Dual-reactivity trans-cyclooctenol probes for sulfenylation in live cells and affinity bioorthogonal chemistry tags for site-selective synthesis of protein-protein and protein-material conjugates
| Author(s) | Scinto, Samuel L. | |
| Date Accessioned | 2023-02-14T14:19:10Z | |
| Date Available | 2023-02-14T14:19:10Z | |
| Publication Date | 2022 | |
| SWORD Update | 2022-09-21T16:08:33Z | |
| Abstract | My research in the Fox lab has focused on new small molecule tetrazine and trans-cyclooctenes (TCOs) for the purpose of carrying out new chemical reactions in biological settings with broad interest to the field of chemical biology and related disciplines. Central to my work is the reaction between TCOs and tetrazines, the fastest known bioorthogonal reaction to date and leveraging their unique structural characteristics for designing new function while preserving their speed and specificity as bioorthogonal reagents. Chapter 1 is intended as a brief overview of bioorthogonal chemistry and the most popular reactions that use non-natural fragment pairs. ☐ Described in Chapter 2 is how we use TCOs as molecules designed to trap sulfenic acids(R-SOH) on proteins in a cellular setting. Sulfenylation (RSH → RSOH) is a post-translational protein modification associated with cellular mechanisms for signal transduction and the regulation of reactive oxygen species. Protein sulfenic acids are challenging to identify and study due to their electrophilic and transient nature. Described here are sulfenic acid modifying transcycloocten-5-ol (SAM-TCO) probes for labeling sulfenic acid functionality in live cells. These probes enable a new mode of capturing sulfenic acids via transannular thioetherification, whereas "ordinary" trans-cyclooctenes react only slowly with sulfenic acids. SAM-TCOs combine with sulfenic acid forms of a model peptide and proteins to form stable adducts. Analogously, SAM-TCO with the selenenic acid form of a model protein leads to a selenoetherification product. Control experiments illustrate the need for the transannulation process coupled with the activated trans-cycloalkene functionality. Bioorthogonal quenching of excess unreacted SAM-TCOs with tetrazines in live cells provides both temporal control and a means of preventing artifacts caused by cellular-lysis. A SAM-TCO biotin conjugate was used to label protein sulfenic acids in live cells, and subsequent quenching by tetrazine prevented further labeling even under harshly oxidizing conditions. A cell-based proteomic study validates the ability of SAM-TCO probes to identify and quantify known sulfenic acid redox proteins as well as targets not captured by dimedone-based probes. ☐ In Chapter 3, I describe the use of pyridyl-tetrazines for applications in protein affinity purification and conjugation. The site-selective functionalization of proteins has broad application in chemical biology but can be limited when mixtures result from incomplete conversion or the formation of protein containing side products. It is shown here that when proteins are covalently tagged with pyridyl-tetrazines, the Ni-IDA resins commonly used for His-tags can be directly used for protein affinity purification. These Affinity Bioorthogonal Chemistry tags (ABC-tags) serve a dual role by enabling affinity-based protein purification through metal chelation and still maintain their rapid kinetics in bioorthogonal reactions. ABC tagging can be applied in conjunction with a range of site-selective bioconjugation methods and is demonstrated for a range of proteins tagged at the C-terminus, N-terminus or at internal positions. ABC-tagged proteins can also be purified from complex mixtures including cell lysate. Importantly, the combination of site-selective conjugation and clean-up with ABC-tagged proteins also allows for facile on-resin reactions for cleanly preparing protein-protein conjugates in minutes. ☐ In Chapter 4, the use of site-selective protein conjugates bearing a tetrazine or TCO can be used for material-protein conjugation applications. These applications include the use of model proteins to visualize and characterize fibers or hydrogels formed through interfacial bioorthogonal characterization. Also described is the use of genetically encoded protease degradable motifs that can be used for cell-mediated release of proteins from hydrogels. The work described and methods used are now routinely being applied in our lab by other members. | |
| Advisor | Fox, Joseph M. | |
| Degree | Ph.D. | |
| Department | University of Delaware, Department of Chemistry and Biochemistry | |
| DOI | https://doi.org/10.58088/d1mq-0r59 | |
| Unique Identifier | 1370012003 | |
| URL | https://udspace.udel.edu/handle/19716/32279 | |
| Language | en | |
| Publisher | University of Delaware | |
| URI | https://login.udel.idm.oclc.org/login?url=https://www.proquest.com/dissertations-theses/dual-reactivity-em-trans-cyclooctenol-probes/docview/2723382417/se-2?accountid=10457 | |
| Keywords | Interfacial bioorthogonal characterization | |
| Keywords | ABC tagging | |
| Keywords | Sulfenic acid functionality | |
| Title | Dual-reactivity trans-cyclooctenol probes for sulfenylation in live cells and affinity bioorthogonal chemistry tags for site-selective synthesis of protein-protein and protein-material conjugates | |
| Type | Thesis |