Industrial chemicals known as polychlorinated biphenyls (PCBs) were widely used for decades until their production was banned worldwide due to their persistence and toxicities to humans and other animals. Upon oxidative metabolism by cytochrome P450, hydroxylated metabolites of PCBs (OHPCBs) are formed. OHPCBs have been shown to competitively displace thyroxine from transthyretin, block normal hormonal activity, and inhibit phenol or family 1 sulfotransferases (SULTs) which catalyze sulfation of thyroid hormones and estrogens. Recently, three OHPCBs were shown to also interact with hydroxysteroid or family 2 sulfotransferases that play a role in the homeostasis of steroid hormones such as dehydroepiandrosterone (DHEA). The objectives of the studies presented in this thesis were to further examine the effects of selected OHPCBs on the activity of human hydroxysteroid sulfotransferase (hSULT2A1), to develop a three-dimensional quantitative structure activity relationship (3D-QSAR) model for OHPCBs as inhibitors of DHEA-sulfation catalyzed by this enzyme, and to investigate the mechanism of inhibition and binding of OHPCBs to hSULT2A1. All 15 OHPCBs examined inhibited the sulfation of 1 μ M [3H] DHEA, catalyzed by hSULT2A1 with IC50 values ranging from 0.6 to 96 μ M. The OHPCBs with a 3, 5-dichloro-4-hydroxy substitution were the most potent inhibitors of DHEA sulfation, and they were also shown to be substrates for hSULT2A1. Eight OHPCBs were substrates for hSULT2A1, and seven were solely inhibitors (i.e. they inhibited the sulfation of DHEA, yet they were not themselves sulfuryl-acceptors in hSULT2A1-catalyzed reactions). A 3D-QSAR model was developed utilizing comparative molecular field analysis (CoMFA). The model fit the data well and also had good predictability. The kinetics of inhibition showed that these OHPCBs were noncompetitive inhibitors of hSULT2A1. Binding studies utilizing the displacement of a fluorescent probe, 8-anilino-1-naphthalene sulfonic acid, revealed that several of the OHPCBs interact either at more than one binding site or with more than one enzyme conformation. Further exploration of this binding by molecular modeling showed that OHPCBs bind similarly to different conformations of the enzyme. This work has helped in our understanding of the roles of sulfotransferases in the metabolism and toxicities of OHPCBs, and it opens new avenues for future work.

Traditional bacterial disease therapies utilize compounds that ultimately kill the target bacteria but it exerts a strong selective pressure on the bacteria to develop multi-drug resistance mutants. The increasing occurrence of resistance in common pathogens has highlighted the need to identify new anti-microbials that target the control of bacterial pathogenicity in a non-extermination manner to reduce the incidence of bacteria resistance. One new strategy exploits the discrete signaling molecules that regulate the various bacterial signaling pathways, which are responsible for the expression of pathogenicity traits. Halogenated furanones (fimbrolides) from the marine red alga, Delisea pulchra have been shown to interfere with the key signaling pathway present in Gram-negative bacteria by competitively displacing the cognate signaling molecule from the transcription protein. This project focused on the design and synthesis of 1,5-dihydropyrrol-2-ones, a new class of fimbrolide derivatives capable of displaying strong antagonistic properties of the fimbrolides. Primary synthetic methodologies examined include the halolactamization of allenamides and the direct lactone-lactam transformation. No doubt, both methodologies yielded the lactam ring, the former failed to introduce the crucial C-5 bromomethylene group essential for bioactivity. A facile high yielding two-step lactone-lactam transformation method was developed and using this method, a wide range of substituted 5-bromomethyl- and 5-dibromomethylene-1,5-dihydropyrrol-2-ones were synthesized. Furthermore, a new class of tricyclic crown-ether type compounds with no literature precedent were discovered. To vary the diversity of the compounds, a related class of compounds, 5,6-dihydroindol-2-ones, were examined. A general versatile method for the synthesis of 7-substituted 5,6-dihydroindol-2-ones was developed. The synthetic strategy proceeds via the established Suzuki-Miyaura cross-coupling reaction of halogenated dihydroindol-2-ones with arylboronic acids/esters. The Suzuki methodology was found to be reliable in furnishing a wide range of 7-substituted products in high yields. A preliminary molecular modeling approach was used to assist in the design of new anti-microbials via the ligand-docking analyses of the TraR and LasR protein. A positive correlation was observed between the docking scores and biological activity and the methodology was further developed into an initial screening tool to filter potential active and non-active compounds. The newly synthesized compounds were analysed for their efficacy in reducing the expression of the Green Fluorescent Protein (GFP) in the presence of natural AHL signaling molecules in an AHL-monitor strain, indicative of the inhibition of bacterial phenotype expression. The dihydropyrrol-2-one class of compounds showed significant biological activity and this highlighted their potential for further development.

This thesis contains quantum chemical models and force field calculations for the RuBisCO isotope effect, the spectral characteristics of the blue-light sensor BLUF and the light harvesting complex II. The work focuses on the influence of the environment on the corresponding systems. For RuBisCO, it was found that the isotopic effect is almost unaffected by the environment. In case of the BLUF domain, an amino acid was found to be important for the UV/vis spectrum, but unaccounted for in experiments so far (Ser41). The residue was shown to be highly mobile and with a systematic influence on the spectral shift of the BLUF domain chromophore (flavin). Finally, for LHCII it was found that small changes in the geometry of a Chlorophyll b/Violaxanthin chromophore pair can have strong influences regarding the light harvesting mechanism. Especially here it was seen that the proper description of the environment can be critical. In conclusion, the environment was observed to be of often unexpected importance for the molecular properties, and it seems not possible to give a reliable estimate on the changes created by the presence of the environment. / Diese Arbeit beinhaltet quantenchemische und molekularmechanische Modelle zum Isotopeneffekt des Enzyms RuBisCO, der spektralen Charakterisierung des Blaulicht-Rezeptors BLUF und dem Lichtsammelkomplex II (LHCII). Es wurden vor allem die Einflüsse der Umgebung auf die entsprechenden Systeme untersucht. Für RuBisCO wurde gefunden, dass der Isotopeneffekt nur marginal von der Umgebung abhängt. Im Falle der BLUF Domäne wurde eine Aminosäure charakterisiert (Ser41), die bis dato experimentell noch nicht beschrieben war. Es wurde festgestellt, dass Ser41 hochmobil ist und einen systematischen Einfluss auf die spektrale Verschiebung des BLUF Chromophors (Flavin) hat. Schließlich wurde bei LHCII festgestellt, dass kleine Veränderungen in der Geometrie eines Chlorophyll b/Violaxanthin Chromophorenpaares bereits massive Einflüsse auf den Mechanismus des Lichtsammelprozesses haben können. Insbesondere hier zeigt sich, wie kritisch die genaue Beschreibung der Umgebung ist. Zusammenfassend wurde beobachtet, dass sich die Umgebung in oft unerwarteter Weise auf die molekularen Eigenschaften auswirken kann und es daher nicht möglich zu sein scheint, die entsprechenden Effekte vorher abzuschätzen.

The Diels-Alder reaction is one of the most powerful synthetic tools in organic chemistry, and asymmetric Diels-Alder catalysis allows for rapid construction of chiral carbon scaffolds. For this reason, considerable effort has been invested in developing efficient and stereoselective organo- and biocatalysts. However, Diels-Alder is a virtually unknown reaction in Nature, and to engineer an enzyme into a Diels-Alderase is therefore a challenging task. Despite several successful designs of catalytic antibodies since the 1980’s, their catalytic activities have remained low, and no true artificial ’Diels-Alderase’ enzyme was reported before 2010. In this thesis, we employ state-of-the-art computational tools to study the mechanism of organocatalyzed Diels-Alder in detail, and to redesign existing enzymes into intermolecular Diels-Alder catalysts. Papers I–IV explore the mechanistic variations when employing increasingly activated reactants and the effect of catalysis. In particular, the relation between the traditionally presumed concerted mechanism and a stepwise pathway, forming one bond at a time, is probed. Papers V–X deal with enzyme design and the computational aspects of predicting catalytic activity. Four novel, computationally designed Diels-Alderase candidates are presented in Papers VI–IX. In Paper X, a new parameterization of the Linear Interaction Energy model for predicting protein-ligand affinities is presented. A general finding in this thesis is that it is difficult to attain large transition state stabilization effects solely by hydrogen bond catalysis. In addition, water (the preferred solvent of enzymes) is well-known for catalyzing Diels- Alder by itself. Therefore, an efficient Diels-Alderase must rely on large binding affinities for the two substrates and preferential binding conformations close to the transition state geometry. In Papers VI–VIII, we co-designed the enzyme active site and substrates in order to achieve the best possible complementarity and maximize binding affinity and pre-organization. Even so, catalysis is limited by the maximum possible stabilization offered by hydrogen bonds, and by the inherently large energy barrier associated with the [4+2] cycloaddition. The stepwise Diels-Alder pathway, proceeding via a zwitterionic intermediate, may offer a productive alternative for enzyme catalysis, since an enzyme active site may be more differentiated towards stabilizing the high-energy states than for the standard mechanism. In Papers I and III, it is demonstrated that a hydrogen bond donor catalyst provides more stabilization of transition states having pronounced charge-transfer character, which shifts the preference towards a stepwise mechanism. Another alternative, explored in Paper IX, is to use an α,β -unsaturated ketone as a ’pro-diene’, and let the enzyme generate the diene in situ by general acid/base catalysis. The results show that the potential reduction in the reaction barrier with such a mechanism is much larger than for conventional Diels-Alder. Moreover, an acid/base-mediated pathway is a better mimic of how natural enzymes function, since remarkably few catalyze their reactions solely by non-covalent interactions. / <p>QC 20120903</p>

In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which was further shown to be substrate dependent (Paper V). A homology structure of Cv-ωTA guided the creation of an enzyme variant showing reduced isopropyl amine inhibition. / QC20100609

In this thesis, we propose a new design of privacy and session control for improving a collaborative molecular modeling CVR system AMMP-VIS [1]. The design mainly addresses the issue of competing user interests and privacy protection coordination. Based on our investigation of AMMP-VIS, we propose a four-level access control structure for collaborative sessions and dynamic action priority specification for manipulations on shared molecular models. Our design allows a single user to participate in multiple simultaneous sessions. Moreover, a messaging system with text chatting and system broadcasting functionality is included. A 2D user interface [2] for easy command invocation is developed in Python. Two other key aspects of system implementation, the collaboration Central deployment and the 2D GUI for control are also discussed. Finally, we describe our system evaluation plan which is based on an improved cognitive walkthrough and heuristic evaluation as well as statistical usage data.

Pentachloronitrobenzene (PCNB) is an organochlorine fungicide used either as seed dressing or for soil treatment. Research was conducted to investigate the microbial reductive transformation of PCNB with cultures developed from a contaminated estuarine sediment. The biotransformation of PCNB to pentachloroaniline (PCA) occurred under all electron accepting conditions tested. Sequential dechlorination of PCA to di- and in some cases to mono-chlorinated anilines occurred under fermentative/methanogenic conditions. Based on the use of inhibitors, methanogens were not involved in the sequential dechlorination of PCA. Based on 16S rRNA gene analysis, among five known dechlorinating bacterial groups tested, only Dehalococcoides was detected in the mixed culture. The sequential dechlorination of PCA was simulated using a branched-chain Michaelis-Menten kinetic model. The dechlorination rate (k) of the chlorinated anilines ranged from 0.25 to 1.19 uM/day and the half-saturation coefficient (KC) ranged from 0.11 to 1.72 uM at an incubation temperature of 22C and pH 6.90.1. Incubation at different temperature and pH values resulted in significant differences in the biotransformation rate and extent of PCNB in the fermentative/methanogenic enrichment culture. Incubation at 35C resulted in significantly different product distribution. The effect of temperature on the PCA dechlorination rate was modeled using an Arrhenius relationship. Dechlorination of PCA and methanogenesis were not observed in cultures amended with completely bioavailable iron sources until all Fe3+ was reduced to Fe2+. In contrast, PCA dechlorination took place at the same time with iron reduction in the same mixed, methanogenic culture amended with a less bioavailable iron source (FeOOH). PCA was sequentially dechlorinated to dichloroanilines in cultures amended with low nitrate concentrations, whereas partial dechlorination of PCA to tetrachloroanilines was observed in cultures amended with high initial nitrate concentrations due to the accumulation of reduced nitrogen species (e.g., NO, N2O). A semi-empirical molecular model (MOPAC/AM1) was used to estimate the thermodynamic and electronic properties of all chlorinated aniline congeners. These values were used to predict the sequential PCA dechlorination pathway and compare to experimentally observed dechlorination reactions. The results of this study have significant environmental implications relative to the fate and transport of PCNB, PCA and its dechlorination products in subsurface systems.

Both clan CA and clan CD proteases have a variety of physiological and pathological roles. In particular, both clans have members who have been implicated in cell death pathways, including apoptosis. Caspases are members of clan CD. Many of the caspase inhibitors used in apoptotic studies have shown cross reactivity with clan CA proteases. Thus, the anti-apoptotic effect of these inhibitors could be due to the broad-spectrum inhibition of a variety of cysteine proteases. Recently, the Powers laboratory designed a new class of inhibitors highly specific for clan CD proteases, aza-peptide epoxides. Initial data showed that this high selectivity could be due to the presence of the aza-residue, and not simply an artifact of substrate specificities. E-64c, an epoxysuccinyl inhibitor, is known to be a highly potent inhibitor of cathepsin B and calpain I. Thus, to determine if these clan CA proteases could tolerate an aza-residue, aza-E-64c and its analogues were synthesized. These inhibitors, termed epoxysuccinyl aza-peptides, were found to be significantly less potent for cathepsin B, calpain I, and papain than their non-aza counterparts, including E-64c. Previous findings have shown that the reactivity and selectivity of aza-peptide epoxides with caspases were significantly influenced by epoxide stereochemistry and the prime side substituent. Thus, this second project involved the systematic study of epoxide stereochemistry effects, prime side substituent effects, and the combined effect of these two variables. All inhibitors were tested with the seven apoptotic caspases: caspases-2, -3, -6, -7, -8, -9, and -10. We found that epoxide stereochemistry, prime side substituent, and also the peptidyl sequence have combined effects on potency and selectivity. In general, the (S,S) stereoisomer is the most potent relative to the (R,R) and (cis) stereochemistries. Modeling studies were done to determine why this is true. Aza-peptide epoxides were also briefly compared to aza-peptide Michael acceptors, another class of inhibitors highly specific for clan CD proteases

Novel, rationally-designed acrylate analogs of various known dipeptide substrates were found to be mechanism-based inactivators of the enzyme peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3). This enzyme is responsible for the rate-limiting and final bioactivation step, a C-terminal amidation of glycine-extended peptides, of a variety of peptide hormones including the potent pro-inflammatory compound Substance P. Protein-ligand docking studies, in tandem with in vitro kinetic analysis of these inactivators, indicated that the rational design of this class of compounds was successful in creating potent competitive inactivators of this enzyme. Pharmacological evaluation, via both acute and chronic models of inflammation in Sprague-Dawley rats, of these compounds indicates that they are highly potent anti-inflammatory agents which ameliorate both acute carrageenan-induced edema and the deleterious effects of chronic adjuvant-induced polyarthritis. Furthermore, these compounds were also able to induce a return toward a more normal phenotype in cancerous WB-Ras epithelial cells, via the interruption of the growth factor-stimulated pathway precipitated by Substance P. Finally, our modeling studies provide a structural basis for both the reaction and subsite stereospecificity of PAM toward its substrates, competitive inhibitors, and mechanism-based inactivators.

Inhibiting protein-protein interactions to achieve a therapeutically desired effect has been a goal in the field of drug discovery for decades. Recently, advances in peptidomimetics have led researches to the use of cyclized peptides to achieve this goal. Cyclization of linear peptides restricts the number of conformations of the peptide, increasing the peptide's affinity to binding to the desired target. Cyclization also stabilizes the peptide, allowing the peptide to be resistant to proteases. This study explores the optimization of solution phase synthesis of an important integrin-mediated cell adhesion cyclic peptide for the therapeutic inhibition of multiple myeloma, cHYD1. cHYD1 was originally synthesized via solid phase peptide synthesis, and the need for a scaled up synthesis version was needed after positive bioactivity results were obtained. Chapter 3 includes the molecular modeling exploration of a possible new mechanism to which cyclized peptides could work, in which, rather than a recognition and non-recognition strand being implemented, a specific directional face is used for protein-protein interaction. This was done with the implementation of an antagonistic cyclic peptide to replace human growth hormone in its interaction with the human growth hormone receptor, and the subsequent di-cyclic peptide agonist.