) and His imidazolium ion (general acid) and that the energy barriers for the nucleophilic addition of Cys thiolate to the thiouronium Cζ atom and His imidazolium ion-assisted elimination from the tetrahedral intermediate are small