The increasing emergence of antibiotic resistant strains of bacteria and fungi is driving the need to increase the production of current antibiotics and produce novel antimicrobial compounds. This study worked to increase the production of cycloheximide and streptomycin antibiotics by co-culturing Streptomyces griseus with other industrially important microbes. 1-3 industrial challenge microbes at a time were added to a culture of S. griseus and allowed to grow for one week in shake flask cultures before harvesting and quantifying antibiotic production. Fifteen different industrial challenge microbes placed in 35 different combinations were used in the study and 17 of these combinations were found to significantly increase antibiotic production after analysis with ANOVA. Antibiotic production was confirmed using bioautograms. Three of the successful different co-cultures were then subjected to a study to see when industrial challenge microbe addition was optimal. Results suggest that the optimal time to add the challenge microbes was 1-3 days following the original S. griseus inoculation. Dead challenge microbes were also added to a culture of S. griseus and it was found that these significantly increased cycloheximide as much as the live co-cultures did. / Department of Biology

Tetrodecamycin (1) is a novel α-(γ-hydroxyacyl) tetronic acid based polyketide antibiotic isolated from the culture broth of Streptomyces nashvillensis MJ885-mF8 by Takeuchi et al. in 1994. Compound 1 shows potent inhibitory activity against Gram-positive bacteria including Bacillus anthracis and methicillin resistant Staphylococcus aureus (MRSA). It was proposed that an Aldol reaction of trans-decalin core 2 and tetronic acid derivative 3 followed by a face selective epoxidation and a subsequent epoxide-opening reaction would deliver the 6,6,7,5-skeleton of tetrodecamycm (1). To investigate this proposal, the silyl enol ether 5 was prepared from cycloheptene 4 in 7 steps. An unusual domino silyl enol ether reaction sequence was observed when the silyl enol ether 5 was submitted to a Diels-Alder reaction. It afforded cycloadduct 6, which was converted to the key intermediate 2 after another 3 steps (Scheme 1). Concurrently, double functionalisation of simple cyclic silyl enol ethers was investigated. Because of some difficulties in reproducing good overall yields to the cycloadduct 6, a second synthetic route was proposed. It was envisaged that a palladium-catalysed oxidative cyclisation or an organoselenium-mediated cyclisation reaction of compound 8 would construct the 6,6,7,5- skeleton 7, which would be converted to tetrodecamycin (1) via dihydroxylation followed by an introduction of the exo-methylene group. The intramolecular Diels-Alder reaction of trienal 11 afforded trans-decalin 10, which was converted to β-keto ester 9 in 2 steps. A Dieckmann-type cyclisation of 9 afforded compound 8 in good yield. However, so far transformation to compound 7 has not been achieved (Scheme 2).

Studies toward synthesis of subunits of sanglifehrin A, an immunosuppressant featuring a highly substituted [5,5]-spirolactam moiety as well as a 22-membered macrocycle are described. The macrolactone contains a peptidic backbone characterized by an unusual [beta]-substituted (S)-piperazic acid and (S)-m-hydroxyphenylalanine units. These studies resulted in the synthesis of advanced intermediate 358 which contains all of the carbon atoms of the C1-C25 macrolactone of sanglifehrin A, and 251 which bears the C31-C41 carbon skeleton of the [5,5]-spirolactam moiety of sanglifehrin A. A Masamune anti-aldol reaction of aldehyde 294 and ester 285 furnished alcohol 295 in a second generation approach to carboxylic acid 242, while a third generation route toward 242 improved the yield and required fewer synthetic steps. An asymmetric, catalytic phase-transfer method was used to introduce an [alpha]-amino function into 331 in the synthesis of (S)-m-hydroxyphenylalanine derivative 244. Assembly of 244, piperazic acid 113 and L-valine derivative 336 into tripeptide 241 using a racemization-free peptide coupling method is described. The synthesis of C31-C37 aldehyde 253 exploited double asymmetric crotylation to set in place the correct configuration of alternating hydroxyl and methyl groups at C33, 34, 35 and 36. / Graduation date: 2013

Many antibiotics are produced as secondary metabolites of Streptomyces species. Commercial production of an antibiotic involves the optimization of environmental parameters, genetic makeup, and medium. Selection of ingredients for both inoculum (seed) and fermentation (production) media must provide for economic production, and easy downstream processing of the compound. Antibiotics are produced as secondary shunt metabolites and represent products that are not essential for primary metabolism of the cell; therefore conditions for their optimal expression may or may not be associated with good growth of the organism. Response Surface Methodology (RSM) is a collection of statistically designed experiments and analyses that directs the investigation of many factors and their interactions. This approach minimizes the number of trials required to identify critical factors and possible synergism between factors. In this research, an antifungal antibiotic produced by an unknown streptomycete collected from soil, was isolated, characterized and identified as cycloheximide. RSM was then used toformulate both a seed and production medium that optimizes cycloheximide biosynethesis. For the seed medium, RSM was used in a three step process: i) full factorial categorical screen of many factors, ii) Plackett-Burman two-level screen of promising factors, and iii) orthogonal central composite design of critical factors. Optimal 24 hour packed cell volume was found with a seed medium containing (g/L): 6.6g soluble starch, 23.4g yeast extract, and Mg K2HPO4. Additionally, the effects of inoculum age and passage on resulting cycloheximide production were studied. It was found that the negative effects of increasing inoculum age and passages on cycloheximide production could be mediated by the composition of the seed medium. For the production medium, RSM analysis of 29 ingredients suggests that an optimal production medium for cycloheximide biosynthesis should contain a combination of starch (40 g/L), corn gluten (17.8 g/L), MgSO4.7H2O (1.16 g/L), and NaCl (6.38 g/L). This final production medium resulted in a cycloheximide titer of 943 µg/ml, a 6-fold improvement in antibiotic production. / Department of Biology

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The biochemical pathways for the formation of the unusual amino acids found in virginiamycin M₁ and A33853 were investigated. Specifically tritiated and carbon 14 labeled serines were incorporated into virginiamycin M₁. (2S)-serine and (2S,3R)-[3-³H] serine were found to be precursors, thus giving evidence of stereochemical control in the formation of the oxazole moiety. This information allowed for postulation of a ring closure pathway. Stereochemical investigations were also carried out on the dehydroproline unit and it was shown that both (R) and (S) prolines were incorporated into the dehydroproline unit. (2S,3R)-[3-³H] proline was synthesized and upon incorporation lost the (3-³H) label as evidence of stereochemical control in the formation of the dehydroproline unit from a saturated precursor. The basic biosynthetic origins of A33853 were investigated by feeding of D-[U-¹⁴C] glucose, sodium [U-¹⁴C] acetate, (S)-[U-¹⁴C] lysine, (S)-[U-¹⁴C] aspartic acid, [carboxyl-¹⁴C] anthranilic acid, and (S)-[5-³H] tryptophan. D-[U-¹⁴C]. Glucose and (S)-[U-¹⁴C] lysine appeared to be the main precursors. ¹³C¹⁵N lysine was synthesized and used to examine the ring closure of the 3-hydroxypicolinic amide ring in virginiamycin S₁. / Ph. D.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

The synthesis of 7-N-acetyldemethyllavendamycin butyl amide (47), 7-Nacetyldemethyllavendamycin isopropyl amide (48), 7-N-acetyldemethyllavendamycin amide of piperidine (49), 7-N-acetyldemethyllavendamycin amide of pyrrolidine (50), 7N-acetyldemethyllavendamycin amide of morpholine (51), demethyllavendamycin butyl amide (52), demethyllavendamycin amide of pyrrolidine (53), and demethyllavendamycin amide of morpholine (54) are described. Pictet Spengler condesation of 7-acetamido-2formylquinoline-5,8-dione (28) with tryptophan butyl amide (66), tryptophan isopropyl amide (67), tryptophan amide of piperidine (68), tryptophan amide of pyrrolidine (69), and tryptophan amide of morpholine (70) in an anisole - pyridine solution directly afforded the five lavendamycin amides 47-51. Compounds 52, 53, and 54 were obtained by hydrolysis of 47, 50, and 51 with 70% H2SO4-H20 solution.Aldehyde 28 was prepared according to the following general procedure.Nitration of 8-hydroxy-2-methylquinoline (30) yielded 8-hydroxy-2-methyl-5,7 dinitroquinoline (31). Compound 31 was then hydrogenated and acylated with acetic anhydride to yield 5,7-diacetamido-2-methyl-8-acetoxyquinoline (33). Compound 33 was oxidized by potassium dichromate to give 7-acetamido-2-methylquinoline-5,8-dione (27). Treatment of 27 with selenium dioxide in refluxing 1,4-dioxane afforded compound 28.Compounds 66, 67, 68, 69, and 70 were synthesized from compounds 61,62, 63, 64, and 65. These compounds were deprotected with ammonium formate in the presence of 10% Palladium on charcoal in methanol under an argon balloon at atmospheric pressure.Compounds 61, 62, 63, 64, and 65 were obtained from 58 with butylamine, isopropylamine, piperidine, pyrrolidine, and morpholine respectively in the presence of triethylamine under an argon balloon at atmospheric pressure.Compound 58 was synthesized by the reaction of N-carbobenzyloxytryptophan, with N-hydroxy succinimide, in the presence of N-dicyclohexylcarbodimide in dried and distilled dioxane under an argon balloon at atmospheric pressure.The structures of the novel compounds 58, 47, 48, 49, 50, 51, 52, 53, and 54 were confirmed by 1H NMR, IR, EIMS, and HRMS.The structures of protected and deprotected amides 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 were also confirmed by 1 H NMR and IR spectroscopy. / Department of Chemistry

A thesis submitted to the Faculty of Science, University of the Witwatersrand Johannesburg. In fulfilment of the requirements for the Degree of Doctor of Philosophy. March 2017 / In this PhD thesis, we report for the first time, new methodology for the synthesis of angucycline antibiotic natural products. In particular, for the synthesis of 1,8-dihydroxy-3methyltetraphene-7,12-dione, commonly known as tetrangulol. We also report on the synthesis of 1,10,12-trimethoxy-8-methylbenzo[c]phenanthridine in our quest to synthesise phenanthroviridone from an intermediate product in the synthesis of tetrangulol. The Suzuki-Miyaura coupling reaction between 1,4,5-(trimethoxynaphthalen-2-yl)boronic acid and 2-iodo-3-methoxy-5-methylbenzaldehyde afforded intermediate, 3-methoxy-5methyl-2-(1,4,5-trimethoxynaphthalen-2-yl)benzaldehyde. Conversion of this benzaldehyde into the alkyne, 2-(2-ethynyl-6-methoxy-4-methylphenyl)-1,4,5-trimethoxynaphthalene was accomplished utilizing the Corey-Fuchs reaction. Exposure of the derived acetylene to a catalytic platinum(II)-mediated ring closure yielded the required tetracyclic aromatic product, 1,7,8,12-tetramethoxy-3-methyltetraphene which was converted into tetrangulol. Exposure of the related 3-methoxy-5-methyl-2-(1,4,5-trimethoxynaphthalen-2-yl)benzaldehyde O-phenyl oxime to microwave irradiation in an ionic liquid yielded 1,10,12-trimethoxy-8methylbenzo[c]phenanthridine, instead of the desired natural product phenanthroviridone. We also report on the unexpected synthesis of the benzonaphthopyranone core found in other classes of angucycline antibiotics from oxygen analogs of 2-naphthylbenzyl alcohols when exposed to N-bromosuccinimide. Treatment of (2-(1,4-dimethoxynaphthalen-2yl)phenyl)methanol and related analogues with N-bromosuccinimide under an oxygen atmosphere afforded 12-methoxy-6H-dibenzo[c,h]chromen-6-one, 2-Methoxy-6Hbenzo[c]chromen-6-one and of 6H-benzo[c]chromen-6-one. An investigation into possible mechanisms for this transformation was also conducted. / LG2017

Thesis (DTech(Chemistry)) --- Cape Technikon, Cape Town, 1996 / A new general synthetic strategy for the synthesis of benzo[c]pyranquinones, with a view to making the route more generally applicable to the synthesis of naturally occurring naphtho[2,3-c]pyranquinones of potential importance as antimicrobial agents, has been developed. This synthetic approach afforded, inter alia, the natural products, is oeleutherin and hongconin (as their racemates) in good overall yield. A new high-yielding synthetic route for the synthesis of 1,5-dimethoxy-4naphthol, 2-allyl-5-methoxy-I,4-naphthoquinone and 3-acetyl-5-methoxy-I,4naphthoquinone, all ofwhich are key intermediates in several laboratory routes to naturally occurring naphtho[2,3-c]pyranquinones, has also been developed. A key-step in their formation is respective methylation, allylation or acetylation of a common intermediate Diels-Alder adduct. A feasible route to a naphtho[2,3-c]pyranone was developed. This model route is envisaged to be generally applicable for the synthesis of higher oxygenated naphtho[2,3-c]pyranones by virtue ofthe nature of the conditions and reagents used in this synthetic route. The target quinones and some of their precursors were evaluated for antimicrobial activity and specificity in vitro. This showed that the benzo[c]pyranquinones have a broader specificity spectrum than their naphtho[2,3-c] or naphtho[2,3-b] analogues.