Phosphonates represent an important class of organophosphorus compounds. Their use as reagents in organic synthesis is prevalent, and there is a plethora of examples of biologically active compounds possessing the phosphonate moiety. To further our exploration of phosphonates as both reagents and biologically active compounds we have developed a one-flask protocol for the direct synthesis of phosphonates from benzylic and allylic alcohols. This transformation is unprecedented and is applicable to a range of substrates. Both electron rich and electron deficient benzylic alcohols react under the conditions developed. Furthermore, good yields are achieved when converting allylic alcohols to the corresponding allylic phosphonates. In at least one case, the one-flask protocol allows for phosphonate formation that was not achievable under the standard conditions. Bisphosphonates represent a significant subclass of phosphonates. Several nitrogenous bisphosphonates have found use in the clinic as treatments for bone-related disease including osteoporosis, and there is speculation that bisphosphonates that are enzyme-specific inhibitors may be used as cancer therapies. To develop our understanding of isoprenoid metabolism, we have prepared a range of bisphosphonates as potential inhibitors of geranylgeranyl pyrophosphate synthase. After much experimentation, an α-amino analog of a potent inhibitor of GGDPS has been synthesized and biological data is forthcoming. Furthermore, a new class of aromatic bisphosphonates, analogs of digeranyl bisphosphonic acid, has been synthesized and assayed. The bioassay results indicate that this series of compounds retains its specificity for the GGDPS enzyme, and that the dialkyl analogues retain much of their potency in the assays in spite of the increased steric bulk of the aromatic substructure. We have also begun the design and synthesis of compounds as potential inhibitors of Rab geranylgeranyl transferase (RGGTase). The lead compound, 3-PEHPC, is documented to inhibit RGGTase selectively, albeit at less than desirable concentrations. Using 3-PEHPC as the model compound we have elected to probe the impact of modifications on the hydrophilic "head" portion of the molecule. Using the phosphonophosphinate functionality as a surrogate for the phosphonocarboxylate moiety we have successfully synthesized digeranyl phosphonophosphinate. Initial assay data indicates that this novel phosphonophosphinate does not act upon GGDPS as does the analogous bisphosphonate substructure. The bioassay data to probe this compound's impact on RGGTase is forthcoming. Given the worldwide impact of tuberculosis infection and the emergence of drug-resistant strains of tuberculosis-causing pathogens, new and potent treatments for tuberculosis are necessary. We have engaged in the synthesis of several compounds as inhibitors of Rv2361c, an enzyme key to cell wall biosynthesis in Mycobacterium tuberculosis, the principle causative agent of tuberculosis in humans. To probe the impact of modifications at the C-9-position of the most potent of our Rv2361c inhibitors, we have made several analogues having phenyl and indole substituents. The in vitro enzyme assay data for the set of compounds has clarified understanding of the essential components of the pharmacophore, and helped to establish the direction for future efforts.

Enzymes are biological catalysts which greatly accelerate the rates of chemical reactions, oftentimes by many orders of magnitude over the uncatalyzed reaction. The remarkable catalytic rate enhancement afforded by enzymes derives ultimately from the structure and chemical properties of the enzyme active sites, which allow enzymes to selectively bind to their substrates and to stabilize high energy chemical species and unstable intermediates along the reaction coordinate. To enhance their catalytic ability, many enzymes have also evolved to require coenzymes for optimal activity. These coenzymes often provide chemical functionality and reactivity that are not accessible by the twenty canonical amino acids and, hence, coenzymes serve to greatly enhance the diversity of chemical reactions that can be mediated by enzymes. The work described in this dissertation focuses on mechanistic studies of two enzymes that use common coenzymes in unusual ways. In the first section of this work, studies will focus on the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2), an essential enzyme in isoprenoid biosynthesis that employs a flavin mononucleotide (FMN) coenzyme for catalysis. In most biological systems, flavin coenzymes mediate electron transfer reactions. However, the IDI-2 catalyzed reaction involves no net redox change, raising questions as to the role of the flavin in the chemical mechanism. The chemical mechanism of IDI-2 will be interrogated with a combination of spectroscopic studies and biochemical techniques. Our studies suggest that the flavin coenzyme of IDI-2 may employ a novel mode of flavin-dependent catalysis involving acid/base chemistry. In the second section of this dissertation, attention will be focused on elucidating the chemical mechanism of 1-aminocyclopropane-1-carboxylate deaminase (ACCD), an enzyme that plays a role in regulating the production of the potent plant hormone, ethylene. ACCD is a pyridoxal-5ʹ-phosphate (PLP)-dependent enzyme that catalyzes a C-C bond cleavage event that is unique among the catalytic cycles of PLP-dependent enzymes. Altogether, our mechanistic studies of IDI-2 and ACCD help to illustrate the catalytic diversity of common coenzymes, and demonstrate that some enzymes have evolved to exploit readily available coenzymes for atypical reactions. / text

Plant sesquiterpene synthases catalyze the conversion of the linear substrate farnesyl diphosphate, FPP, into a remarkable array of secondary metabolites. These secondary metabolites in turn mediate a number of important interactions between plants and their environment, such as plant-plant, plant-insect and plant-pathogen interactions. Given the relative biological importance of sesquiterpenes and their use in numerous practical applications, the current thesis was directed towards developing a better understanding of the mechanisms employed by sesquiterpene synthases in the biosynthesis of such a diverse class of compounds. Substrate preference for sesquiterpene synthases initially isolated from Nicotiana tabacum (TEAS), Hyoscyamus muticus (HPS) and Artemisia annuna (ADS) were optimized with regards to a divalent metal ion requirement. Surprisingly, careful titration with manganese stimulated bona fide synthase activity with the native 15-carbon substrate farnesyl diphopshate (FPP) as well as with the 10-carbon substrate geranyl diphosphate (GPP). Reaction product analysis suggested that the GPP could be used to investigate early steps in the catalytic cascade of these enzymes. To investigate how structural features of the sesquiterpene synthases translate into enzymatic traits, a series of substrate and active site residue contacts maps were developed and used in a comparative approach to identify residues that might direct product specificity. The role and contribution of several of these residues to catalysis and product specificity were subsequently tested by the creation of site-directed mutants. One series of mutants was demonstrated to change the reaction product to a novel sesquiterpene, 4-epi-eremophilene, and while another series successfully transmutated TEAS into a HPS-like enzyme. This is the first report of a rational redesign of product specificity for any terpene synthase. The contact map provides a basis for the prediction of specific configurations of amino acids that might be necessary for as yet uncharacterized sesquiterpene synthases from natural sources. This prediction was tested by the subsequent isolation and validation that valencene synthase, a synthase from citrus, did indeed have the amino acid configuration as predicted. Lastly, an in vitro system was developed for analyzing the interaction between sesquiterpene synthases and the corresponding terpene hydroxylase. Development of this in vitro system is presented as a new important tool in further defining those biochemical features giving rise to the biological diversity of sesquiterpenes.

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.