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Towards a multi-epitope cell imaging technique assisted by metal-chelating polymers and SEM-EDS
Thesis (MSc)--Stellenbosch University, 2018.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2018
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| _version_ | 1867613902357921792 |
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| access_status_str | Open Access |
| author | Dreyer, Rudolf Petrus Gerhardus |
| author2 | Klumperman, Bert |
| author_browse | Dreyer, Rudolf Petrus Gerhardus Klumperman, Bert |
| author_facet | Klumperman, Bert Dreyer, Rudolf Petrus Gerhardus |
| author_sort | Dreyer, Rudolf Petrus Gerhardus |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2018. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/103865 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:43:31.605Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2018 |
| publishDateRange | 2018 |
| publishDateSort | 2018 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/103865 Towards a multi-epitope cell imaging technique assisted by metal-chelating polymers and SEM-EDS Dreyer, Rudolf Petrus Gerhardus Klumperman, Bert Pfukwa, Rueben Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science. Cell-imaging Epitopes Metal chelate affinity chromatography Fragmentation (Chemistry) Thesis (MSc)--Stellenbosch University, 2018. ENGLISH ABSTRACT: This thesis reports the design, synthesis and characterization of a novel trithiocarbonate chain transfer agent for reversible addition-fragmentation chain-transfer (RAFT) polymerization. The RAFT agent is designed with benzylguanine (BG) functionality built into the R-group. The rationale behind the RAFT agent design is to develop a facile route to the synthesis of polymers with α-BG functionality to be used as labels for SNAP-tag – a modified version of the hAGT enzyme with the ability to bind various BG-modified substrates. The RAFT agent with BG functionality was successfully synthesized by the copper(I)- assisted azide-alkyne cycloaddition reaction of but-3-yn-2-yl carbonotrithioate and an O6-(p- azidobenzyl)guanine derivative which we developed. The RAFT agent was used in the synthesis of poly(tert-butyl acrylate) (PtBA) showing good control and a low dispersity, and we were able to confirm the α-BG end-functionality of the polymer. The synthesis of α-BG-functional PtBA was followed by a series of side-chain modifications, whilst retaining the α-BG chain-end. The first step was the deprotection of the butyl ester group to form poly(acrylic acid) (PAA), followed by a coupling reaction with t-BOC- ethylenediamine and the subsequent deprotection of the BOC groups to yield an amine- functional polymer. The final step was the attachment of a DTPA ligand to each repeat unit to form α-BG functional poly(DTPA) as a polymer easily capable of metal-chelation. Finally, we show that ω-chain-end modification of the polymer can be achieved by aminolysis of the ω-trithiocarbonate end-group followed by a thiol-Michael addition click reaction of a maleimide functional fluorescent dye, without affecting the α-BG chain-end. The thesis reports the successful synthesis of a novel BG-functional RAFT agent, followed by preliminary investigations towards the development of α-BG-functional and ω- fluorescently labelled metal-chelating polymers (MCPs) which we aim to use in the development of a high resolution, spatial cell imaging technique assisted by SEM-EDS ENGLISH ABSTRACT: Hierdie tesis rapporteer die ontwerp, sintese en karakterisering van 'n nuwe trithiocarbonate ketting oordrag agent vir omkeerbare byvoeging-fragmentasie ketting-oordrag (RAFT) polimerisasie. Die RAFT agent is ontwerp met benzylguanine (BG) funksionaliteit by die R- groep ingebou. Die rasionaal agter die ontwerp is die ontwikkeling van ‘n eenvoudige roete tot die sintese van polimere met α-BG funksionaliteit as merkers vir SNAP-tag – 'n gewysigde weergawe van die hAGT ensiem met die vermoë om te bind met BG- gemodifiseerde molekules. Die RAFT agent met BG funksionaliteit was suksesvol gesintetiseer deur die koper (I) - geassiseerde azide-alkyne sikloaddisie reaksie van but-3-yn-2-yl carbonotrithioate en 'n O6- (p-azidobenzyl) guanine afgeleide wat ons ontwikkel het. Die RAFT agent was gebruik in die sintese van poly(tert-butyl acrylate) (PtBA) en toon goeie beheer en 'n lae ketinglengte verspreiding. Ons het die teenwoordigheid van die α-BG eindgroep van die polimeer bevestig. Die sintese van α-BG-funksionele PtBA is gevolg deur 'n reeks kant-ketting modifikasies, met focus op die behoud van die α-BG eindgroep. Die eerste stap was die hidroliese van die butiel ester groep om poly(acrylic acid) (PAA) te vorm. Dit was gevolg deur 'n koppeling reaksie met t-BOC-ethylenediamine en die daaropvolgende verwydering van die BOC groepe om 'n amien-funksionele polimeer te vorm. Die finale stap was die aanheging van 'n DTPA ligand op elke herhaaleenheid om α-BG funksionele poly(DTPA) te vorm as 'n polimeer wat maklik met ‘n verskeidenheid metale kan bind. Hierna wys ons dat modifikasies van die ω- ketting-einde van die polimeer bereikbaar is deur aminoliese van die ω-trithiocarbonate einde-groep gevolg deur 'n thiol-Michael-byvoeging klik reaksie van 'n maleimide funksionele fluoresserende molekule, sonder om die α-BG ketting-einde te affekteer. n opsomming rapporteer hierdie tesis die suksesvolle sintese van 'n BG-funksionele RAFT agent, gevolg deur voorlopige ondersoeke na die ontwikkeling van α-BG-funksionele en ω- fluoresserende metaal-chelaterende polimere (MCPs). Ons mik om hierdie te gebruik in die ontwikkeling van 'n hoë resolusie, ruimtelike cell imaging tegniek gehelp deur SEM-EDS. Masters 2018-02-26T08:52:37Z 2018-04-09T11:46:11Z 2021-03-23T03:00:09Z 2018-03 Thesis http://hdl.handle.net/10019.1/103865 en_ZA Stellenbosch University 70 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Cell-imaging Epitopes Metal chelate affinity chromatography Fragmentation (Chemistry) Dreyer, Rudolf Petrus Gerhardus Towards a multi-epitope cell imaging technique assisted by metal-chelating polymers and SEM-EDS |
| title | Towards a multi-epitope cell imaging technique assisted by metal-chelating polymers and SEM-EDS |
| title_full | Towards a multi-epitope cell imaging technique assisted by metal-chelating polymers and SEM-EDS |
| title_fullStr | Towards a multi-epitope cell imaging technique assisted by metal-chelating polymers and SEM-EDS |
| title_full_unstemmed | Towards a multi-epitope cell imaging technique assisted by metal-chelating polymers and SEM-EDS |
| title_short | Towards a multi-epitope cell imaging technique assisted by metal-chelating polymers and SEM-EDS |
| title_sort | towards a multi epitope cell imaging technique assisted by metal chelating polymers and sem eds |
| topic | Cell-imaging Epitopes Metal chelate affinity chromatography Fragmentation (Chemistry) |
| url | http://hdl.handle.net/10019.1/103865 |
| work_keys_str_mv | AT dreyerrudolfpetrusgerhardus towardsamultiepitopecellimagingtechniqueassistedbymetalchelatingpolymersandsemeds |