Biotransformations of water insoluble substrates in aqueous, two-phase and encapsulated systems /

The biotransformation of water insoluble substrates by mammalian and bacterial cells has been problematic, since these whole cell reactions are primarily performed in an aqueous environment The implementation of a twophase or encapsulated system has the advantages of providing a low water system along with the physiological environment the cells require to sustain themselves. Encapsulation of mammalian cells by formation of polyamide capsules via interfacial polymerization illustrated that the cells could not survive this type of encapsulation process. Biotransformation of the steroid spironolactone [3] by human kidney carcinoma cells was performed in a substrate-encapsulated system, yielding canrenone [4] in 70% yield. Encapsulation of nitrile-metabolizing Rhodococcus rhodochrous cells using a polyamide membrane yielded leaky capsules, but biotransformation of 2-(4- chlorophenyl)-3-methylbutyronitrile (CPIN) [6] in a free cell system yielded CPIN amide [7] in 40% yield and 94% ee. A two-phase biotransformation of CPIN consisting of a 5:1 ratio of tris buffer, pH 7.2 to octane respectively, gave CPIN acid [8] in 30% yield and 97% ee. It was concluded that Rhodococcus rhodochrous ATCC 17895 contained a nonselective nitrile hydratase and a highly selective amidase enzyme.

The investigation of the reduction mechanism of the 5a-reductase enzyme of Penicillium decumbens /

The 5a-reductase of Penicillium decumbens ATCC 10436 was used as a model for the mammalian enzyme to investigate the mechanism of reduction of testosterone to 5adihydrotestosterone . The purpose of this study was to search for specific 5a-reductase inhibitors which antagonize prostate cancer . In a whole-cell biotransformation mode, this organism reduced testosterone (1) to 5a-dihydrosteroids (8) and 5aandrostane- 3, 17-dione (9) in yields of 28% and 37% respectively. Control experiments have shown that 5aandrostane- 3, 17-dione (9) can be produced from the corresponding alcohol (8) in a subsequent reaction separate from that catalysed by the 5a-reductase enzyme . Androst-4- ene-3, 17-dione (2) is reduced to give only (9) with a recovery of 80% The stereochemistry of the reduction was determined by 500 MHz ^H NMR analysis of the products resulting from the deuterium labelled substrates. The results were obtained by an analysis of the NOE difference spectra, double-quantum filtered phase sensitive COSY 2-D spectra, and ^^c-Ir 2-D shift correlation spectra of deuterium labelled products. According to the unambiguous assignment of the signals due to H-4a and H-4Ii in 5a-dihydro steroids, the NMR data show clearly that addition of hydrogen to the 4{5)K bond has occurred in a trans manner at positions 413 and 5a. To Study the reduction mechanism of this enzyme, several substrates were prepared as following; 3-methyleneandrost-4-en- 17fi-ol(3), androst-4-en-17i5-ol(5) , androst-4-en-3ii, 17fi-diol (6) and 4, 5ii-epoxyandrostane-3, 17-dione (7) . Results suggest that this enzyme system requires an oxygen atom at the 3-position of the steroid in order to bind the substrate. Furthermore, the mechanism of this 5a-reductase may proceed via direct addition of hydrogen at the 4,5 position without involvement of a carbonyl group as an intermediate.

Enantiodivergent chemoenzymatic synthesis of codeine

The present thesis describes our latest results in the chemistry of morphine alkaloids. An enantiodivergent synthesis of codeine utilizing a cis-cyclohexadiene diol derived from microbial whole cell oxidation of ~-bromoethylbenzene,as starting material is discussed. The total synthesis of (+)-codeine in 14 steps featuring a Mitsunobu inversion and two intramolecular Heck cyclizations is presented. Investigation of a regioselective nucleophilic opening of a homochiral vinyl oxirane, which led to a total synthesis of the natural isomer of codeine, is detailed. Furthermore, described herein are novel methodologies designed for the transformation of naturally occurring opiates into medicinally relevant derivatives. Two studies on the conversion of thebaine into the commercially available analgesic hydrocodone, two novel ·transition metal catalyzed N-demethylation procedures for opioids, and the development of a catalytic protocol for N-demethylation and Nacylation of morphine and tropane alkaloids are presented. In addition, reactions of a menthol-based version of the Burgess reagent with epoxides are discussed. The synthetic utility of this novel chiral derivative of the Burgess reagent was demonstrated by an enantiodivergent formal total synthesis of balanol. ii

Applications of dihydroarenediols to chemoenzymatic synthesis : approaches to total synthesis of morphine alkaloids

The present studies describe, as a primary goal, our recent progess toward the synthesis of morphine alkaloids from aromatic precursors. Model substrates were synthesized which allowed investigation into Diels-Alder, radical cascade, and palladium-catalyzed bond-forming reactions as possible routes to the morphine alkaloid skeleton. As a secondary objective, three separate series of aromatic substrates were subjected to whole-cell oxidation with Escherichia coli JM 109 (pDTG601), a recombinant organism over-expressing the enzyme toluene dioxygenase. Included in this study were bromothioanisoles, dibromobenzenes, and cyclopropylbenzene derivatives. The products of oxidation were characterized by chemical conversion to known intermediates. The synthetic utility of one of these bacterial metabolites, derived from oxidation of o-dibromobenezene, was demonstrated by chemical conversion to (-)conduritol E.

Immuno-cytochemical localization of glycoproteins involved in recognition and attachment in a mycoparatism

Polyclonal antibodies prepared against the two glycoproteins (Mr 100 and 85 kDa) involved in recognition and attachment of the mycoparasite, Piptocephalis virginiana, to its hosts, Mortierella pusilla and Phascolomyces articulosus, susceptible and resistant, respectively, were employed to localize the antigens at their cell surfaces. Indirect immunocytochemical technique using secondary antibodies labelled with either FITC or gold particles as probes, were used. FITC-Iabelled antibodies revealed a discontinous pattern of fluorescence on the hyphae of MortlerelLa pusilla and no fluorescence on the hyphae of Phascolomyces articulosus. Intensity of fluorescence was high in the germinating spores of both the fungi. Fluoresence could be observed on P. articulosus hyphae pretreated with a commercial proteinase. Fluorescence was not observed on either hyphae or germinating spores of the nonhost M0 r tie re11 a ca ndelabrum and the mycoparasite P. virginiana. Antibodies labelled with gold conjugate showed a different pattern of antigen localization on the hyphal walls of the susceptible and resistant hosts. Patches of gold particles were observed allover the whole cell wall of the susceptible host but only on the inner cell wall layer of the resistant host. Cell wall fragments of the susceptible host but not those of the resistant host, previously incubated with the antibodies inhibited attachment of the mycoparasite. Implications of preferential localization of the antigen in the resistant host and its absence in the nonhost are described.

Development of thermally stable versions of the Burgess Reagent : approaches to the chemoenzymatic total synthesis of morphine

The present studies describe our recent work on expanding the use of the Burgess reagent and its reaction with oxiranes. Several new variants of the Burgess reagent and its chiral auxiliary version were evaluated for their thermal stability by NMR spectroscopy. Three new versions of the reagent were synthesized and their stability was determined. The reactivity of all five Burgess reagents was compared in a dehydration reaction and reactions with epoxides and diols. Progress toward a chemoenzymatic synthesis of morphine is also included in this report. The synthesis began with the whole cell oxidation of bromobenzene by Escherichia coli JMI09(pDTG601). The preparation of several precursors for a key step involving the lohnson-Claisen rearrangement and progress toward the total synthesis are described.

The influence of skeletal muscle cell volume on the regulation of carbohydrate uptake and muscle metabolism

This study investigated the regulation of carbohydrate metabolism and glucose uptake through changes in skeletal muscle cell volume. Using an established invitro isolated whole muscle model, soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected from male rats and incubated in an organ bath containing Sigma medium-199 with 8 mM D-glucose altered to target osmolality (hypo-osmotic: HYPO, iso-osmotic: ISO, hyper-osmotic: HYPER; 190, 290, 400 mmol/kg). Muscles were divided into two groups; metabolite (MM) and uptake (MU). MM (N=48) were incubated for 60 minutes and were then immediately flash frozen. MU (N=24) were incubated for 30 minutes and then the extracellular fluid was exchanged for media containing ^H-glucose and ^'*C-mannitol and incubated for another 30 minutes. After the incubation, the muscles were freeze clamped. Results demonstrated a relative water decrease and increase in HYPER and HYPO, respectively. EDL and SOL glucose uptakes were found to be significantly greater in HYPER conditions. The HYPER condition resulted in significant alterations in muscle metabolite concentrations (lower glycogen, elevated lactate, and G-6-P) suggesting a catabolic cell state, and an increase in glycogen synthase transformation when compared to the HYPO group. In conclusion, skeletal muscle cell volume alters rates of glucose uptake with further alterations in muscle metabolites and glycogen synthase transformation.

Skeletal muscle fibre-type comparison of whole tissue and subcellular membrane phospholipids and fatty acids

Membranes are dynamic structures that affect cell structure and function. Compositional changes ofmembranes have been shown with the application of a perturbation; however these are limited to whole tissue analysis. The purpose of this thesis was to compare the phospholipid (PL) fatty acid (FA) composition of rat whole muscle (Wm) to 1) purified and non-purified subsarcolemmal (SS) mitochondria in soleus, plantaris, and red gastrocnemius, and 2) sarcolemma, transverse-tubules, SS and intermyofibrillar (IMF) mitochondria fix)m whole hindlimb. The major findings were that 1) contamination significantly altered the PL FA composition of the SS mitochondrial membrane fraction, 2) Wm and SS mitochondria compositions differed between muscle types, and 3) Wm did not accurately reflect the PL FA composition of any isolated subcellular membranes, with each being unique from each other. As such, the relevancy of the trends reported in the literature of the effects of perturbations on Wm may be limited.

The effect of extracellular osmolality on cell volume and resting skeletal muscle metabolism

The purpose of the current investigation was to establish an in-l'itro skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated. whole muscle (SOL and EDL) was dissected from Long Evans rats and incubated for 60 min in Sigma Medium-199 (resting tension (lg). bubbled with 95:5% 02:C02, 30 ± 2°C, and pH 7.4). Media osmolality was altered to simulate hypo-osmotic (190 ± 10 Osm) (HYPO) or hyper-osmotic conditions (400 ± 10 Osm) (HYPER) while an iso-osmotic condition (290± 1 0 Osm) (CON) served as a control (n= 17.19.17). Following incubation, relative muscle water content decreased with HYPER and increased with HYPO in both muscle types (p<0.05). The cross-sectional area of HYPO SOL type I and type II fibres increased (p<0.05) while the EDL type 11 fibre area decreased in HYPER and increascd from HYPO exposure. Furthermore, HYPER exposure in both muscles lead to decreased ATP and phosphocreatine (p<0.05) and increased creatine and lactate (p<0.05) compared to CON. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acutc alterations in muscle water content and resting muscle metabolism.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Engineering an improved adenovirus packaging cell line

Recombinant human adenovirus (Ad) vectors are being extensively explored for their use in gene therapy and recombinant vaccines. Ad vectors are attractive for many reasons, including the fact that (1) they are relatively safe, based on their use as live oral vaccines, (2) they can accept large transgene inserts, (3) they can infect dividing and postmitotic cells, and (4) they can be produced to high titers. However, there are also a number of major problems associated with Ad vectors, including transient foreign gene expression due to host cellular immune responses, problems with humoral immunity, and the creation of replication competent adenoviruses (RCA). Most Ad vectors contain deletions in the E1 region that allow for insertion of a transgene. However, the E1 gene products are required for replication and thus must be supplied in trans by a helper ceillille that will allow for the growth and packaging of the defective virus. For this purpose the 293 cell line (Graham et al., 1977) is used most often; however, homologous recombination between the vector and the cell line often results in the generation of RCA. The presence of RCA in batches of adenoviral vectors for clinical use is a safety risk because tlley . may result in the mobilization and spread of the replication-defective vector viruses, and in significant tissue damage and pathogenicity. The present research focused on the alteration of the 293 cell line such that RCA formation can be eliminated. The strategy to modify the 293 cells involved the removal of the first 380 bp of the adenovirus genome through the process of homologous recombination. The first step towards this goal involved identifying and cloning the left-end cellular-viral jUl1ction from 293 cells to assemble sequences required for homologous recombination. Polymerase chain reaction (PCR) was performed to clone the junction, and the clone was verified through sequencing. The plasn1id PAM2 was then constructed, which served as the targeting cassette used to modify the 293 cells. The cassette consisted of (1) the cellular-viral junction as the left-end region of homology, (2) the neo gene to use for positive selection upon tranfection into 293 cells, (3) the adenoviral genome from bp 380 to bp 3438 as the right-end region of homology, and (4) the HSV-tk gene to use for negative selection. The plasmid PAM2 was linearized to produce a double strand break outside the region of homology, and transfected into 293 cells using the calcium-phosphate technique. Cells were first selected for their resistance to the drug G418, and subsequently for their resistance to the drug Gancyclovir (GANC). From 17 transfections, 100 pools of G418f and GANCf cells were picked using cloning lings and expanded for screening. Genomic DNA was isolated from the pools and screened for the presence of the 380 bps using PCR. Ten of the most promising pools were diluted to single cells and expanded in order to isolate homogeneous cell lines. From these, an additional 100 G41Sf and GANef foci were screened. These preliminary screening results appear promising for the detection of the desired cell line. Future work would include further cloning and purification of the promising cell lines that have potentially undergone homologous recombination, in order to isolate a homogeneous cell line of interest.

Cell-selective modulation of the neuromuscular system in Drosophila

The capacity for all living cells to sense and interact with their environment is a necessity for life. In highly evolved, eukaryotic species, like humans, signalling mechanisms are necessary to regulate the function and survival of all cells in the organism. Synchronizing systemic signalling systems at the cellular, organ and whole-organism level is a formidable task, and for most species requires a large number of signalling molecules and their receptors. One of the major types of signalling molecules used throughout the animal kingdom are modulatory substances (e.x. hormones and peptides). Modulators can act as chemical transmitters, facilitating communication at chemical synapses. There are hundreds of circulating modulators within the mammalian system, but the reason for so many remains a mystery. Recent work with the fruit fly, Drosophila melanogaster demonstrated the capacity for peptides to modulate synaptic transmission in a neuron-specific manner, suggesting that peptides are not simply redundant, but rather may have highly specific roles. Thus, the diversity of peptides may reflect cell-specific functions. The main objective of my doctoral thesis was to examine the extent to which neuromodulator substances and their receptors modulate synaptic transmission at a cell-specific level using D. melanogaster. Using three different modulatory substances, i) octopamine - a biogenic amine released from motor neuron terminals, ii) DPKQDFMRFa - a neuropeptide secreted into circulation, and iii) Proctolin - a pentapeptide released both from motor neuron terminals and into circulation, I was able to investigate not only the capacity of these various substances to work in a cell-selective manner, but also examine the different mechanisms of action and how modulatory substances work in concert to execute systemic functionality . The results support the idea that modulatory substances act in a circuit-selective manner in the central nervous system and in the periphery in order to coordinate and synchronize physiologically and behaviourally relevant outputs. The findings contribute as to why the nervous system encodes so many modulatory substances.