Browsing by Author "Govender, Thavendran."

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Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)
(2011) Moothoo-Padayachie, Anushka.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Govender, Thavendran.; Govender, Patrick.
In clinical diagnosis and fermentation industries there is a need for a method that allows for the differentiation of yeast to the strain level (biotyping). The ideal biotyping method should be accurate, simple, rapid and cost-effective, and capable of testing a large number of yeast isolates. Matrix assisted laser desorption/ionization time of flight mass spectrometry has emerged as a powerful biotyping tool for the identification of bacteria and clinical yeast isolates, mainly Candida. It has been found that the MALDI-TOF MS signals from yeast are harder to obtain than from bacteria. It has been reported by several research studies that a cell lysis step is required to obtain a mass spectral signal for clinical Candida strains. To date an optimized sample preparation protocol has not been devised for the biotyping of S. cerevisiae strains. Studies on the identification of yeast using MALDI-TOF MS have focused primarily on clinical Candida yeast isolates but have included very few S. cerevisiae strains. Furthermore these yeast identification studies using MALDI-TOF MS have only achieved identification to the species and not strain level. A major limiting attribute of MALDI-TOF MS for the accurate identification of microbes, is its dependency on a comprehensive mass spectral database. Bruker Daltonics is a pioneer and leader in providing innovative life science tools based on mass spectrometry thus the Bruker Daltonics mass spectral database and state-of-the-art instruments and accompanying software were selected for this study. The Bruker Daltonics mass spectral database currently holds three thousand seven hundred and forty microorganisms of which only a mere seven are S. cerevisiae strains. Initially in this study, a number of parameters of a generic ethanol/formic acid protein extraction procedure as originally described by Bruker Daltoincs were considered in the development of a sample preparation protocol that yielded characteristic and highly reproducible MALDI-TOF mass spectra. The parameters considered included cell number, alcohol fixation, matrix solution and media. It was found that using the optimized sample preparation protocol unique and highly reproducible mass spectral profiles were obtained for all three S. cerevisiae strains. Multivariate analysis confirmed that the differences between all three S. cerevisiae strains were statistically significant. For quality assurance, the spectra of the three strains were sent for evaluation by Bruker Daltonics and were deemed suitable for the purpose of biotyping. The newly created ethanol/formic acid extraction procedure was used to generate an S. cerevisiae mass spectral database comprising of forty-five S. cerevisiae strains within a local context but also of global significance. The accuracy of the mass spectral database was assessed using blind coded S. cerevisiae strains obtained from the Agricultural Research Council Infruitec-Nietvoorbij (Institute for Deciduous Fruit, Vines and Wine), Stellenbosch, South Africa. It was found that S. cerevisiae identification to the species and more importantly strain level was achievable with relatively good accuracy. To determine the potential application of MALDI-TOF MS as an accurate method for S. cerevisiae strain identification in industry, blind coded S. cerevisiae strains were obtained from Natal Cane Products and subjected to MALDITOF MS analysis. It was found that four of the pure cultures submitted were correctly identified to the strain level and the three S. cerevisiae strains incorrectly identified may have been contaminants or the result of incorrect optimization conditions for the fermentation. Thus MALDITOF MS was shown to be an accurate identification tool, that may also be used to detect contaminants or incorrect environmental conditions which can result in substantial losses.
Computational studies of pentacycloundecane peptide based HIV-1 protease inhibitors.
(2013) Chibi, Buyisile.; Soliman, Mahmoud Elsayed Soliman.; Kruger, Hendrik Gerhardus.; Govender, Thavendran.; Maguire, Glenn Eamonn Mitchel.
Abstract available in PDF.
Design, synthesis and biological screening of potential anticancer Smac peptidomimetics to target XIAP.
Chetty, Thashini.; Kruger, Hendrik Gerhardus.; Govender, Thavendran.; Govender, Patrick.
Abstract available from print copy only.
Design, synthesis and pharmaceutical application of novel polycyclic 'cage' diamines.
(2010) Onajole, Oluseye Kehinde.; Govender, Patrick.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Govender, Thavendran.
Despite over 50 centuries of living with the disease, tuberculosis (TB) still remains one of the oldest and deadliest diseases known to man and is gradually becoming a serious threat to the human race. According to the 2009 Global tuberculosis control report of the World Health Organisation (WHO), it is estimated that about 9.4 million incident cases of TB occurred globally. Of these cases an estimated 1.4 million were HIV positive of which 78 % were in the African region while 13 % are located in the South-East Asia Region. An estimate of 1.3 million deaths was reportedly caused by TB among HIV negative people. South Africa has the highest percentage of HIV patients living with tuberculosis. The design, synthesis and evaluation of novel polycyclic ‘cage’ amines for their pharmaceutical profiles are presented in this thesis. In this project a total of 12 novel intermediates and 31 novel products were synthesised. A thorough NMR elucidation of the various structures was also pursued. This study was motivated by the reported discovery of SQ109 by Sequella. SQ109 (N-Geranyl- N’-(2-adamantyl)ethane-1,2-diamine) shares the same 1,2 ethylenediamine pharmacophore with ethambutol (EMB), a commercial TB-drug. SQ109 also possess remarkable activity against MDR-TB which includes the EMB resistant strain suggesting that SQ109 is a new anti-TB drug and not an EMB analogue. SQ109 comprises of a polycyclic adamantane moiety, an isoprenyl moiety and a diamine. This study had three main aims, namely (a) the design and synthesis of novel polycyclic ‘cage’ amines derivatives; the polycyclic ‘cage’ moieties investigated in this study includes adamantane, trishomocubane, oxa-pentacycloundecane, aza-pentacycloundecane, pentacyclodecane and pentacycloundecane, (b) structural elucidation (using 2D NMR techniques) of synthesized novel polycyclic ‘cage’ amine derivatives (c) the anti-mycobacterial screening of novel polycyclic ‘cage’ amines derivatives against H37Rv, MDR (multi-drug resistant) and XDR (extensively-drug resistant) strains of Mycobacteria tuberculosis and (d) the anti-bacterial and anti-fungal screening of selected novel polycyclic ‘cage’ amine derivatives. Furthermore, the design, synthesis and NMR elucidation of a family of similar novel PCU diamine ligands are also reported herein. The aim of these ligands is to complex and transport copper ions to the sites of inflammation caused by arthritus. The known pharmaceutical properties of polycyclic ‘cage’ compounds such as their ability to cross membranes due to improved drug lipophilicity makes them suitable candidates for such a study. This project stems from a logic collaboration between UKZN, UCT (University of Cape Town) and CPUT (Cape Peninsula University of Technology) to test some of these cage diamines for activity against arthritus. Experimental work in this aspect is performed by the group of Prof. Graham E. Jackson (UCT) and Dr. Sebusi Odisitse (UPUT).
Design, synthesis and screening of novel PCU-peptide/peptoid derived HIV protease inhibitors.
(2011) Makatini, Maya Mellisa.; Govender, Patrick.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Govender, Thavendran.
The AIDS epidemic in Africa has reached dramatic proportions. Of the 42 million people infected with HIV worldwide, 30 million are in Africa. Current available therapies have begun to transform this fatal disease into a chronic condition but there are still major obstacles that have resulted in a great demand for new and better drugs. The aim of this study was to synthesize novel and effective HIV protease inhibitors. This work describes the first account of pentacycloundecane (PCU)-peptide and peptoid based protease inhibitors. These inhibitors are proposed to bind the wild type C-South African HIV protease (C-SA) catalytic site via the norstatine or dihydroxylethelene type functional group of the PCU. The desired compounds were synthesized by the coupling of the peptides and peptoids to the PCU cage which resulted in a series of promising and structurally diverse HIV-1 protease inhibitors. The inhibitors were characterized by Nuclear Magnetic Resonance (NMR) and evaluated against the wild type C-SA enzyme for its ability to inhibit 50 % of the enzyme’s activity (IC50). Two of the compounds reported herein, inhibited the enzyme activity at concentrations less than 80 nM. NMR investigations indicated that the activity was related to the chirality of the PCU moiety and its ability to induce conformations of the coupled peptide side chain. Employing the new Efficient Adiabatic Symmetrized Rotating Overhauser Effect Spectroscopy (EASY-ROESY) technique enabled us to obtain vital information about the 3D structure of these small linear peptides and peptoids in solution. This technique is the first example describing the successful through space correlations of such small peptides. Furthermore, docking and a combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics MD simulation at the AM1 semi empirical level mirrored the observed NMR results and the experimental IC50 activity profile of the considered inhibitors. The combination of these experimental and theoretical methods provided a powerful insight into the interaction mode of these cage peptide and peptoid inhibitors with the enzyme.
Effectiveness of selected surface disinfectants in the dental clinic – a report from a tertiary training facility in KwaZulu-Natal.
(2016) Deulkar, Swati Ahay.; Singh, Shenuka.; Govender, Thavendran.
Optimal infection control practice forms the cornerstone of quality oral health care delivery in any dental setting. There is very little published evidence on dental infection control practices in South Africa. In addition there is a paucity of evidence that specifically examined the efficacy of commonly used surface disinfectants in oral health clinical settings. The aim of this study was to determine the effectiveness of selected surface disinfectants on specific dental environmental surfaces in an identified public oral health training facility in KwaZulu-Natal. The objectives included the identification and classification of environmental areas that are at risk for cross-contamination in the dental clinic, and comparison of the microbial count at specified times of the day after the use of three surface disinfectants. This was a prevalence (cross-sectional), descriptive research study with a non-experimental design. Data collection included the application of three commonly used surface disinfectants (Chlorine®, Ethanol and Glutaraldehyde) on identified dental environmental surfaces in a public sector dental clinic facility in KwaZulu-Natal. The clinic consists of seventeen dental units that are numbered from one to seventeen. Systematic random sampling technique was used to select selected every second chair for the study (Dental Unit number: number: 1, 3, 5, 7, 9, 11, 13, 15, 17). The dental clinical environment was then divided into four zones: 1): the working area around the dental operator/assistant (chair, head rest, arm rest, foot rest, dental hand pieces, overhead light source, air water syringe tip, spittoon, suction hose, based of dental chair, dental stool, foot control, instrument counter and handle); 2): the area behind chair (wash basin, computer monitor, window, wall, table top, dust bin and taps): 3): the area away from chair (computer processing unit, telephone and floor); and 4): the reception area (patient chairs and reception table top). The swab samples were collected at specific time intervals (7am, 9am, 11am, and at 16.00) using a charcoal swab. Chlorine, Ethanol (70% in water) and Glutaraldehyde (2%) disinfectants were applied separately on the identified nine dental units by using a spray method. Use of the MALDE-TOF spectrometer enabled the mass spectra to be acquired and the bacteria to be identified. Out of the 312 samples taken, 262 (84%) were shown to be bacterial culture positive. More than seven microbial species were identified in which staphylococci, Bacillus species and fungi were present. The most contaminated areas in the dental environment were the area around the chair (86.5 %) and away from chair (92%). The results indicate that Chlorine® was not active against several bacteria because 92% samples had positive growth at the end of the day. Only 56 % of the samples using Ethanol were positive in the morning but the microbial growth increased to 96 % by the end of the day. The use of Glutaraldehyde indicated that 52% of samples were positive at 9 am but that 82% were found to be positive at the end of day. The bacterial survival rate was found to be less with the use of Glutaraldehyde. The study suggests that there was an association between frequency of cleaning, the type of disinfectants used and the microbial count on the specified dental environmental surfaces in the identified oral health facility. The findings therefore indicate that disinfection processes at the identified dental centre are inadequate, sub-optimal and could contribute to the infection chain. There is an urgent need to review the current infection control procedures and protocols, including a review of the type of surface disinfectants used. The frequency of disinfection (damp-dusting and housekeeping) must be reviewed, given the number of patients that are seen on a daily basis. It is also important that simple procedures such as awareness of hand hygiene practices are implemented and prioritized. There should also be dedicated infection control monitoring and evaluation processes.
Enzymatic activation of a peptide functionalised gold nanoparticle system for prodrug delivery.
Pietersen, Lauren Kara.; Govender, Thavendran.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Govender, Patrick.
Traditional chemotherapeutic drugs are often restricted by severe side effects and a lack of specificity. An approach aimed at improving the selectivity of cancer drugs includes the use of prodrugs that can be selectively activated in tumour tissue (tumour-activated prodrugs). Peptide prodrugs cleavable by proteases in the tumour environment have been widely explored to improve the therapeutic index of cytotoxic drugs. In this study we demonstrate that the cell surface proteases over expressed in tumour cells are capable of cleaving the peptide CKAFKRK attached to gold nanoparticles (GNPs). This proteolic cleavage exposes the potential cytotoxic “agent” to the tumour cell. As these proteases are not over expressed in healthy cells, the prodrug will take much longer to be activated. A simple system is developed in this study as a proof of concept by replacing the cytotoxic agent with a fluorophore. An enhanced fluorescence emission is expected on exposure of the peptide functionalised GNP to the tumour cells, whereas a constant level of emission is anticipated on exposure to the healthy cell line.
LC-MS/MS method development and validation for simultaneous quantification of first-line HIV drugs and second-line TB drugs in rat plasma.
(2018) Malinga, Thembeka Hlengiwe.; Govender, Thavendran.; Baijnath, Sooraj.; Naicker, Tricia.; Kruger, Hendrik Gerhardus.
Tuberculosis (TB) and human immunodeficiency virus (HIV) co-infection continues to be a major public health concern, worldwide. HIV infection has increased the TB incidence over the past twenty years, making it hard to eliminate TB. At the same time, TB continues to be responsible for approximately 30% of deaths among HIV-infected individuals. Emtricitabine (FTC), efavirenz (EFV), and tenofovir (TFV) are constituents of a one-day-pill, AtriplaTM, which was approved in 2006 by the Food and Drug Administration (FDA). AtriplaTM is a triple combination anti-HIV drug that provides an efficient dosing plan. Streptomycin (STR), kanamycin (KAN), and ofloxacin (OFL) are second-line anti-TB drugs used to treat multidrug-resistant/ extensively drug-resistant tuberculosis (MDR/XDR-TB). The worldwide increase in the prevalence of anti-TB drugs resistance is of concern to researchers since it remains one of the most significant threats to the community. Co-prescription of anti-HIV and anti-TB drugs poses a challenge of drug-drug interactions, which causes adverse effects. Therapeutic drug monitoring (TDM) seems to be the tool for a solution to these problems since it personalizes doses thus reducing drug toxicity. LC-MS/MS methods with short run times are required to produce effective TDM studies. Therefore, this study aimed to evaluate the new Ascentis Express column technologies [pentafluorophenylpropyl (F5), octadecyl (C18), biphenyl, and reversed phase amide (RP-Amide)] and their applicability to the simultaneous quantification of current first-line anti-HIV treatment Atripla. It also aimed to develop, optimize and validate a liquid-chromatography tandem mass spectrometry (LC-MS/MS) methods for the simultaneous quantification of anti-HIV drugs (FTC, EFV, and TFV) and second-line anti-TB drugs (STR, KAN, and OFL) in rat plasma for the usage of TDM. The currently used HPLC columns have longer run times making them impractical in a point of care environment since the number of patients and diseases is globally increasing. There are also no or very few studies regarding the LC-MS/MS method of simultaneous HIV and TB drugs for HIV positive TB patients. The biphenyl column showed consistency and optimum performance with regard to the number of theoretical plates, resolution and peak asymmetry factor. It showed good separation and overall effectiveness. However, this does not rule out other columns for other purposes intended to be accomplished. The LC-MS/MS method developed for the simultaneous quantification of anti-HIV drugs and second-line anti-TB drugs was short to eleven minutes and met all the recommendations by the European Medicines Agency (EMA) guidelines for bioanalytical method validation. The new HPLC column matrices (F5, C18, biphenyl, and RP-Amide) offer various benefits such as the potential of saving solvents and short runtimes, essential in TDM studies. Therefore, the usage of the new HPLC column technologies iv will be beneficial in a point of care environment in terms of saving time and money. The LC-MS/MS method validated in this study can be used in clinical trials and in the simultaneous determination of the effective plasma concentrations of anti-TB and anti-HIV drugs, making it a strong candidate for TDM in a point of care setting.
Mass spectrometric imaging for Tuberculosis drug development.
(2017) Baijnath, Sooraj.; Govender, Thavendran.; Naicker, Tricia.; Kruger, Hendrik Gerhardus.
For many years, Tuberculosis (TB) has plagued the human race claiming millions, if not billions, of lives. With the advent of short-course chemotherapy TB has become a manageable disease, however in recent times Mycobacterium tuberculosis has developed resistance to a number of established and trusted antibiotics. This coupled with severe forms of extra-pulmonary TB, has placed significant emphasis on the development of new anti-TB agents. The drug development process is a long and costly affair, with less than 1% of new drugs reaching clinical trials. This is where molecular imaging, in particular mass spectrometry imaging (MSI), is fast becoming a promising tool in the evaluation of drug candidates. MSI can be used to streamline the drug development process by fast tracking areas of target identification, target quantification, pharmacokinetics, drug distribution and tissue localization. MSI possesses some distinct advantages in terms of sample preparation and the lack of the need for radiolabeling, making it the ideal technique for in vivo tissue drug distribution studies. The objectives of this study were to demonstrate the value of MSI in the development and evaluation of new and existing TB antibiotics, focusing on central nervous system (CNS) manifestations of the disease. In order to achieve these objectives, two of the most promising antimycobacterial agents, clofazimine (CFZ) and linezolid (LIN), were selected. Initially, the distribution of these agents in a healthy animal model was investigated, since these would represent the minimum tissue concentrations achievable. The single-dose study for both drugs were similar, in that there was poor penetration into the brain after a 100mg/kg dose in a healthy murine and rodent model, respectively. A four-week multiple dose study was conducted, each of the antibacterials showed excellent accumulation in the CNS, with preference to specific areas of the brain, demonstrating the neuroprotective potential of these drugs (Chapters 2 and 3). For the effective evaluation of anti-TB drugs, the lung has to be taken into consideration since this is the primary site of M.tb infections. However, the lung poses problems in terms of sample preparation for MSI. Since the lung is responsible for gaseous exchange, it is made up of a number of air-filled spaces that are kept “open” by a fine balance in pressure, inside and outside the lung. When this balance is disturbed, such as when the thoracic cavity is pierced, to collect tissue, the lung collapses. This results in distortion of tissue structure and subsequent distribution information can be misleading. For this reason, we evaluated various established cryoprotectants as lung inflation media. This inflation procedure would main structural integrity of the lung and provide accurate tissue distribution data. From the cryoprotective agents tested in this experiment we found that 10% DMSO was ideal, in terms of structural preservation and accurate drug distribution (Chapter 4). As part of this series of experiments other anti-bacterial agents were also evaluated, to demonstrate the value of MSI in drug development. These drugs also appear in the antibiotic pipeline; tetracyclines, tigecycline (TIG) and doxycycline (DOX), rifampicin (RIF), gatifloxacin (GAT) and pretomanid (PA-824). The findings were very interesting in that each agent displayed a unique pattern of distribution, this is due to the chemical nature of these drugs and their interaction with the blood-brain-barrier (BBB). In addition to this, we have demonstrated how MSI can be used to determine various aspects of drug-tissue interaction for drug development. MSI was used to prove that the chemical properties of a drug do not always govern its movement across the BBB. RIF is a large drug molecule that one would not expect to permeate the brain, however this experiment has demonstrated its time-dependent distribution in the brain (Chapter 5). The results show how the tetracyclines have widespread tissue distribution in the brain, which contributes to their efficacy in the treatment of brain damage (Chapters 6 and 7). This technique was also used to understand how GAT enters the brain and contributes to the proven neurotoxicity of the flouroquinolones (Chapter 8). In the final chapter, we showed how MSI can be used in the tissue evaluation of novel antibiotics, such as pretomanid (Chapter 9). These findings emphasize the need to evaluate the drug distribution of antibiotics, since pathogens manifest themselves in different areas of the brain and cause damage. This information will be invaluable in our pursuit of effective treatments to CNS diseases and disorders, allowing medical practitioners to develop more targeted treatment programmes.
Organocatalyzed synthesis of carbapenem β-lactam cores and monobactam.
Khanyase, Sibusiso Blessing.; Naicker, Tricia.; Govender, Thavendran.
Organocatalysis has emerged as a new powerful methodology for the catalytic production of enantiomerically pure organic compounds. The main aim of this work was to develop organocatalyzed routes to novel β-lactam derivatives. In chapter 2, the first organocatalyzed C-C bond forming reactions have been performed on the carbapenem core 1, was the Aldol reaction, with various aldehydes to afford the corresponding products in good yields (up to 76%) and excellent diastereoselectivities (up to 99:1 ratios). Next, the Mannich reaction was evaluated with different amines and aldehydes. The products were obtained with modest chemical efficiency (up to 55%) and excellent diastereoselectivities (up to 99:1 ratios) as with the Aldol reaction. The reactivity of the carbapenem core 1 was also evaluated in the Michael addition reaction with electrophilic olefins. Chapter 3 includes the full substrate scope of organocatalytic asymmetric Michael addition transformations on the carbapenem core 1 reported. Good yields (up to 67%) and some excellent diastereoselectivities (up to 92:8 ratios) were obtained with L-proline as the organocatalyst. We have also demonstrated the possibility to effectively convert the Michael products to monobactams through a retro-Dieckmann reaction under basic conditions, thereby leading to another highly valued class of β-lactam antibiotics. Chapter 4 is the summary of the thesis.
Proline n-oxide : manipulation of the 3D conformation of linear peptides.
(2013) Farahani, Majid Darestani.; Maguire, Glenn Eamonn Mitchel.; Govender, Thavendran.; Kruger, Hendrik Gerhardus.; Arvidsson, Per Ingemar.; Albericio, Fernando.
During the last the few years, the number of peptide pharmaceutical drugs reaching the market has notably increased. Drug based peptides have many advantages, such as high potency of action and limited off-target side effects that are not present in most small molecules. However, oral bioavailability of peptides is a major obstacle that hinders the development of more therapeutic formulations. Physiochemical properties of peptides, such as short plasma half-life, sensitivity to enzyme degradation and the tendency to undergo aggregation, are some of the main reasons for the lack of bioavailability. Enhancement of bioavailability can be achieved when the flexibility of peptides is reduced to a more defined stereochemical structure. There are some approaches that can reduce the flexibility of peptide backbones to a more defined structure e.g. application of peptidomimetics and cyclic peptides. Here, we have investigated another new tool that can lead us to the same goal. The purpose of this study was to investigate the possibility of modulating the secondary structure of short peptides. This was envisaged via the application of a proline N-oxide moiety and by N-methylation of the peptide backbone. The major objectives for this study were: 1. To synthesise a series of tetrapeptides to investigate the effect of proline N-oxide on their secondary structure. 2. The effect of N-methylation of the amide bond on isomerization of the N-oxide peptide backbone in terms of cis- and trans-isomers and how it directs the potential hydrogen bonding interactions 3. To study the effect of side chain bulkiness on the potential of hydrogen bonding interactions of the selected peptides. 4. Analyses of the formed secondary structures with advanced NMR techniques. 5. To perform a thermal coefficient NMR study on these molecules in order to obtain an improved understanding of the nature of hydrogen bond interactions of these peptides.
A study of chiral pentacyclo-undecane derived macrocycles and ligands.
(2004) Govender, Thavendran.
Several chiral macrocyclic crown ethers and related analogues have shown to be capable of
Synthesis and evaluation of novel tetrahydroisoquinoline organocatalysts in asymmetric catalysis.
(2012) Naicker, Tricia.; Govender, Thavendran.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Arvidsson, Per Ingemar.
Organocatalysis has rapidly expanded in the last decade to encompass a wide variety of small organic molecules that are capable of either activating substrates or transforming them into more reactive forms. The aim of this study was to develop novel chiral organocatalysts based on the tetrahydroisoquinoline backbone and evaluate them on asymmetric reactions. Three organocatalytic modes of activation have been investigated for C-C bond forming asymmetric reactions. In chapter 2, for the first time organocatalysts bearing a secondary nitrogen within a cyclohexane ring were evaluated in the asymmetric Diels–Alder reaction. These catalysts were tested over a range of dienes and dienophiles and displayed promising chemical conversions of up to 100 % with up to 64 % ee when triflic acid was employed as the cocatalyst. Density functional theory computational studies and 2D NMR spectroscopy were used to determine the structure of the intermediate iminium ion formed between the most efficient catalyst and cinnamaldehyde. Chapter 3 includes a series of novel tetrahydroisoquinoline chiral N-oxide organocatalysts and their evaluation in the asymmetric allylation reaction of aromatic and α-β-unsaturated aldehydes with allyltrichlorosilane. The chiral homoallyl products were obtained with good chemical efficiency (up to 93 % yield) and high enantioselectivity (up to 91 % ee) under mild reaction conditions (23 °C). Chapter 4 is the simple and practical microwave-assisted synthesis of new tetrahydroisquinoline guanidine organocatalysts and their evaluation in the asymmetric Michael addition reaction of malonates and β-ketoesters with nitro-olefins. In addition, a novel microwave assisted procedure of introducing the guanidine unit onto amino amide derivatives is reported. The chiral products were obtained with quantitative chemical efficiency (up to 99 % yield) and excellent enantioselectivity (up to 97 % ee). Chapter 5 is a collection of all X-ray crystal structures that were published from novel compounds synthesized pertaining to Chapters 2-4, it contains 15 published crystal structures while Chapters 3-4 contain 3 other X-ray crystal structures. It should be noted that with the exception of the introduction and Chapter 4 (submitted for publication), the remaining chapters of this thesis have been published in international peer reviewed journals. In the next section (DECLARATION 2 – PUBLICATIONS) a precise description of my contribution to each of the publications/chapters is provided.
Synthesis and evaluation of peptides for radiopharmaceutical applications.
(2017) Dutta, Jyotibon.; Govender, Thavendran.; Naicker, Tricia.; Kruger, Hendrik Gerhardus.
Bacterial infection is considered as one of the major threats to human life as well as to the global economy; especially with the increasing number of new multidrug resistant strains. Timely as well as accurate diagnosis of these infections significantly affect the treatment strategies and the prognosis of the disease. Until now, isolation and culturing of the organism is considered to be the gold standard for bacterial infection diagnosis. Conversely, this method is time consuming and labor-intensive. However, with the exponential development in the area of radiopharmaceutics, new imaging probes for the diagnosis of bacterial infection are emerging. The aim of this study focuses particularly on the development of potential radiotracers for imaging of bacterial infection. In this thesis, several topics related to bacterial infection imaging were explored. These topics can be categorized into sections namely; a review on the synthetic approaches of existing potential probes for bacterial imaging, novel on and off resin synthesis of a bifunctional chelator NODASA with a model peptide and lastly, an efficient method for the synthesis of LL37 and NODAGA-LL37 along with its evaluation for bacterial specificity. The first concern about radio tracers is the requirement for an ideal radiopharmaceutical for direct imaging of bacteria. The prime aim of the first part of this thesis is to evaluate the current approaches used for the synthesis of radiolabelled probes for bacterial infection identification as it is clear that such a review will be timeous. In this regard, a review of published work was carried out on the clinical as well as preclinical available probes. Furthermore, existing radiolabelling procedures and suggested mechanisms of radio tracer uptake is also discussed. These molecular probes comprises of leukocytes, antibodies, small molecules, peptides, antibiotics, macrolides, vitamins, oligomers and siderophores. Bifunctional chelators (BFCs) are one of the key elements of a successful radiotracer, which act as a linker between the tracer moiety and the radio isotope. One of the major aims of this study is to develop a method for the synthesis of bifunctional metal chelator NODASA, a potential chelator for radiolabelling. Herein, a facile economic on and off resin method for the synthesis of potential bifunctional chelator “NODASA” functionalized peptide is presented. The seven step synthesis was initiated with a Michael addition reaction between monomethyl fumarate and 1,4,7-triazacyclononane. The final product of NODASA functionalized peptide was obtained with an isolated yield of 84%. A potential human antimicrobial peptide LL37 possesses impending therapeutic values due to its close association with the immune system. Efforts to synthesize LL37 efficiently is one of the goals of this study. In this regard, a highly efficient and optimized methodology for the synthesis of LL37 on solid phase using microwave energy was developed. During this method development it was concluded that uronium coupling reagents along with standard conditions were inadequate for the synthesis of 20th amino acid residue onwards. Val and Ile amino acid coupling was revealed as the key problematic reaction in the segmentation approach of synthesizing the peptide. It was also found that DIC/OxymaPure in THF is a better combination of reagents for this coupling. The synthesized peptide was further verified for its antimicrobial activity. In the last part of this study the aim was to explore the radiolabelling potential of LL37 and its usability as a radiotracer. For this purpose LL37 was functionalized with bifunctional chelator NODAGA. NODASA-LL37 was also labelled with cold/hot gallium successfully. This complex was further evaluated in vitro for its bacterial selectivity over mammalian cell line. With the rapid development of bacterial resistance and the development of “super bugs” there is an urgent need for diagnostic tools which can provide a faster and efficient detection of pathogenic microorganisms. Radiopharmaceutics is an ideal candidate to solve this problem, especially due to its high selectivity, sensitivity and non-invasive nature. In this thesis, an effort was put forward to answer the critical questions regarding the development of synthetic methods, purification and characterization of the antimicrobial peptide LL37. Firstly, a combination of different coupling reagents were used for the optimization of the synthesis, from this study we were able to conclude that the DIC/OxymaPure is better than the HBTU/DIPEA and HATU/DIPEA systems. This method can now be utilized for the large scale production of LL37. In addition to this, a facile seven step method for the synthesis of the bifunctional chelator NODASA functionalized peptide was developed. In this study a NODASA functionalized peptide was conjugated with cold gallium which, demonstrating it as a potential PET agent for molecular imaging. This route offers a simple and inexpensive alternative to commercially available NODASA and can be coupled with various other peptides. Finally, LL37 was also functionalized with the chelator, NODAGA, and subsequently labelled with natGa. This complex showed significant affinity towards bacterial cells in comparison with mammalian cells and providing evidence that NODAGA-LL37 could be a potential radiotracer for bacterial infection imaging.
Synthesis of camphor derived ligands for applications in asymmetric catalysis.
(2009) Boyle, Grant Alexander.; Govender, Thavendran.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.
Chiral monoterpenes such as camphor have been widely used in the development of asymmetric catalysts with varying degrees of success. Pyridyl N-donor ligands derived from camphor have been extensively studied and have proven to be very successful. C3 pendant pyridyl alcohol ligands have been neglected until this study. Herein the synthesis of a series of six novel C3 pendant ligands is described. The ligands were synthesised in six steps (seven for ligand 4) using R-(+)-camphor as the starting material. Two alternative methods for the synthesis were investigated with the second method (Method B) proving to be superior. Several difficulties with regards to regioisomers and diastereomers were overcome in establishing the procedure for the synthesis of the ligands. The final compounds were successfully synthesised in moderate yields with absolute regio- and stereo-control. The ligands were evaluated as chiral catalysts in a series of different reactions. The first of these was the alkylation of a series of aldehydes using diethylzinc. This reaction was investigated in order to compare the efficacy of the novel compounds to previous camphor derived pyridyl alcohol ligands. All previous molecules of this type have been evaluated as catalysts in this reaction with varying degrees of success. The novel ligands successfully catalysed this reaction with moderate to good enantioselectivity (up to 85% ee). The results obtained showed these compounds to be significantly superior to a previous analogous C2 pendant β-amino alcohol reported in literature. The results were also comparable to other camphor derived pyridyl alcohol ligands reported previously. The synthesis of ligands 1-4 as well as their evaluation as catalysts in the alkylation of aldehydes with diethylzinc is discussed in detail in chapter 2 (Paper 1). The second reaction in which the ligands were evaluated was the Henry (Nitroaldol) reaction. This reaction has not seen many camphor derived ligands applied as catalysts. Two additional derivatives (5-6) were synthesized and all the compounds were screened as catalysts in this reaction. The ligands successfully catalyzed the reaction with good to excellent yields but only moderate selectivity (up to 56% ee). The details of this evaluation are discussed in chapter 3 (Paper 2). The final reaction in which the ligands were evaluated was the Diels-Alder reaction of 2- acrolyloxazolidinone with cyclopentadiene. The reaction was again successfully catalysed in moderate to good yields with good endo:exo selectivity but fairly poor stereoselectivity (up to 43% ee). Computational models of the proposed complexes were developed in order to explain the poor observed selectivity. The details of this study are reported in chapter 4 (Paper 3). Chapter 5 (Paper 4) involves a NMR and computational investigation of some of the ligands. Complete NMR elucidation using 2D NMR techniques were carried out for the selected ligands. Optimisation of the ligands using high level DFT calculations was carried out in order to aid in the visualisation of potential through space interactions within each molecule.
Synthesis of novel cage amino acid analogues.
(2001) Govender, Thavendran.; Kruger, Hendrik Gerhardus.; Hariprakasha, H. K.
Amino acids are important building blocks for the synthesis of a large number of biologically active compounds and drugs. Amino acids with the pentacyclo-undecane (1) and trishomocubane (2) frameworks fall into the class of conformationally constrained non-natural amino acids. Conformationally constrained amino acids are found in many naturally occurring, biologically active compounds. It was found that incorporating cage structures into drugs induces a range of positive effects: promotes transport across the cell membrane, drugs can be designed to target the central nervous system, increased receptor site specificity, and retards metabolic degradation. In the light of this, it was decided to investigate the incorporation of cage amino acids into peptides. A synthetic route has been established for the efficient synthesis of amino acids 1 and 2, and for their incorporation into peptides. Several chiral macrocyclic crown ethers and related analogues have been shown to be capable of forming complexes enantioselectively with chiral organic ammonium salts. The design and synthesis of host chiral macrocycles which are able to distinguish between the enantiomers of guest organic ammonium salts is of interest in the areas that include synthesis of enzymes, electrodes for specific ions or molecules, drugs targeted for specific sites, and enantiomer separation. A synthetic procedure has been established for the synthesis of cage annulated chiral crown ethers derived from amino acids. The advantage of using cage compounds in crown ethers is due to increased rigidity, increased solubility in non-polar solvents and increased chirality. Various techniques for the determination of enantiomeric recognition have been studied and include NMR spectroscopy, fluorescence emission spectroscopy and computational methods. The cage crown ether 3 represents a typical example of these new cage annulated, chiral crown ethers.
Synthesis of novel tetrahydroisoquinoline chiral ligands for application in asymmetric transfer hydrogenation.
(2010) Peters, Byron Kennedy.; Govender, Thavendran.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.
Several tetrahydroisoquinoline (TIQ) diamine derivatives were prepared for use as ligands in asymmetric transfer hydrogenation (ATH) of acetophenone of which 17 intermediates and the eight target ligands were novel compounds. The initial design followed that of Noyori, who presented the efficiency of his monotosylated diamine in ATH. A series of eight novel secondary amine derivatives (78a-g and 88) were prepared with substituents that influenced the electronics and the sterics of and around the nitrogen donor. Ligand 71 was shown to have no activity for the ATH of acetophenone. It was apparent from experimental observations that a balance between the electronic and steric characteristics of the substituent was necessary to facilitate activity. It was found that ligand 78d possessing a benzyl group, had the greatest activity (81 % conv.). The greatest selectivity was obtained with ligand 78f (77 % ee) having a chiral phenylmethyl substituent. It was discovered in the case of the active diamine ligands that an optimised 1500 equivalents of water was required in order to demonstrate any enantioselectivity. The exact role of the water has never been ascertained, although there are many publications in which the effect of water has been examined. The most active metal precursor was also investigated and [RhCl2(Cp*)]2 was found to be the best for these TIQ diamine ligands in the specified model reactions. This work has recently been accepted for publication and has established criteria for further rational design on this system.
Synthesis of polycyclic hydantoin derivitaves and peptides.
(2008) Albasheer, Mohamed Saadaldin Altaib.; Govender, Thavendran.; Kruger, Hendrik Gerhardus.
Cyclic cage compounds have attracted much attention in pharmaceutical studies. The lipophilic nature of these compounds plays an important role in facilitating the crossing of the cellular membranes, including the blood brain barrier (BBB) and the central nervous system (CNS). Several adamantane and pentacyclo[5.4.0.02,6.03,10.05,9]undecane (PCU) derivatives have shown great potential as antiviral, antibacterial and neuroprotective compounds. The aim of this study includes the synthesis of hydantoin derivatives of adamantane and PCU as anticonvulsant compounds. Fosphentoin sodium (Cerebyx) 48 is a commercial anticonvulsant drug. Structurally, compound 51 and 52 are similar to Cerebyx 48, where the two phenyl groups have been replaced with PCU or adamantane skeleton respectively. The cage skeleton should increase the lipophilic character of the drug whilst the phosphate group should retain the water solubility of the substrate. The attempted synthesis of these compounds is described in Chapter 2. The PCU hydantoin is readily converted to the PCU amino acid. The synthesis of the PCU amino acid 41 and its Fmoc derivative 106 is described in Chapter 3. This compound was incorporated into small peptides, namely Ala-Ala-Ala-PCU-Ala-Ala-Ala-Fmoc and Ala-Val- PCU-Ile for future testing as a potential anti-cancer agent. NMR studies of these peptides are also reported.
Synthesis of tetrahydroisoquinoline (TIQ) ligands and their applications in ayymmetric catalysis.
(2010) Chakka, Sai Kumar.; Govender, Thavendran.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.
A series of 88 novel tetrahydroisoquinoline (TIQ) compounds have been synthesised for applications in asymmetric catalysis. Several chiral TIQ ligands, possessing N,O and N,N donor atoms, have been prepared and evaluated for the catalytic asymmetric transfer hydrogenation (ATH) of pro-chiral ketones. The highest selectivity obtained for the asymmetric transfer hydrogenation of acetophenone with the N,O donor atom ligands was >99 % ee at low temperatures in iso-propanol with [Ru(p-cymene)Cl2]2 as a pre-catalyst. The observed enantioselectivity was supported by theoretical calculations using the Jaguar interphase program (Paper I). An enantioselectivity of 70 % was obtained with the ligands possessing the N,N donor atoms with the observation that water played a significant role in the enantioselectivity of the ATH reaction of acetophenone (Paper II). An investigation into the usefulness of the TIQ scaffold with other donor atoms was also undertaken. A series of novel P,N oxazoline ligands were synthesised and coordinated to Iridium BArF. These complexes were screened as chiral catalysts for the high pressure asymmetric hydrogenation of unsymmetrical olefins. The reactions proceeded readily at ambient temperature and provided selectivities of up to >91 % ee with excellent conversion rates (>99 %) for the benchmark reactions. Based upon these favorable results, the ligands providing the best results were further screened on a variety of functionalized and unfunctionalised olefins (Paper III). The success of the TIQ backbone in ATH reactions prompted an investigation into its applications in carbon-carbon (C-C) bond forming reactions. The Henry (nitroaldol) reaction is an important C-C bond forming reaction with the chiral oxazoline class of ligands being widely utilised. A series of novel, chiral TIQ oxazoline ligands were synthesised and complexed to various metals (Cu, Sc, Co, Zn, Ni and Mn). These complexes were screened as chiral catalysts in the asymmetric Henry reaction. The highest enantioselectivity (>77 %) was obtained when Cu(OAc)2was employed as a pre-catalyst and iso-propanol as a solvent (Paper IV). The final chapter deals with carbon-sulphur bond formations facilitated by the conjugate addition of thioglycolate to various chalcones. A series of novel, chiral TIQ N-oxide ligands were synthesised and complexed to lanthanum. The complexes were screened for activity against the benchmark reaction and an enantioselectivity of 81 % was obtained.
Use of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to detect carbapenem-resistant Enterobacteriaceae.
Somboro, Anou Dit Moise.; Govender, Thavendran.; Essack, Sabiha Yusuf.; Kruger, Hendrik Gerhardus.
Antimicrobial drug resistance is increasing worldwide at a frightening rate and is found among different species of bacteria causing nosocomial and community-acquired infections. Carbapenem-resistant Enterobacteriaceae (CRE’s) are one of the multi-drug resistant bacteria increasingly reported in the past decade, leaving the health care system unable to control the spread of antibiotic-resistant infections. A rapid and efficient method to identify drug resistant bacteria could advance the containment of this scourge. We thus investigated the use of MALDI-TOF MS technology, recognized as an accurate method for microorganism identification, as a potential tool for the detection of CRE based on the protein profiling for specific fingerprints. From 24 hours sub-cultured bacterial isolates known to carry and/or express single genes of a variety of carbapenemases, proteins were extracted using the formic acid ethanol extraction method and subjected to MS-based identification by MALDI-TOF MS Biotyper technology. The mass spectrometry spectra were then analyzed employing the programmed software flexAnalysis version 3.4 build 70 and ClinProTools version 2.2 build 83 to detect carbapenemases. The spectra were subjected to visual inspection for specific peaks characterization. Despite their close relationship, the method was able to differentiate between CRE and susceptible bacteria. The differences within carbapenemases were better distinguishable by visual observation of specific biomarker peaks and the modified main spectrum (MSP) method demonstrated that these differences could be more obvious by focusing on matchless low intensity peaks. Although the differentiation of CRE remains a challenge using MALDI Biotyper MS based on the protein profiling, according to our data and pending further investigation in clinical isolates of CRE, MALDI-TOF MS has potential as a diagnostic tool for the rapid detection of these multi-drug resistant bacteria.