Browsing by Author "Naicker, Tricia."

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Computationally aided direct electrophilic trifluoromethylation and trifluoromethylthiolation via sulfonimidamides.
(2020) Chetty, Lloyd Christopher.; Naicker, Tricia.; Kruger, Hendrik Gerhardus.
Fluorination chemistry is of interest due to fluorine being recognized as a crucial element in pharmaceuticals, and agrochemicals, with 30% of new small molecule drugs incorporating fluorine. The trifluoromethylation and trifluoromethylthiolation of the active pharmaceutical ingredients formed the basis for the modern trend of fluorination of pharmaceutical compounds. The incorporation of a trifluoromethyl group (CF3) into an organic molecule has a significant effect on its lipophilicity, permeability, and metabolic stability. Radical mediated trifluoromethylation facilitated by photoredox catalysis offers mild and highly selective reaction conditions. While there are several commercially available trifluoromethylation reagents, some limitations include the use of gaseous, volatile, and expensive reagents. Therefore, the development of cheaper and safer trifluoromethylation reagents is crucial. The utilisation of computational chemistry can facilitate the design of new potential agents. This study focused on the computational design and thereafter, the synthesis of sulfonimidamides as potential radical trifluoromethylation agents via photoredox catalysis. Despite all efforts to synthesise trifluoromethylated sulfonimidamides being unsuccessful, the synthesis and characterisation of precsuors compounds 5a-d were successful and resulted in 6 novel X-ray crystal structures. In addition, a simple yet efficient computational method for calculating redox potentials was developed. The decision was then to synthesise trifluoromethylthiolated sulfonimidamides based on the success of sulfonamides as trifluoromethylthiolating agents. The trifluoromethylthio group (SCF3) has attracted particular interest in medicinal chemistry due to its remarkable lipophilicity. Due to its high lipophilicity and strong electronwithdrawing ability, the SCF3 greatly improves the pharmacokinetic properties of lead compounds. Among the various electrophilic reagents available, N-SCF3 reagents are the most utilised. Previously developed reagents require a strong Brønsted or Lewis acid for activation of the reaction. To address this problem, the second part of this study focused on the computational design and thereafter synthesis of more efficient sulfonimidamide based electrophilic trifluoromethylthiolation agents. Sulfonimidamides 5c, f were successfully trifluoromethylthiolated, resulting in the corresponding N-trifluoromethylthio sulfonimidamides 7c, f. Novel X-ray crystal structures for 5e and 5f are also obtained. The computationally calculated SCF3 electrophilic donation potential of sulfonimidamides 7c, e, f revealed that sulfonimidamide 7c possessed the greatest potential for donation (36.51 Kcal mol-1) and has the potential to be more electrophilic than previously applied delivering agents (ranging from 9.8-59.1 Kcal mol-1). Therefore, sulfonimidamide 7c was chosen as the donating agent for the further electrophilic trifluoromethylthiolation of ethyl cyanoacetate and 2,4-dimethylpyrrole. The results from the trifluoromethylthiolation model reactions indicated that sulfonimidamide 7c is a potentially new SCF3 donating agent, due to the trifluoromethylthio group leaving from sulfonimidamide 7c as confirmed by crude 19F NMR and LC-MS analysis. However, further method optimisation is required and is ongoing to determine the substrate scope and reaction conditions. Various characterisation techniques were used to confirm the chemical synthesis of the compounds which include liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), high resolution mass spectrometry (HRMS), X-ray powder diffraction (XRD), and infrared spectrometry (IR). A potential future recommendation for the N-trifluoromethylthiolation of the sp2 type nitrogen’s and N-trifluoromethylation of sulfonimidamides is the use of trifluoromethylthiolated and trifluoromethylated amines for the amination of the sulfonimidoyl chlorides.
Evaluation of the pharmacokinetics of ketamine for the treatment of major depressive disorder.
(2018) Naidoo, Vivian Campbell.; Baijnath, Sooraj.; Naicker, Tricia.; Naidoo, Panjasaram.; Kruger, Hendrik Gerhardus.
Recent reports have demonstrated ketamine’s potential use in the treatment of major depressive disorder (MDD), as it elicits potent antidepressant effects via a different mechanism compared to conventional antidepressants. Ketamine’s hypothesized antidepressant effect is elicited by a neurochemical cascade involving the antagonization of the N-methyl-D-aspartate (NMDA) receptors and the subsequent activation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors; resulting in the disinhibition of glutamate signalling due to the suppression of tonic glutamate input into the GABAergic interneurons, providing rapid symptomatic relief as opposed to the two-week delay with conventional treatments. There is a large escalation in the number of individuals being diagnosed with treatment resistant depression (TRD) even after numerous trials on conventional antidepressant therapy. Health care professionals are now resorting to unconventional treatments, such as ketamine’s off-label use, to achieve therapeutic outcomes and provide symptomatic relief. MDD’s increasing prevalence has been associated with significant public health costs and morbidity rates and therefore alternative, effective treatments are now essential. Many reports have been published on the intranasal (IN) efficacy of ketamine in the treatment of major depressive disorder, however there have been no studies investigating the effects on the route of administration in drug delivery to the brain. The purpose of this study was to investigate pharmacokinetics of ketamine following oral, intraperitoneal and intranasal administration. A dose of 15mg/kg (body weight) was administered to healthy male Sprague-Dawley rats, and ketamine concentrations were quantified in both plasma and brain tissue homogenates at time intervals of 5, 15, 30, 60, 120, 240 minutes post-treatment. The results showed that with intraperitoneal administration, concentrations of 524,58 ng/mL and 352,06 ng/mL, were achieved in plasma and brain tissue, respectively. Surprisingly, IN administration which is believed to favour drug delivery to the brain only exhibited moderate levels post administration; whereas, oral administration produced significantly lower levels due to extensive first-pass metabolism of ketamine in the liver and intestines. These results show that parenteral administration should be used for the administration of ketamine in the treatment of MDD. The findings of the study provide a platform for future investigations assessing alternative routes of administration of ketamine; and its use in clinical practice for the treatment of MDD. This paves the way forward to optimize treatment and provide symptomatic relief were conventional antidepressants have failed those suffering with MDD.
Evaluation of the use of cannabidiol in the treatment of anxiety related disorders by assessing changes in neurotransmitter levels and expression of CREB/BDNF in the rodent brain.
(2020) Haripershad, Advaitaa Meera.; Baijnath, Sooraj.; Naicker, Tricia.
Anxiety and anxiety-related disorders are common psychiatric disorders that are responsible for high disease burden. The pathogenesis of anxiety involves dysfunction in the limbic brain regions including the amygdala, prefrontal cortex, and hippocampus. The current pharmacological treatments for anxiety target the modulation of the activity monoamine neurotransmitters such as dopamine, serotonin, gamma-aminobutyric acid, norepinephrine and glutamate. These neurotransmitters are key in the regulation of the maladaptive responses of anxiety. Primary pharmacotherapies demonstrate limitations in drug efficacy as well as adverse side effects, highlighting the need for novel therapeutics for anxiety and anxiety-related disorders. Cannabidiol (CBD), a non-psychoactive cannabinoid from the Cannabis sativa plant, has been considered a potential anxiolytic treatment as a result of its interaction in the endocannabinoid system, which regulates synaptic plasticity and neuronal activity implicated in the anxiety response. The therapeutic potential of CBD against neuropsychiatric disorders have been reported in preclinical and clinical studies. Since the global increase in cannabis legalization, there remains a need to supplement the available literature related to the neural effect of cannabis use on behavioural, neurochemical and biochemical changes. There are gaps in the knowledge of the pharmacokinetics and behavioural effects of CBD. This study will contribute to increasing the knowledge of the effect of cannabis on neurotransmitters and molecular changes in the brain. In this thesis, chapter 1 is a literature review focusing on the neurobiology and pharmacological treatments of anxiety, cannabidiol as a treatment for anxiety, and the neurotransmitters and genes implicated in anxiety. In addition to this, chapter 1 also reviews the theory of the experimental processes performed in this study. Chapter 2 is the publication “Evaluation of the use of cannabidiol in the treatment of anxiety related disorders by assessing changes in neurotransmitter levels and expression of CREB/BDNF in the rodent brain” submitted to The Journal of Neuroscience Research. Chapter 3 is the summary and conclusion of the thesis.
The in vitro and in vivo efficacy of novel metallo-β- lactamase inhibitors co-administered with meropenem to target CREs.
(2022) Reddy, Nakita.; Naicker, Tricia.
The evolution and phenotypic expression of metallo β-lactamase genes across the world has led to the escalated transmission rates of carbapenem resistance. The effect has crippled the already impaired healthcare system, with the emergence of COVID-19 exacerbating the crisis further. Our plight for a solution to combat antimicrobial resistance has not been greater. One strategy to tackle this non-susceptibility is the development of metallo-β-lactamase inhibitors that can neutralize the metallo-β-lactamase enzyme, thereby allowing the carbapenem antibiotic to elicit its function on the microorganism. Currently, there is no FDA-approved metallo-β-lactamase inhibitor to meet the clinical challenges of drug resistance. In a desperate need to find a candidate drug, research has been initiated into the discovery and development of biologically active inhibitors. Therefore, this thesis focuses on the advances made by our research group, the Catalysis and Peptide Research Unit, in developing novel β-lactam derived inhibitors; NOTA, NO3PY, BP- 1, 6,10 and 14, that re-sensitize the microbe to the efficacy of meropenem. The in vitro and in vivo activities of the initial chelators, NOTA and NO3PY, were evaluated as potential metallo-β-lactamase inhibitors (MBLIs) against metallo-β-lactamase (MBL) resistant bacteria. Time-kill studies showed that NOTA and NO3PY restored the efficacy of meropenem against all bacterial strains tested. A murine infection model was then used to study both metal chelators’ in vivo pharmacokinetics and efficacy. NO3PY displayed poor bioavailability at the selected doses using a validated LC-MS/MS method, therefore discouraging the in vivo efficacy evaluation. NOTA showed good bioavailability; hence, the in vivo efficacy was determined in a murine thigh infection model. The co-administration of meropenem and NOTA (100 mg/kg.bw each) significantly decreased the colony-forming units of K. pneumoniae NDM over an eight-hour treatment period. The findings suggested that chelators, such as NOTA, hold strong potential for use as an MBLI in treating CRE infections; however, further preclinical development was needed to improve the pharmacokinetic properties of these agents to increase their bioavailability and tissue distribution. With this information, our group derivatized NOTA by coupling it to a β-lactam to create the BP series of novel MBLIs. The results generated by the BP compounds have proven to interact synergistically with meropenem, by restoring the MIC of meropenem to therapeutically acceptable concentrations (< 2 mg/L) that concur with the breakpoints outlined by CLSI. In addition, the bactericidal activity of the re-sensitized meropenem was evident in the time-kill study over 24 hours. Cytotoxicity assays were further conducted to study the inhibitors, with an outcome in favor of safe administration in vivo. The metallo-β-lactamase inhibitors reported herein have demonstrated good potency against NDM-1 and VIM-2 metallo-β-lactamases with a Ki of 25-97μM. Since the BP compounds are metal chelators that function as metallo-β- lactamase inhibitors, it was important to determine the binding specificity of the BP compounds to a physiologically relevant zinc-harboring enzyme, glyoxylase II. At concentrations of up to 500 μM of BP, the activity of glyoxylase II remained unhindered. This confirmed the hypothesis of BP specificity to be exclusive to NDM-1 and VIM-2 metallo-β-lactamases. These findings prompted further interest in the binding exhibited by BP and led to additional studies to address the binding interactions of BP with the metallo-β-lactamases through quenching and computational experiments. Fluorescent quenching experiments investigating the Ka of BP indicated that a higher binding affinity was noted for NDM-1 compared to VIM-2 MBLs, thus implying a stronger interaction with NDM-1. Molecular docking and dynamic simulation experiments shed light on the BPs’ mode of action, showing the interaction of the chelators’ carboxylic moiety with the Zn 2+ ions in the MBLs structure. In favor of this BP series as functional inhibitors, in vivo efficacy was explored in a murine infection model (BP1 and BP10). In Klebsiella pneumoniae NDM infected mice, BP co-administered with meropenem was efficacious in reducing the bacterial load by > 3 log10 units’ post-infection, compared to meropenem monotherapy. These findings validate our strategy for derivatizing NOTA into the series of the BPs, as the bioavailability of NOTA, when coupled to a cephalosporin, improved the overall in vivo efficacy, and allowed the drug to be quantified in plasma under the same conditions previously used. This study clearly indicated the influence of the BP compounds in reducing the bacterial burden and the success of employing combination therapy as a treatment alternative. Moreover, the outcome of this preclinical development represents a solid foundation, whereby we can build on our existing knowledge. In aligning with our research goals of alleviating the threat of antimicrobial resistance, coupling β-lactams to a cyclic zinc chelator offers a safe and efficacious solution to meet the calamity that plagues our healthcare system.
Investigation into the synthetic potential of ethenesulfonyl fluoride via homogenous catalysis.
(2017) Govender, Kimberleigh Bianca.; Naicker, Tricia.
Sulfonyl fluorides are becoming increasingly attractive in both chemistry and biology. Not only does this functional group inhibit several proteins, but several advantages over its Br and Cl counterparts, makes it a useful synthon and “click reagent” for organic synthesis. Despite this, there are very few methods to easily obtain various sulfonyl fluoride derivatives. One particular sulfonyl fluoride containing compound that has received considerable attention in recent literature is ethenesulfonyl fluoride (ESF). This molecule has been used to attach the sulfonyl fluoride moiety onto larger, more complex molecules. Thus, the main objective of this thesis was to investigate some unexplored avenues of the application of ESF in homogenous catalysis in order to easily prepare sulfonyl fluoride derivatives. The transformations and target materials selected would thus be both versatile and indispensable tools for organic synthesis. To achieve this aim, the use of a transition metal catalyzed reaction was first employed. A palladium catalyzed oxidative Heck coupling reaction was developed using ESF as the substrate. After screening various palladium catalysts, oxidants and bases, a mixture of Pd(OAc)2, Cu(OAc)2 and LiOAc resulted in an effective combination that provided the desired products in good yields (up to 80 %) under mild conditions. The generality of this method was demonstrated by the effective reactivity maintained when screened against a diversity of boronic acids. The usefulness of the products generated from this method was demonstrated by the subsequent development of a novel one-pot preparation of β-sultams. These motifs have otherwise remained elusive to organic synthesis emphasizing the importance of this new approach (Chapter 2). The application of the oxidative Heck method was further investigated as a key step in the preparation of Naratriptan, an effective and popular drug for the treatment of migraines. The attachment of ESF onto the desired indole scaffold was successful, however the remaining proposed steps proved to require additional optimization. It was found that when the β-sultam was treated under basic conditions, a novel ring opening reaction of the β-sultam occurred, which resulted in the formation of vinyl sulfonamides. It was concluded that as a future prospect, hydrogenation of the vinyl sulfonamides be carried out prior to the Aldol condensation in the preparation of Naratriptan (Chapter 3). To fully apply the ESF to homogenous catalysis and to obtain more sulfonyl fluoride derivatives, it’s amiability to organocatalysis was also explored. ESF was used as a substrate in a number of Michael reactions in which organocatalysts with various modes of action were applied. It was found that reacting the ESF with methyl 2-oxocyclopentanecarboxylate in the presence of a chiral thiourea catalyst (hydrogen bonding catalysis), showed the best reactivity and yield (96 %). Determining the enantioselectivity of the reaction proved difficult by chiral HPLC, however when the optical rotation was measured, it was found that the reaction was enantioselective (Chapter 4). A crystal of the product was grown and analysed, which confirmed the synthesis of the racemate (Chapter 5). This proved to be the first instance of an organocatalyzed reaction where ESF was used as a substrate. These results have confirmed the hypothesis of using ESF within organocatalysis and has led to new opportunities in the search for new sulfonyl fluoride derivatives.
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.
Sub/supercritical fluid chromatography purification of biologics.
(2020) Govender, Kamini.; Naicker, Tricia.; Kruger, Hendrik Gerhardus.
Peptide and protein drugs are highly versatile with numerous therapeutic properties such as anti-cancer, anti-diabetic, anti-hypertensive, and anti-microbial; which are therefore ideal candidates for the development of next-generation drugs. This is exemplified using these drugs for the treatment of diseases such as diabetes. Diabetes is one of the most prevalent non-communicable diseases worldwide. The rapid increase in the number of diabetic patients globally places a burden on current insulin manufacturers. The traditional reversed-phase high-performance liquid chromatography (RP-HPLC) purification methods of insulin and peptides are problematic, tedious with long run times of approximately 50 minutes, low yields employ harsh solvents such as acetonitrile, which has a negative impact on the environment. There is a need for a greener process for the purification of insulin and peptides. Sub/supercritical fluid chromatography (SFC) can provide the solution since it utilises greener mobile phases such as carbon dioxide (CO2) and methanol, which can be recycled. However, there is a paucity of knowledge regarding the SFC purification of human insulin and peptides. Therefore, this research study aimed to provide an efficient, innovative approach for the biosynthesis of human insulin and the SFC purification of biosynthesised human insulin, as well as the extension into the SFC purification of peptides. The background of these topics is presented in Chapter One. Chapter two (manuscript one) presents the development of a novel and more efficient method of human insulin biosynthesis in Escherichia coli (E. coli). Several of the conventional steps were eliminated. The crude biosynthesised protein sequence was verified using protein sequencing, which had a 100% similarity to the human insulin sequence. The biological activity of the biosynthesised human insulin was tested in vitro using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The biosynthesis of human insulin was conducted on a laboratory-scale basis; future studies should investigate scaling up of this method. Chapter three (manuscript two) was based on the SFC purification of the commercially available standard sample of human insulin and a crude biosynthesised sample of human insulin. The SFC purified standard the biosynthesised human insulin samples were detected and quantified using liquid chromatographymass spectrometry (LC-MS) and protein sequencing techniques. SFC columns, i.e., silica, 2’ ethyl pyridine, diol-HILIC, and the pentafluoro phenyl (PFP), were evaluated to determine the ideal column. The PFP column gave the best results since it displayed good peak shapes, resolution, retention factors, retention times, and the least relative standard deviation in comparison to the other columns. Therefore, the aforementioned column was selected for further analysis using the biosynthesised human insulin, whereby a column efficiency test was conducted on a semi-preparative scale, yielding 84% recovery. Subsequently, the biological activities of the SFC purified standard sample of human insulin and biosynthesised version were tested in vitro using a MTT assay. The results indicated that the biological activities of the standard and biosynthesised human insulin derivatives were retained subsequent to SFC purification. The biological activities were highly significant, with a p-value of < 0.0001. From chapter three, band broadening and phase separation peaks were experienced during SFC purification of the commercially available standard sample of human insulin and biosynthesised human insulin. Therefore, in Chapter four (manuscript three), a SFC purification method was developed to purify peptides at an analytical scale. A tetrapeptide [insulin β chain peptide (15-18)], octapeptide [angiotensin II], nonapeptide [insulin β chain peptide (15-23)] were purified using four SFC columns, i.e., PFP, diolHILIC, silica, and 2’ ethyl pyridine. Subsequently, the 2’ ethyl pyridine column was selected for further analysis based on the reproducibility, peak shapes, efficient separations and retention factors. The three peptides were monitored using LC-MS analysis. The successful peptide recoveries ranged from 80-102%. Chapter five pertains to the summary and conclusion drawn from the study and reflects on possible future endeavours. The present study was successful in providing a more affordable and innovative approach for the biosynthesis of human insulin. The work also successfully developed a rapid, greener, and more efficient method of SFC purifying biosynthesised human insulin and peptides as opposed to conventional HPLC purification methods. As far as we are aware, this study is the first of its kind to purify biosynthesised human insulin and this combination of peptides using SFC purification techniques. Future research studies can focus on the SFC purification of larger protein molecules and consider the use of custom columns and or other modifiers for the improvement of the isolation of other highly sought after biologics within the pharmaceutical industry.
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 novel pentacyclo-undecane chiral ligands for application in asymmetric catalysis
(2008) Naicker, Tricia.
There is enormous interest in the design and development of efficient chiral ligands for asymmetric catalysis, as a result, this field has become one of the most popular areas of research in organic chemistry. This project involved the investigation of the novel chiral pentacyclo-undecane (PCU) diol 54a, PCU bisimine 87 and PCU bis(oxazoline) 100 type ligands. The PCU diol ligand was synthesized, but proved to be difficult to obtain enantiomerically pure which hindered further investigation into this type of ligand. The PCU bisimine ligand 87 was synthesized. However due to its instability it was not further pursued. Synthesis of the PCU bis(oxazoline) ligand 100 was successful. This ligand was complexed to various metal salts and its efficiency as a chiral Lewis acid catalyst was evaluated on the asymmetric Diels-Alder reaction between 3-acryloyloxazolidin- 2-one 52 and cyclopentadiene 33. The anhydrous magnesium perchlorate ligand complex emerged as the best catalyst providing the endo-cycloadduct product 53 in 81 % enantiomeric excess at -40 oC. Optimizations of the possible conformations of the magnesium complex of ligand 100 with the substrate 52 were performed using Density Functional Theory (DFT) calculations. The more energetically favoured complex conformation was established. The Re-face of the dienophile which was less hindered produced the product consistent with the experimentally observed product 16. Based on the calculated bond lengths from the computational model binding of the ether oxygen on the PCU moiety to magnesium was observed. All the novel compounds were fully characterized using NMR, IR and mass spectroscopy as the main tools.