Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells

Chen, Jianwei; Jiang, Xiqian; Carroll, Shaina L.; Huang, Jia; Wang, Jin

doi:10.1021/acs.orglett.5b02910

Cited by 73 publications

(46 citation statements)

References 14 publications

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“…For the addition–elimination mechanism, the most logical set‐up is as follows. The addition reaction is probably reversible, as proposed in the literature, whereas the elimination reaction is irreversible . Therefore, k 3 is the rate‐determining step.…”

Section: Resultsmentioning

confidence: 61%

Stability of strigolactone analog GR24 toward nucleophiles

Halouzka

Tarkowski

Zwanenburg

et al. 2017

Pest Management Science

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Section: Resultsmentioning

confidence: 61%

Stability of strigolactone analog GR24 toward nucleophiles

Halouzka

Tarkowski

Zwanenburg

et al. 2017

Pest Management Science

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“…Reversibility of Michael additions has been shown for different addends and adducts (35,36). For example, reversible Michael addition of a thiol to a fluorescent probe was shown to blue shift the absorbance and fluorescence spectra, allowing the measurement of glutathione concentration inside living cells (37).…”

Section: Resultsmentioning

confidence: 99%

Efficient switching of mCherry fluorescence using chemical caging

Cloin

Zitter

Salas

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantummechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores. fluorescent proteins | mCherry | localization microscopy | β-mercaptoethanol | photoactivation

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“…Based on density functional theory (DFT) theoretical calculations of the thermodynamics and kinetics of thiol-Michael addition reactions, the authors develop an improved reversible GSH probe 2. [11] This probe shows faster kinetics than probe 1 in both the forward and reverse reactions. Thus, probe 2 is successfully applied to detect reversible changes of GSH concentrations upon biological stimulation in single cells for the first time.…”

Section: Thiol-michael Additionmentioning

confidence: 94%

Reversible Reaction‐Based Fluorescent Probes for Dynamic Sensing and Bioimaging

2020

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Diverse chemical species such as ions and molecules exist within living cells and organisms undergoing dynamic changes in their local environment by a web of continuously interacting reactions. Reaction‐based fluorescent probes with a highly sought reversible feature can provide a real‐time monitor of the concentration dynamics (increases and decreases) of such chemical species, thus ideally suited to understand the physiological function, and pathogenic mechanisms of corresponding bio‐species in the regulation of cellular function and disease progression. This review summarizes the current methods for constructing reversible reaction‐based fluorescent probes. The sensing mechanisms and biological applications of these probes are also discussed. The representative examples reported recently are categorized according to the type of reversible chemical reactions utilized: nucleophilic additions (Michael additions, chromophore reactions), nucleophilic addition‐condensation reactions, and redox reactions. Finally, we present the potential challenges and suggestions for developing probes based on dynamic and reversible covalent bond formation reactions.

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Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells

Cited by 73 publications

References 14 publications

Stability of strigolactone analog GR24 toward nucleophiles

Stability of strigolactone analog GR24 toward nucleophiles

Efficient switching of mCherry fluorescence using chemical caging

Reversible Reaction‐Based Fluorescent Probes for Dynamic Sensing and Bioimaging

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