Design and synthesis of nitrogen containing heterocyclic fragments via isocyanide chemistry and their biological evaluation as potential inhibitors of HIV-1 replication

A thesis submitted to the Faculty of Science University of the Witwatersrand Johannesburg in fulfilment for the requirements of the degree of Doctor of philosophy, 2017Five membered nitrogen-containing heterocycles exhibit a wide range of biological activities. In this study we set out to demonstrate the utility of isocyanides in the synthesis of these heterocycles, and to identify hit scaffolds demonstrating the ability to disrupt important HIV-1host protein-protein interactions. In this project study, isocyanide synthons were used to generate different libraries of five membered nitrogen-containing heterocycles. In the first part of the project (Chapter 2), para-toluenesulfonylmethyl isocyanide (TosMIC) was reacted with N(arylidene)alkylamines (generated from aryl aldehydes and primary amines) under basic conditions using both a conventional and microwave-assisted van Leusen approach to generate a series of novel 5-aryl-1H-imidazole compounds. This family of 25 imidazole-based compounds were assessed for biological activity in an HIV-1 IN-LEDGF/p75 AlphaScreen assay and six compounds were found to be inhibitors of this interaction. In order to improve the potency of the hit fragments, a carboxylic acid functionality was introduced at the imidazole moiety of the hit compound to give novel 1,5-diaryl-1H-imidazole-4-carboxylic acid compounds as second generation of compounds. Biological evaluation of these carboxylic acid scaffolds in the HIV-1 IN-LEDGF/p75 AlphaScreen assay identified compounds exhibiting more than 50% inhibition, with four inhibitors surpassing the percentage inhibition of the hit scaffold. In the second part of the thesis (Chapter 3), TosMIC was reacted with various aryl aldehydes under basic conditions via a microwave-assisted van Leusen reaction to afford a set of 5-aryl-1,3oxazole derivatives. However, attempts to prepare similar scaffolds in acetonitrile afforded the oxazoline intermediates instead, which were easily converted into the corresponding 5-aryl-1,3oxazoles in one additional step. This family of 12 compounds did not significantly inhibit the HIV-1 IN-LEDGF/p75 AlphaScreen assay and the binding of antiVpu to Vpu. To improve the percentage inhibition of the original scaffolds, insertion of an N-aryl-carboxamide at the 4position of the oxazole moiety was carried out. Initial, attempts via formation of 2-isocyano-Naryl-acetamide intermediates and oxidation of N,5-aryl-4,5-dihydrooxazole-4-carboxamide intermediates met with failure. However, this second generation of novel N,5-diaryl-4carboxamide-1,3-oxazole derivatives were then synthesized successfully using benzoyl chloride and ethyl isocyanide as starting reagents. Evaluation of the desired 11 novel N,5-diaryl-4carboxamide-1,3-oxazoles in the HIV-1 IN-LEDGF/p75 assay showed that at least two compounds with the N-aryl-4-carboxamide moiety showed increased inhibition potency at 100 μM relative to the first generation of oxazoles. In the third part of the study (Chapter 4), TosMIC was reacted with ethyl 2(arylideneamino)acylate intermediates (generated from simple amino acids) under basic conditions via a van Leusen reaction to give a family of novel 5-aryl-1H-imidazoyl acylates. However, isolation of adducts derived from D-leucine proved to be impossible. A series of 5aryl-1H-imidazoyl-acylates derived from glycine were further transformed into two novel sets of 5-aryl-1H-imidazoyl-acetic acid and 5-aryl-1H-imidazoyl-acetohydrazide compounds. This family of 25 novel 5-aryl-1H-imidazo-yl-based compounds was screened for biological activity. In direct HIV-1 IN-LEDGF/p75 AlphaScreen assay, one compound was found to be active while seven compounds were identified as inhibitors of HIV-1 Vpu- host BST-2 interactions. To improve the percentage inhibition of these compounds in the HIV-1 IN-LEDGF/p75 AlphaScreen, N'arylidene and carboxamide groups were introduced which resulted in the generation of two sets of novel (E)-N'-arylidene-2-(5-aryl-1H-imidazol-1-yl)acetohydrazides and, N,5-diaryl-2-(1Himidazol-1-yl)acetamide compounds. From this library, at least three compounds with the 4-tertbutylphenyl motif were found to be a disruptor of HIV-1 IN and LEDGF/p75 interactions. In the fourth part of the study (Chapter 5), aryl isocyanide synthons were reacted with 2-haloarylethanone oximes to afford a series of 3-aryl-5-amino-isoxazole derivatives in poor yields. The poor yields were attributed to the low reactivity of the isocyanide and instability of the nitrosoalkene generated in situ from 2-halo-arylethanone oxime intermediates. Better yields of 3aryl-5-amino-isoxazoles were obtained when methyl isocyanoacetate was used instead of aryl isocyanide. Additionally, treatment of 2-halo-arylethanone oxime intermediates with potassium cyanide in MeOH afforded 3-aryl-isoxazol-5-amine derivative in varying yields. From this library, 10 isoxazole-based compounds were biologically evaluated in an HIV-1 IN – LEDGF/p75 interaction assay at 100 μM and, with the exception of two compounds, all the compounds displayed marginal activities. All compounds showing significant inhibitory activity in one of the protein-protein interaction assays were tested for activity in a cell based anti-HIV-1 assay. Unfortunately, none of the compounds was found to be active in this cell based assay. Lastly in Chapter 6, the synthesized libraries of the five membered nitrogen containing heterocyclic compounds were assessed for their antimicrobial activity using MIC methods. At least four compounds exhibited moderate antimicrobial activity against gram positive bacterial strains and a yeast strain.XL201

The bioaccumulation of platinum (IV) from aqueous solution using sulphate reducing bacteria: role of a hydrogenase enzyme

The enzymatic reduction of a high-valence form of metals to a low-valence reduced form has been proposed as a strategy to treat water contaminated with a range of metals and radionuclides. Metal reduction by sulphate reducing bacteria (SRB) is carried out either chemically (involving reduction by hydrogen sulphide) or enzymatically (involving redox enzymes such as the hydrogenases). While reduction of metal ions by hydrogen sulphide is well known, the enzymatic mechanism for metal reduction is poorly understood. The aims of this study were to investigate the role of SRB in facilitating platinum removal, and to investigate the role of a hydrogenase in platinum reduction in vitro. In order to avoid precipitation of platinum as platinum sulphide, a resting (non-growing) mixed SRB culture was used. The maximum initial concentration of platinum (IV), which SRB can effectively remove from solution was shown to be 50 mg.l⁻¹. Electron donor studies showed high platinum (IV) uptake in the presence of hydrogen, suggesting that platinum (IV) uptake from solution by SRB requires careful optimization with respect to the correct electron donor. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis indicated that platinum was being precipitated in the periplasm, a major area of hydrogenase activity in SRB. Purification of the hydrogenase by ammonium sulphate precipitation (65%), Toyopearl-Super Q 650S ion exchange and Sephacry 1 S-100 size exclusion chromatography revealed that the hydrogenase was monomeric with a molecular weight of 58 KDa, when analyzed by 12% SDS-PAGE. The purified hydrogenase showed optimal temperature and pH at 35°C and 7.5 respectively, and a poor thermal stability. In vitro investigation of platinum reduction by purified hydrogenase from mixed SRB culture showed that hydrogenase reduces platinum only in the presence of hydrogen. Major platinum (IV) reduction was observed when hydrogenase was incubated with cytochrome C₃ (physiological electron carrier in vivo) under hydrogen. The same observations were also noted with industrial effluent. Collectively these findings suggest that in vitro platinum reduction is mediated by hydrogenase with a concerted action of cytochrome C₃ required to shuttle the electron from hydrogenase

Studies towards the synthesis of novel, coumarin-based HIV-1 protease inhibitors

A series of the Baylis-Hillman adducts have been obtained by reacting protected O-benzylated and unprotected substituted salicylaldehydes with methyl acrylate or tertbutyl acrylate, respectively, using DABCO as catalyst. Treatment of the Baylis-Hillman adducts with HCl in a mixture of acetic acid and acetic anhydride afforded the corresponding 3-(chloromethyl)coumarin derivatives with yields of up to 94%. Similar use of HI afforded the corresponding 3-(iodomethyl)coumarins but, depending on the reaction time, the reduced 3-methyl analogues could also be obtained. Arbuzov reactions of the 3-(halomethyl)coumarin derivatives have been undertaken to afford 4-phosphorylated and 1’-phosphorylated derivatives, regioselectivity being dependent on the halide-leaving group. The 3-(chloromethyl)coumarin derivatives have been subjected to nucleophilic (SN) attack by benzylamine to give the corresponding 3- [(benzylamino)methyl]coumarin derivatives in yields of up to 74%. Further treatment of the 3-[(benzylamino)methyl]coumarin derivatives with chloroacetyl chloride afforded the chloroacetamide derivatives, which exhibit hindered rotation about the amine C(O)-N bond. The acetamide derivatives have also been subjected to Arbuzov reaction conditions to afford the phosphorylated derivatives in yields of up to 86%. In a preliminary modelling study, hydrolysed analogues of the synthesized phosphorylated derivatives have been docked into the active site of the HIV-1 protease enzyme using the Cerius-2 Ligandfit software module to provide an insight into potential receptor-ligand hydrogen bonding interactions

Exploring Viral Diversity in a Unique South African Soil Habitat

Abstract The Kogelberg Biosphere Reserve in the Cape Floral Kingdom in South Africa is known for its unique plant biodiversity. The potential presence of unique microbial and viral biodiversity associated with this unique plant biodiversity led us to explore the fynbos soil using metaviromic techniques. In this study, metaviromes of a soil community from the Kogelberg Biosphere Reserve has been characterised in detail for the first time. Metaviromic DNA was recovered from soil and sequenced by Next Generation Sequencing. The MetaVir, MG-RAST and VIROME bioinformatics pipelines were used to analyse taxonomic composition, phylogenetic and functional assessments of the sequences. Taxonomic composition revealed members of the order Caudovirales, in particular the family Siphoviridae, as prevalent in the soil samples and other compared viromes. Functional analysis and other metaviromes showed a relatively high frequency of phage-related and structural proteins. Phylogenetic analysis of PolB, PolB2, terL and T7gp17 genes indicated that many viral sequences are closely related to the order Caudovirales, while the remainder were distinct from known isolates. The use of single virome which only includes double stranded DNA viruses limits this study. Novel phage sequences were detected, presenting an opportunity for future studies aimed at targeting novel genetic resources for applied biotechnology

A novel dimeric exoglucanase (GH5_38)

An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked β-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards β-glucan, with a Km of 7.9 mg/mL and a kcat of 117.2 s−1, compared to lichenin which had a Km of 21.5 mg/mL and a kcat of 70.0 s−1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% β-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography–mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit

The interplay of descriptor-based computational analysis with pharmacophore modeling builds the basis for a novel classification scheme for feruloyl esterases

One of the most intriguing groups of enzymes, the feruloyl esterases (FAEs), is ubiquitous in both simple and complex organisms. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing high-added value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production and partial characterization of FAEs from fungi, while much less is known about FAEs of bacterial or plant origin. Initial classification studies on FAEs were restricted on sequence similarity and substrate specificity on just four model substrates and considered only a handful of FAEs belonging to the fungal kingdom. This study centers on the descriptor-based classification and structural analysis of experimentally verified and putative FAEs; nevertheless, the framework presented here is applicable to every poorly characterized enzyme family. 365 FAE-related sequences of fungal, bacterial and plantae origin were collected and they were clustered using Self Organizing Maps followed by k-means clustering into distinct groups based on amino acid composition and physico-chemical composition descriptors derived from the respective amino acid sequence. A Support Vector Machine model was subsequently constructed for the classification of new FAEs into the pre-assigned clusters. The model successfully recognized 98.2% of the training sequences and all the sequences of the blind test. The underlying functionality of the 12 proposed FAE families was validated against a combination of prediction tools and published experimental data. Another important aspect of the present work involves the development of pharmacophore models for the new FAE families, for which sufficient information on known substrates existed. Knowing the pharmacophoric features of a small molecule that are essential for binding to the members of a certain family opens a window of opportunities for tailored applications of FAEs

Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36

A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a Vmax of 2.03 × 10−9 s −1 and a Km of 206 μM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70°C and the enzyme had a half life at 60°C of 20.8 h

The application of omics in ruminant production: a review in the tropical and sub-tropical animal production context

The demand for animal products (e.g. dairy and beef) in tropical regions is expected to increase in parallel with the public demand for sustainable practices, due to factors such as population growth and climate change. The necessity to increase animal production output must be achieved with better management and production technologies. For this to happen, novel research methodologies, animal selection and postgenomic tools play a pivotal role. Indeed, improving breeder selection programs, the quality of meat and dairy products as well as animal health will contribute to higher sustainability and productivity. This would surely benefit regions where resource quality and quantity are increasingly unstable, and research is still very incipient, which is the case of many regions in the tropics. The purpose of this review is to demonstrate how omics-based approaches play a major role in animal science, particularly concerning ruminant production systems and research associated to the tropics and developing countriesinfo:eu-repo/semantics/acceptedVersio