Chemical tools for detecting cysteine sulfenic acid

Author: Lisa Alcock

Alcock, Lisa, 2019 Chemical tools for detecting cysteine sulfenic acid, Flinders University, College of Science and Engineering

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The study of oxidative stress is a complex problem in biological systems. Cysteines are susceptible to oxidation by reactive oxygen species (ROS) to the cysteine sulfenic acid, which is often considered a biomarker of oxidative stress. Oxidation of cysteine residues has become recognised as a major regulatory route for protein function as well as a key player in signalling pathways. Discovering these nuances in cysteine reactivity, particularly sulfenic acids, will assist in exploring their purpose within the cellular environment. An arsenal of chemical probes for the detection of cysteine sulfenic acids is at the disposal of researchers, however issues such as chemoselectivity, reactivity, and reversibility have hindered their potential for sulfenic acid detection. This research explores the application of a new chemical probe, norbornene, to further study cysteine sulfenic acid formation in cells and address the current caveats in detection methods.

In this thesis, norbornene derivatives were found to react selectively with the sulfenic acid functional group on a small molecule cysteine model as analysed by NMR and LC-MS. This reaction proceeded with fast kinetics through a strain promoted ligation to give a stable and distinguishable sulfoxide product. Other reported cysteine sulfenic acid probes failed to trap the sulfenic acid generated in the small molecule cysteine model, highlighting the superior kinetics and reactivity of the norbornene scaffold. These results provided the foundational evidence for the continued pursuit of norbornene probes as a contender in the study of sulfenic acid formation in living cells.

Norbornene probes were able to react selectively with protein sulfenic acids in a similar manner and with broad reactivity across a range of proteins. Using both commercially available and synthesised norbornene derivatives, this demonstrated for the first time the ability of norbornene to react with sulfenic acids on purified proteins and protein mixtures. Comparison to a commonly used dimedone derivative revealed some interesting questions about its selectivity. Extending this further, norbornene probes were applied to live cell systems and found to selectively trap only the sulfenic acid modified proteins. This result provided the information needed to introduce these norbornene probes as tools to study proteome-wide cellular oxidative stress.

Application of the norbornene probe to living cells under oxidative stress revealed new protein targets of cysteine oxidation along with numerous previously identified protein sulfenic acids through proteomics analysis. Further examination of these protein hits in a future study could provide useful insight into how they are affected during oxidative stress, and how this may play a wider role in regulation or disease.

The work presented in this thesis has provided the core experimental reasoning for implementing norbornene as chemical tools to study cysteine sulfenic acid formation on the proteome-wide cellular scale. The results presented provide constructive insight for the field of cysteine redox chemistry and could lead to further discoveries into unravelling the specific roles of the identified protein sulfenic acids regarding oxidative stress.

Keywords: cysteine sulfenic acid, norbornene, chemical probe, oxidative stress, cysteine oxidation, cysteine redox chemistry

Subject: Chemistry thesis

Thesis type: Doctor of Philosophy
Completed: 2019
School: College of Science and Engineering
Supervisor: Dr Justin Chalker