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RAFT polymerization as a scaffold for the synthesis of SNAP-tag labels
Thesis (PhD)--Stellenbosch University, 2022.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613986810232832 |
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| access_status_str | Open Access |
| author | Dreyer, Rudolf Petrus Gerhardus |
| author2 | Klumperman, Lubertus |
| author_browse | Dreyer, Rudolf Petrus Gerhardus Klumperman, Lubertus |
| author_facet | Klumperman, Lubertus Dreyer, Rudolf Petrus Gerhardus |
| author_sort | Dreyer, Rudolf Petrus Gerhardus |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/125160 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:44:52.037Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/125160 RAFT polymerization as a scaffold for the synthesis of SNAP-tag labels Dreyer, Rudolf Petrus Gerhardus Klumperman, Lubertus Pfukwa, Rueben Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science. Bioconjugates Fluorescent labeling Polymers -- Synthesis UCTD Thesis (PhD)--Stellenbosch University, 2022. ENGLISH ABSTRACT: This thesis reports the design, synthesis, and characterization of a series of novel chain transfer agents for the synthesis of polymeric SNAP-tag labels by reversible addition-fragmentation chain-transfer (RAFT) polymerization. The RAFT agents are designed with an O6-benzylguanine (BG) moiety built into the R-group to impart α-BG functionality to the synthesized polymers. The SNAP-tag enzyme reacts specifically and covalently with BG-derivatives to label SNAP-tag fusion proteins. Our goal is to form polymeric bioconjugates between SNAP-tag fusion proteins and RAFT-synthesized α-BG functional polymers. The first RAFT agent (BG-T-R1) was successfully synthesized and showed good control in the synthesis of poly(tert-butyl acrylate) (PtBA). The synthesis of α-BG-PtBA was followed by a series of side-chain modifications to form a BG functional metal chelating polymer (MCP), α-BG-P(DTPA). After attempting SNAP-tag conjugation, we found that the use of trifluoroacetic acid (TFA) in the side-chain modification steps destroyed the BG-functionality of the polymer. The polymer system was changed to poly(pentafluorophenyl acrylate) (P(PFPA)). P(PFPA) side chains can be modified by reaction with an amine to form a MCP, however we were also unable to achieve SNAP-tag conjugation using polymers based on the P(PFPA) scaffold. To overcome suspected steric hinderance, the RAFT agent was modified by inserting a methylene spacer between the phenyl ring and triazole linker (BG-T-R2). After using the RAFT agent to synthesize P(PFPA) and modification of the polymer to water-soluble poly((2-hydroxypropyl)acrylamide) (P(HPAm)) we were still unable to achieve SNAP-tag conjugation. The RAFT agent was then adapted to have a xanthate Z-group (BG-X-R2) which controls the polymerization of N-vinylpyrrolidone to form α-BG-poly(N-vinylpyrrolidone) (α-BG-PVP). The ω-chain end of α-BG-PVP underwent aminolysis and the resulting thiol was reacted with a maleimide functional fluorophore to form α-BG-ω-Alexa488-PVP35. Using fluorescence imaging we were able to confirm SNAP-tag conjugation with the ω-modified polymer and could assume that polymers without the fluorophore had also conjugated. Finally, we developed a fluorogenic RAFT agent (BGAN-X-R) with which we planned to follow the SNAP-tag conjugation kinetics of various chain lengths of α-BGAN functional polymers. The system was not as efficient as desired, but the work has laid the foundation for future investigations. AFRIKAANSE OPSOMMING: Hierdie tesis rapporteer die ontwerp, sintese en karakterisering van 'n reeks nuwe kettingoordragmiddels vir die sintese van polimeriese SNAP-tag etikette deur omkeerbare addisie-fragmentasie kettingoordrag (RAFT) polimerisasie. Die RAFT-middels is ontwerp met 'n O6-bensielguanien (BG) in die R-groep ingebou om α-BG-funksionaliteit aan gesintetiseerde polimere te verleen. Die SNAP-tag-ensiem reageer spesifiek en kovalent met BG-afgeleides om SNAP-tag-fusieproteïene te merk. Ons doel is om polimeriese biokonjugate te vorm tussen SNAP-merker-fusieproteïene en RAFT-gesintetiseerde α-BG funksionele polimere. Die eerste RAFT-middel (BG-T-R1) is suksesvol gesintetiseer en het goeie beheer in die sintese van poli(tert-butielakrilaat) (PtBA) getoon. Die sintese van α-BG-PtBA is gevolg deur 'n reeks syketting modifikasies om 'n BG funksionele metaal chelerende polimeer (MCP), α-BG-P(DTPA) te vorm. Nadat ons SNAP-tag-vervoeging probeer het, het ons gevind dat die gebruik van trifluoroasynsuur (TFA) in die sykettingmodifikasiestappe die BG-funksionaliteit van die polimeer vernietig het. Die polimeerstelsel is verander na poli(pentafluorfenielakrilaat) (P(PFPA)). P(PFPA)-sykettings kan gemodifiseer word deur reaksie met 'n amien om 'n MCP te vorm, maar ons kon ook nie SNAP-tag-vervoeging te bereik deur met hierdie polimere nie. Om vermoedelike steriese verhindering te oorkom, is die RAFT-middel gemodifiseer deur 'n metileenspasieerder tussen die fenielring en triasoolskakelaar (BG-T-R2) in te voeg. Na die gebruik van die RAFT-middel om P(PFPA) te sintetiseer en die modifikasie van die polimeer na wateroplosbare poli((2-hidroksipropiel)akrielamied) (P(HPAm)), was ons SNAP-tag-vervoeging steeds onsuksesvol. Die RAFT-middel is dan aangepas om 'n xanthaat Z-groep (BG-X-R2) te hê om die polimerisasie van N-vinielpirrolidon te beheer en α-BG-poli(N-vinielpirrolidon) (α-BG-PVP) te vorm. Die ω-ketting einde van α-BG-PVP het aminolise ondergaan en die resulterende tiol is met 'n maleïmied funksionele fluorofoor gereageer om α-BG-ω-Alexa488-PVP35 te vorm. Met behulp van fluoressensie beelding kon ons SNAP-tag vervoeging met die ω-gemodifiseerde polimeer bevestig en aanvaar dat polimere sonder die fluorofoor ook gekonjugeer het. Laastens het ons 'n fluorogeniese RAFT-middel (BGAN-X-R) ontwikkel waarmee ons beplan het om die SNAP-tag konjugasiekinetika van verskeie kettinglengtes van α-BGAN funksionele polimere te volg. Die stelsel was nie so doeltreffend soos verlang nie, maar die werk het die grondslag gelê vir toekomstige ondersoeke. Doctoral 2022-01-14T07:39:11Z 2022-04-29T12:56:38Z 2022-01-14T07:39:11Z 2022-01 Thesis http://hdl.handle.net/10019.1/125160 en Stellenbosch University xv, 127 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Bioconjugates Fluorescent labeling Polymers -- Synthesis UCTD Dreyer, Rudolf Petrus Gerhardus RAFT polymerization as a scaffold for the synthesis of SNAP-tag labels |
| title | RAFT polymerization as a scaffold for the synthesis of SNAP-tag labels |
| title_full | RAFT polymerization as a scaffold for the synthesis of SNAP-tag labels |
| title_fullStr | RAFT polymerization as a scaffold for the synthesis of SNAP-tag labels |
| title_full_unstemmed | RAFT polymerization as a scaffold for the synthesis of SNAP-tag labels |
| title_short | RAFT polymerization as a scaffold for the synthesis of SNAP-tag labels |
| title_sort | raft polymerization as a scaffold for the synthesis of snap tag labels |
| topic | Bioconjugates Fluorescent labeling Polymers -- Synthesis UCTD |
| url | http://hdl.handle.net/10019.1/125160 |
| work_keys_str_mv | AT dreyerrudolfpetrusgerhardus raftpolymerizationasascaffoldforthesynthesisofsnaptaglabels |