Synthesis and evaluation of peptides for radiopharmaceutical applications.

Doctoral Degree. University of KwaZulu-Natal, Durban.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