Discovering Monoterpene Catalysis Inside Nanocapsules with Multiscale Modeling and Experiments

Large-scale production of natural products, such as terpenes, presents a significant scientific and technological challenge. One promising approach to tackle this problem is chemical synthesis inside nanocapsules, although enzyme-like control of such chemistry has not yet been achieved. In order to better understand the complex chemistry inside nanocapsules, we design a multiscale nanoreactor simulation approach. The nanoreactor simulation protocol consists of hybrid quantum mechanics-molecular mechanics-based high temperature Langevin molecular dynamics simulations. Using this approach we model the tail-to-head formation of monoterpenes inside a resorcin[4]arene-based capsule (capsule I ). We provide a rationale for the experimentally observed kinetics of monoterpene product formation and product distribution using capsule I , and we explain why additional stable monoterpenes, like camphene, are not observed. On the basis of the in-capsule I simulations, and mechanistic insights, we propose that feeding the capsule with pinene can yield camphene, and this proposal is verified experimentally. This suggests that the capsule may direct the dynamic reaction cascades by virtue of π-cation interactions

Enantioselective, Lewis base-catalyzed transformations: I. Polyene sulfenocyclization (preparative and mechanistic aspects) II. Sulfenofunctionalization of alkenyl boronates enabled by 1,2-boronate migration

This thesis covers two independent projects which are united under the umbrella of Lewis base catalysis. Following an overview of the key principles behind Lewis base catalysis and how it is used to enhance the electrophilicity of Lewis acids (Chapter 1), the bulk of this thesis will focus on the development of a catalytic, enantioselective sulfenocyclization of polyenes (Chapter 2). Sulfenyl group transfer from a highly reactive, cationic, Lewis acid-base adduct to an unactivated alkene generates a cyclic thiiranium ion, which serves as the initiating event for a highly stereoselective polyene cyclization that is terminated by arenes or phenols. This reaction was enabled by the identification of hexafluoroisopropyl alcohol (HFIP) as a superior solvent which dramatically improves site selectivity of thiiranium ion generation. A broad substrate scope is demonstrated, and the tricyclic products are isolated in good yield and enantioselectivity. Furthermore, a number of functional group interconversions (FGIs) of the resulting thioether moiety are demonstrated. This method is employed for the concise, enantioselective syntheses of the natural products (+)-ferruginol and (+)-hinokiol. Additionally, investigations into the sulfenocyclization of trienes to form even more complex products are disclosed. Preliminary mechanistic experiments to elucidate the rate-determining step of the catalytic cycle and the order in each reaction component were also performed. Chapter 3 of this thesis will cover the development of a Lewis base-catalyzed, enantioselective carbosulfenylation of alkenylboronate complexes which is enabled by a 1,2-boronate migration. The generation of “iranium” ions from alkenylboronates triggers a diastereospecific, ring-opening migration of an alkyl or aryl group to form 1,2-difunctionalized organoboron compounds. This strategy was employed together with Lewis base-catalyzed, enantioselective sulfenyl group transfer to ultimately afford chiral, non-racemic alkylboronic esters in generally high yield, high enantioselectivity, and perfect diastereospecificity. The products of the transformation are useful synthetic intermediates, and a number of useful FGIs are demonstrated

Untersuchungen zur Substratspezifität von Squalen-Hopen Zyklasen (SHCs)

Cyclic terpenoids form a large group of natural products with various biological functions. About 60,000 different cyclic terpenoids have been identified by now, containing scaffolds of ten to more than 30 carbon atoms. Among this huge amount of different cyclic compounds, there are many well-known flavors and fragrances, such as menthol or limonene, compounds which are widely used for pharmaceutical purposes like the antitumor compound Taxol and the anti-malaria agent artemisinin, or common membrane constituents and hormones such as the sterols. All of these natural products are derived from cyclization reactions of few linear precursor molecules catalyzed by terpenoid cyclases. The main focus of the present work rests on one very interesting family of the terpenoid cyclases, the squalene-hopene cyclases (SHCs). Among the over 300 annotated SHCs, most extensive studies had been carried out characterizing the SHC from the thermophilic bacterium Alicyclobacillus acidocaldarius (AacSHC), solving the crystal structure and the complex cyclization mechanism of the C30 precursor squalene into the pentacyclic products hopene and hopanol. This reaction constitutes one of the most complex reaction mechanisms found in nature, including the stereospecific formation of nine stereocenters and 13 covalent bonds.13 Besides AacSHC, several SHCs were partially characterized in previous works. Our interest was triggered by the ethanol and sugar tolerant strain Zymomonas mobilis which is known as one of the most potent hopanoid producers. Zymomonas mobilis contains two genes encoding for SHCs: ZmoSHC1 and the formerly partially characterized ZmoSHC2. It could be shown that the SHCs are also capable of cyclizing other linear terpenoids. For example, it was found that truncated squalene analogs were accepted as substrates by AacSHC and also the alcohol homofarnesol could be converted into the corresponding cyclic ether ambroxan.16–19 These results indicate that SHCs represent a promising family for catalysis of very different and complex cyclization reactions. Thus, we decided to investigate the SHCs’ potentials regarding their substrate specificities. In order to characterize the squalene-hopene cyclases ZmoSHC1 and ZmoSHC2 and compare them with AacSHC especially regarding their biocatalytic activities towards unnatural substrates, the SHCs were cloned and heterologously expressed in Escherichia coli. Functional expression was confirmed by conversion of the natural substrate squalene. For the direct comparison, a protocol for partial purification of the membrane-anchored SHCs was elaborated and optimized. For this partial purification as well as for the conversion of the hydrophobic substrates in aqueous milieu a suitable detergent had to be selected. ZmoSHC1 was characterized in more detail, retrieving information about pH- and temperature-dependence of the activity and the biocatalytic stability over a long period of time as well as inhibitory effects. All of the three enzymes were tested with unnatural substrates of C10-C18 carbon chain lengths. A special focus was laid on substrates containing functional groups such as hydroxyl , carboxy- or keto-groups expected to participate in the cyclization reaction, as shown for the hydroxyl-group of homofarnesol. Several of the substrates were accepted and cyclic products were generated. Interestingly, the functional groups were integrated in the final ring closure and to products with new properties were obtained. Homofarnesol conversion yielding the cyclic ether ambroxan, which is known as a expensive and rare flavor compound, was observed as reported in the literature. Also the corresponding carboxylic acid, homofarnesoic acid, could be converted into the cyclic lactone sclareolide. The C15 tertiary alcohol nerolidol was accepted as substrate and the bicyclic ether caparrapioxide was formed. Lastly, two ketones were accepted as substrates leading to cyclic enol ether products. Within the present work, all of these new products were characterized after preparative biotransformation and product isolation. Not only the facts that these substrates are much shorter than the natural substrate squalene and possess different functional groups which take part in the cyclization reaction and that useful products containing new properties are formed, but also the different activities of the SHCs towards these substrates are remarkable. Thus, it could be shown that ZmoSHC1 exhibits special biocatalytic properties, as the substrate activity pattern was unexpected. While squalene was converted very poorly, good activity was found towards the reaction of homofarnesol to ambroxan. All of the other substrates were converted in low but significant rates into the corresponding cyclic products. A completely different substrate activity pattern was observed using AacSHC as biocatalyst. Besides very good squalene conversion, much lower activities towards all of the other substrates were found. Using ZmoSHC2, only very low conversion rates were found for squalene and farnesylacetone and no conversion of any of the other substrates. Based on these observations, it can be concluded that ZmoSHC1 represents a versatile biocatalyst for complex cyclization reactions, as it shows unexpected substrate activity towards other substrates than squalene. In the present work, these and further detailed results are described. Besides the examination of the SHCs’ activities towards different substrates there were also several mutants created in order to find explanations for the differences between the SHCs regarding their substrate activities. This characterization of the triterpenoid cyclase ZmoSHC1 and discussion of their special properties leads to new conclusions about the potential of SHCs to serve as potent biocatalysts for new reactions.Die zyklischen Terpenoide stellen eine große Gruppe von Naturstoffen mit verschiedensten biologischen Funktionen dar. Bis heute konnten etwa 60.000 verschiedene zyklische Terpenoide, die aus Gerüsten von zehn bis über 30 Kohlenstoffatomen aufgebaut sind, identifiziert werden. Unter dieser großen Anzahl von zyklischen Stoffen finden sich viele bekannte Duft- und Aromastoffe, wie zum Beispiel Menthol oder Limonen, Verbindungen, die als pharmazeutisch wirksame Inhaltsstoffe in Medikamenten Anwendung finden, wie etwa der gegen Tumor wirksame Stoff Taxol oder das gegen Malaria angewendete Artemisinin oder auch die als Membranbestandteile und Hormone bekannten Steroide. All diese interessanten Naturstoffe werden durch Zyklisierung von wenigen linearen Vorläufermolekülen gebildet. Diese Zyklisierungsreaktionen werden von Terpenoid Zyklasen katalysiert. Der Schwerpunkt der vorliegenden Arbeit liegt auf einer Unterfamilie dieser Enzymgruppe der Terpenoid Zyklasen, den Squalen-Hopen Zyklasen (SHCs). Unter den über 300 annotierten SHCs wurde die SHC vom thermophilen Bakterium Alicyclobacillus acidocaldarius (AacSHC) am besten untersucht. Neben der Kristallstruktur wurde auch der komplexe Mechanismus aufgeklärt, nach dem das lineare C30 Substrat Squalen zu den pentazyklischen Produkten Hopen und Hopanol zyklisiert wird. Der Mechanismus dieser Reaktion, bei der neun Stereozentren und 13 kovalente C-C Bindungen spezifisch entstehen, gilt als einer der komplexesten, die man in der Chemie der Naturstoffe bislang entdecken konnte. Neben AacSHC wurden in vorangehenden Arbeiten auch einige andere SHCs teilweise charakterisiert. Von besonderem Interesse ist hierbei der gegen hohe Alkohol- und Zuckerkonzentrationen tolerante Stamm Zymomonas mobilis, der als einer der besten Hopanoid-produzierenden Bakterienstämme bekannt ist und zwei Gene enthält, die für SHCs codieren: ZmoSHC1 und die in vorherigen Arbeiten partiell charakterisierte ZmoSHC2. Es war gezeigt worden, dass SHCs neben dem natürlichen Substrat Squalen auch einige andere lineare Terpenoide als Substrate akzeptieren und diese zyklisieren. Zum Beispiel konnten verkürzte Squalen-Analoga von AacSHC zyklisiert werden und auch der C16-Alkohol Homofarnesol wurde in den entsprechenden zyklischen Ether Ambroxan umgesetzt. Diese Ergebnisse ließen darauf schließen, dass die SHCs eine vielversprechende Enzymfamilie zur Katalyse von sehr verschiedenen, komplexen Zyklisierungsreaktionen darstellen könnten und deswegen entschieden wir uns dazu, die Substratbreite der SHCs näher zu untersuchen. Um die Squalen-Hopen Zyklasen ZmoSHC1 und ZmoSHC2 zu charakterisieren und ihre biokatalytischen Aktivitäten mit der von AacSHC vergleichen zu können, wurden die für diese Enzyme codierenden Gene kloniert und heterolog in Escherichia coli exprimiert. Die Expression funktioneller Enzyme wurde durch Umsetzung des natürlichen Substrates Squalen bestätigt. Um die Enzyme direkt miteinander vergleichen zu können, wurde ein Protokoll für die partielle Aufreinigung der membrangebundenen SHCs ausgearbeitet und optimiert. Für diese Aufreinigung sowie für die Umsetzung der hydrophoben Substrate in wässrigem Milieu musste ein geeignetes Detergenz verwendet werden. ZmoSHC1 wurde des Weiteren näher charakterisiert, wobei die pH- und Temperaturabhängigkeit der katalytischen Aktivität, die biokatalytische Stabilität des Enzyms über eine längere Zeitdauer sowie Inhibierungseffekte untersucht wurden. Die drei Enzyme wurden auf Aktivität gegenüber unnatürlichen Substraten mit C-Kettenlängen von C10-C18 getestet. Ein besonderer Fokus wurde hierbei auf Substrate gelegt, die funktionelle Gruppen enthalten, wie zum Beispiel Hydroxyl-, Carboxy- oder Ketogruppen, die, wie für die Hydroxylgruppe von Homofarnesol gezeigt, an der Zyklisierungsreaktion teilnehmen könnten. Interessanterweise wurden diese funktionellen Gruppen in den finalen Ring der polyzyklischen Produkte integriert, wodurch Produkte mit neuen, attraktiven Eigenschaften entstanden. Homofarnesol konnte in den zyklischen Ether und bekannten Duftstoff Ambroxan umgesetzt werden. Die entsprechende Carbonsäure Homofarnesolsäure wurde ebenfalls als Substrat akzeptiert und es wurde das zyklische Lakton Sclareolid erhalten. Der tertiäre C15 Alkohol Nerolidol wurde zum bizyklischen Caparrapioxid umgesetzt. Des Weiteren wurden auch zwei Ketone als Substrate akzeptiert und in zyklische Enolether umgesetzt. Im Rahmen der vorliegenden Arbeit wurden die neuen Produkte nach präparativer Biotransformation und Aufreinigung charakterisiert. Nicht nur die Tatsache, dass diese Substrate sehr viel kürzere Kohlenstoff-Ketten als das „natürliche“ Substrat Squalen besitzen und über verschiedene funktionelle Gruppen verfügen, die an den Zyklisierungsreaktionen teilhaben und zu interessanten Produkten umgesetzt werden, sondern auch die unterschiedlichen Aktivitäten der SHCs gegenüber dieser Substrate sind bemerkenswert. Es konnte gezeigt werden, dass ZmoSHC1 über besondere Eigenschaften verfügt, da unerwartete Umsetzungsraten bei der Katalyse mit diesem Enzym bestimmt wurden. Während die Zyklisierung von Squalen von ZmoSHC1 nur sehr gering katalysiert wurde, wurde eine gute Aktivität gegenüber der Reaktion von Homofarnesol zu Ambroxan ermittelt. Alle anderen beschriebenen Substrate wurden in geringen, aber signifikanten Raten umgesetzt. Ein vollkommen anderes Aktivitäts-Muster wurde bei Umsetzungen mit AacSHC erhalten. Hier wurde neben sehr guter Umsetzung von Squalen eine viel geringere Aktivität gegenüber allen anderen Substraten bestimmt. Vom Enzym ZmoSHC2 wurden nur Squalen und Farnesylaceton mit sehr geringer Aktivität als Substrate akzeptiert, alle anderen Substrate wurden nicht umgesetzt. Anhand dieser Ergebnisse kann gefolgert werden, dass SHCs als vielseitige Biokatalysatoren für komplexe Zyklisierungsreaktionen verwendet werden können, da diese Enzyme eine unerwartete Substrataktivität mit anderen Substraten als Squalen zeigen. In der vorliegenden Arbeit werden diese und weitere Ergebnisse im Detail beschrieben. Neben der erwähnten Untersuchung der Aktivität der verschiedenen SHCs gegenüber unterschiedlichen Substraten wurden auch Mutanten hergestellt und untersucht, die zu einer Erklärung der Aktivitätsunterschiede zwischen den verschiedenen Squalen-Hopen Zyklasen verhelfen sollten. Diese Charakterisierung der Triterpen Zyklasen und die Diskussion ihrer besonderen Eigenschaften führen zu neuen Schlussfolgerungen über das Potenzial von SHCs, als fähige Biokatalysatoren für noch nie gezeigte Reaktionen eingesetzt werden zu können

Halocyclizations and cycloisomerizations of 1,6-diynes, and sequence-defined, self-replicating polymers

The exploration of chemical space in search of molecules that perturb or mimic biological systems is essential to understanding human biology. The first part of this dissertation (Chapters 1-4) describes efforts to aid in the exploration of biologically relevant space through the invention of new π-cyclization methodologies. This strategy can be viewed as part of a “top-down” approach to investigating biology: the construction of small molecule drugs or probes which modify the behavior of the existing system. The second part of this dissertation (Chapter 5) describes preliminary efforts to expand life-like chemical space beyond the nucleic acids of DNA and RNA. This can be viewed as a “bottom-up” approach to biology: the construction of systems which mimic the features of biochemical processes. In part one, cyclizations of nitrogen tethered 1,6-diynes were developed as a means to new heterocyclic scaffolds. A GaX3 promoted halocyclization transformed the acyclic diynes into tetrahydropyridine rings with exocyclic vinyl halides. In the presence of strong acid, the tetrahydropyridine products were further cyclized to tetrahydroindenopyridine scaffolds. These scaffolds were then diversified through Pd(0)-catalyzed cross-coupling reactions of the vinyl halide, and modifications to the tethering amino nitrogen. Subsequently, a Brønsted acid-catalyzed cyclization was developed, transforming N-sufonyl tethered bis-aryl 1,6-diynes to dihydroindenopyridines. Using unsymmetric bis-aryl diynes, the regio- and chemoselectivity of this Brønsted acid-catalyzed cyclization was investigated, and compared to the GaX3 Lewis acid promoted cyclization developed previously. The regiochemical preference of the initial cyclization step was found to be reversed under the two different conditions. In part two, a means to sequence-defined synthetic polymers which emulate the information storage and self-replication abilities of nucleic acid-based biopolymers was designed. Information was encoded in two dimers as a specific sequence of aniline and benzaldehyde subunits, which were linked together by a diethynyl benzene backbone. These dimers functioned as a template for the synthesis of new dimers with a complementary sequence. Unpolymerized ethynylaniline and ethynylbenzaldehyde monomers, associated to a polymer template by reversible imine bonds, were polymerized via Sonogashira cross coupling with diiodobenzene. Under the same set of conditions, the sequence of two parent dimers was transferred to the daughters

Towards the Total Synthesis of (+/-)-Gephyrotoxin 287C

Studies directed toward the total synthesis of (+/-)-Gephyrotoxin 287C are described herein. Even though the target alkaloid has been synthesized four times to date, it still presents an interesting synthetic challenge. Our ultimate goal is to develop an efficient enantioselective synthetic route utilizing oxazolone chemistry in order to demonstrate its utility as a dienophilic component in intramolecular Diels-Alder reactions. This is part of a greater ongoing study aimed at a range of suitable alkaloid targets. Chapter 1 provides introduction to frog toxins as secondary metabolites and subsequently shifts to the target alkaloid. (+/-)-Gephyrotoxin 287C was originally described by Daly and Witkop in 1974 following isolation from the skin of the southwestern Columbian poison arrow frog Dendrobates histrionicus.29 Its discovery, isolation structure elucidation and biological activity are discussed. Chapter 2 delivers detailed accounts of four total and seven formal synthesis of the target alkaloid to date. Previous synthetic works were an abundant source of valuable ideas that expedited our synthetic efforts. Chapter 3 describes overall retrosynthetic approach to (+/-)-Gephyrotoxin 287C buoyed by successful completion of (+/-)-2-epi-Pumiliotoxin C in our lab.164 The initial stage of the synthesis (formation of cis-cycloadduct) mimics approach developed by Dr. Thongsornkleeb164, a post-doc in our laboratory, but diverges entirely afterwards. Chapter 4 presents the synthetic methods employed to tackle the challenge of (+/-)-Gephyrotoxin 287C synthesis. It begins with preparation of the key triene followed by experimental and theoretical studies of intramolecular [4 + 2] Diels-Alder cycloaddition to generate desired cis-decahydroquinoline framework. Afterwards it splits into a model study of formation of the pyrrolidine C-ring via tandem elimination/ring closure and cyclopropanation studies. The bulk of presented work, however, is dedicated to rather simple, one might say, cyclopropane rupture. In spite of numerous attempts and subsequent model studies which proved vain reiteratively the last to be or not to be struggle provided genuinely desired ring rupture in regiospecific manner. Chapter 5 concludes completed synthetic efforts and offers a detailed plan for future studies, partially completed, that would lead to the total synthesis of (+/-)-Gephyrotoxin 287C

Establishing a Comprehensive Toolbox for Isotopic Labelling Studies on Terpene Synthases

The cumulative doctoral thesis "Establishing a Comprehensive Toolbox for Isotopic Labelling Studies on Terpene Synthases" describes the synthesis and application of isotopically labelled compounds for the systematic in vitro investigation of recombinant terpene synthases to target both cyclisation mechanism and product structure. Methodically, the known approach of enantioselectively deuterated oligoprenyl diphosphate substrates was further developed by the addition of 13C-labelling, which led to a more sensitive detection of the labelled product by NMR. With a stereochemical anchor of known absolute configuration installed in the substrate and untouched by the enzymatic cyclisation mechanism, it is possible to infer the absolute configuration of the terpene product by following the incorporation of deuterium into the diastereotopic hydrogen positions. By combining chemical and enzymatic synthesis, it was finally possible to label every methylene group of the common terpene precursors by 13C and 2H in an enantioselective fashion. These extensions improve both feasibility and robustness of this method, which contributes to the challenging structure elucidation of terpene natural products, including their difficult to address absolute configurations. Depending on the cyclisation mechanism, also the stereochemical course of hydrogen movements can be delineated. Connected to the expanding labelling possibilities, several newly identified terpene synthases from bacteria and fungi have been addressed covering various aspects of their catalysis such as substrate or product specificity, repetitive mechanistic motifs and stereochemical issues. The structural variety of the known and newly identified natural products thereby inspired further studies like tailored labelling experiments, site-directed mutagenesis, chemical modifications and the investigation of EI-MS fragmentation mechanisms. With few publications dealing with other aspects of natural product chemistry such as fungal aromatic volatiles, lignin degradation and selected aspects of the secondary metabolism of marine Roseobacter group bacteria also being included in this work, the main focus lays on a deepened understanding of terpene synthase reactions. The isotopically labelled substrates introduced in this study thereby represent a valuable experimental tool towards a comprehensive picture of these astonishing enzymes that create the largest group of natural products

Diffusion of tin from TEC-8 conductive glass into mesoporous titanium dioxide in dye sensitized solar cells

The photoanode of a dye sensitized solar cell is typically a mesoporous titanium dioxide thin film adhered to a conductive glass plate. In the case of TEC-8 glass, an approximately 500 nm film of tin oxide provides the conductivity of this substrate. During the calcining step of photoanode fabrication, tin diffuses into the titanium dioxide layer. Scanning Electron Microscopy and Electron Dispersion Microscopy are used to analyze quantitatively the diffusion of tin through the photoanode. At temperatures (400 to 600 °C) and times (30 to 90 min) typically employed in the calcinations of titanium dioxide layers for dye sensitized solar cells, tin is observed to diffuse through several micrometers of the photoanode. The transport of tin is reasonably described using Fick\u27s Law of Diffusion through a semi-infinite medium with a fixed tin concentration at the interface. Numerical modeling allows for extraction of mass transport parameters that will be important in assessing the degree to which tin diffusion influences the performance of dye sensitized solar cells

Design Synthesis and Evaluation Of Diterpenones As Potent Chemopreventive Agents For Aflatoxin B1 Induced Carcinogenesis In Human Liver Cells

DESIGN, SYNTHESIS, AND EVALUATION OF DITERPENONES AS POTENT CHEMOPREVENTIVE AGENTS FOR AFB1 INDUCED CARCINOGENISIS IN HUMAN LIVER CELLS By Miguel A. Zuniga, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Major Director: Qibing Zhou, Ph.D Assistant Professor Department of Chemistry Terpene quinone methides (TPQMs) have been isolated from a variety of plants and show broad activities against bacteria, fungi, and cancerous cell lines. The biological activity has been attributed to the reactive electrophilic QM moiety, this structural feature has long been recognized as an intermediate in organic synthesis and in certain biosynthetic processes. It has been shown that quinone methide structures play a key role in the chemistry of several classes of antibiotic drugs and antitumor compounds such as mitomycin C and anthracyclines. The goal of this study was to understand the basis of QM bioactivity so that terpene catechols as analogs of natural TPQMs precursors can be designed as effective chemopreventive agents.In order to investigate the oxidation pathway of terpene QM precursors, a homoconjugated diterpene catechol was synthesized. A review of the literature revealed that Cu2+ -induced oxidation of simple catechols proceeds through a two-step one electron transfer process, and o-quinone is the sole oxidation product. In contrast, our studies showed direct p-QM formation from a diterpene catechol and no o-quinone oxidation products were observed. Furthermore, the Cu2+-induced oxidation pathway of our homoconjugated diterpene catechol revealed multiple p-QM formations under aqueous conditions. The implications of these findings suggest that terpene QM precursors can cause extensive DNA damage through in situ generated hydroxyl radicals or by DNA alkylations with p-QMs. To elucidate the Cu2+-induced DNA damage mechanism, a series of catechol analogues of natural terpene QM precursors were designed to investigate potential effects of stereochemistry, substitutiional, and functional groups on nucleobase alkylation and production of reactive oxygen species. The results of these tests suggested that production of ROS was the dominant mechanism for the observed DNA damage in the Cu2+-induced oxidation regardless of stereo and structural differences of catechols or subsequent oxidation products as QM or quinone. From the DNA damage study we found that the presence of NADH significantly enhanced the extent of DNA damage by oxidation of these catechols. More specifically, in the case of alkene catechols, our results showed that DNA damage was independent of the concentration of catechols, thus providing ample evidence for production of ROS through the redox cycle of catechols/quinones. Additional support for the formation of hydroxyl radical and futile redox cycling was clearly demonstrated by comparison of the fragmentation pattern with that of a Fenton reaction. The identify of the ROS was also shown to be in the form of a Cu(I)OOH complex by radical scavenging and metal chelation experiments. Cis-terpenones were first shown to have chemoprotective activity by Dr. Zhou and colleagues. In collaboration with their efforts to identify the mode of action of cis-terpenones, another project to achieve an isotope labeled cis-terpenone was undertaken. The isotope study was employed to obtain and experimentally demonstrate the feasibility of incorporating a radioactive label in cis-terpenone for the future studies of cis-terpenone metabolism. An analysis of the deuterium labeled cis-terpenone from the isotope exchange reaction showed that the isotope was being incorporated into multiple positions through scattering processes. This non-radioactive isotope study made it possible to optimize the conditions prior to using a radioactive tritium label, which will be a requisite for future metabolic studies.As an extension of this work, a structure activity relationship (SARs) study was undertaken with a focus on improving the physiochemical properties and chemical stability of cis-terpenones. The primary purpose of this study was to attempt to explain the reason for the observed protective effects of cis-terpenones against AFB1. Considerable efforts were made to introduce an unsaturated double bond in the structure of cis-terpenone by an intramolecular Pd (II) catalyzed Heck reaction. Unfortunately, this method was unsuccessful and which was attributed to the disconnection of our starting material during the formation of an enolate intermediate. A second model study to generate desired coumarin and ditepene related structures was investigated with a Diels Alder [4+2] cycloaddition reaction. After numerous attempts, we found that the successfull synthesis of these compounds was highly dependent on the temperature, solvent, and the use of stabilizers in the reaction. Finally, the targeted diterpene analogs were screened for protective effects against AFB1 by the MTT cell viability assay. However, these preliminary data showed that additional structural features and key modifications are still required to better correlate the structure with the mechanism of chemoprotection

TOWARDS PHORBOXAZOLE B: THE C20-C32 FRAGMENT

Phorboxazole A and phorboxazole B are two potent cytostatic polyketides isolated from Phorbas marine sponge found in the Indian Ocean. Because of their excellent cytostatic activity and unprecedented structure phorboxazoles have been a very attractive target for synthetic chemists and eleven total syntheses have been reported. A novel and efficient synthesis of the C20-C32 core fragment of phorboxazoles has been developed. Key steps were: an enantioselective aldol reaction, a diastereoselective crotylation and, a diastereoselective oxy-Michael reaction. The synthesis was 7 steps long with an overall yield of 31%. A stereodivergent oxy-Michael reaction was further investigated in a computational study and analogue study

Chemistry & Chemical Biology 2013 APR Self-Study & Documents

UNM Chemistry & Chemical Biology APR self-study report, review team report, response to review report, and initial action plan for Spring 2013, fulfilling requirements of the Higher Learning Commission