Electron‐withdrawing inductive effects enhanced strategy for protein thiol sensing and blocking agent design

Abstract

It is a great challenge to discover novel chemical reactions suitable for biological analysis in a living system. The development of novel protein thiol blocking agents is a crucial need for exploring protein thiol functions in protein refolding, signal transduction, and redox regulation. We are always keen on seeking novel chemical reactions applied to endogenous biological macromolecules or protein thiol sensing, blocking, and labeling. In the present work, we have successfully developed a novel agent to block protein thiol by enhanced electron‐withdrawing inductive effects. This sensing and blocking process was detailedly monitored by UV‐vis, fluorescent spectra, and SDS‐Page gel separation. The spectral studies demonstrated that the agent could react ultrafastly with thiol within seconds at μM level. Furthermore, fluorescent imaging in cells and in vivo was further used for the validation of its ability to sensing and blocking thiol, providing evidence of downregulated protein thiols in Parkinson's disease. The enhanced electron‐withdrawing inductive effect strategy in this work may provide a general guideline for designing protein thiol agent.

INTRODUCTION

Numerous disease occurrence are keeping close relationships with improper post-translational modifications caused by oxidative stress derived from external changes, such as physical factors and environmental contaminants. Developing novel chemical toolbox for exploring post-translational modifications of biological molecules is of vital importance to understand the physiological and pathological processes in the living system. Thereinto, exploring effective methods applied in the living systems to unveil the biological functions of macromolecules in redox regulation is meaningful. One of the key objectives is developing the proper chemical toolbox for bio-thiols. As one kind of antioxidants, the bio-thiols, including small molecule thiols and thiol residues in proteins, play vital roles in maintaining redox homeostasis to defense against oxidative stress. The consumption of the bio-thiols would form a series of different oxidative intermediates, such as disulfide, sulfenic acid, sulfinic acid, and sulfonic acid. Therefore, discovering new chemical reactions that occurred under mild conditions and can be applied in living systems to monitor cellular events is the important breakthrough. Up to now, a large number of organic small molecules have been developed by decorating functional recognition groups onto various fluorophores and acted as fluorescent probes for this purpose. In combination with fluorescent microscopy, small organic fluorescent molecules exhibit indispensable advantages for insight into biological events, especially in the aspects of non-destructive imaging and visualization monitoring. However, the crucial factor in probe design is to discover chemical reactions with high specificity toward the target and reasonably tune the optical performances.

In recent years, we are always trying to seek novel chemical reactions to unveil the biological functions of macromolecules in redox regulation. We have designed a series of small organic molecule probes for monitoring the activity of biological macromolecules based on their enzyme catalytic characteristics, such as the first thioredoxin reductase probe TRFS-green and the methionine sulfoxide reductase probes (Msr-blue, Msr-Ratio and Msr-TFMCM). Based on our previous studies, we have constructed a methyl sulfoxide library (Figure ), showing that the methyl sulfoxide group is a good leaving group when it met a nucleophilic reagent in a push-pull conjugate system, viz. A molecule containing electron withdrawing group (EWG) − π system − electron donating group (EDG). Inspired by this, we hypothesized that sulfone might have a stronger leaving ability than the sulfoxide group. Having deeply investigated the topic, we found that only several sulfone-based organic compounds were developed for labeling or sensing thiols. However, these compounds suffered from different disadvantages, mainly including a large proportion of organic solvents, low reaction activity, and poor optical properties. Xian et al. developed methylsulfonyl benzothiazole for protein thiol blocking, which required more than 20 min to complete this process in a mixed aqueous solution containing a large proportion of organic solvents (THF/PBS, 1:2). Fang and Kong's labs synthesized naphthalimide-based methylsulfonyl compounds for labeling or sensing biothiols. However, the reaction time of this kind of methylsulfonyl compounds with thiols needs a longer response time (more than half an hour) at the level of mmol/L. Therefore, it is a great challenge to elevate the response rate and optical property by tuning the leaving ability of sulfone group in a push-pull conjugate system.

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To solve the problems, we selected several typical fluorophores containing π-conjugated system uniformly furnished with methylsulfonyl groups to construct a small library, including phenylbenzothiazole, cyanine, naphthalimide, chromone, and coumarin (Figure ). All the compounds were simply synthesized within two-steps and fully characterized in supporting information. Screening by spectral studies (Figure ), we found the type of conjugated system in coumarin skeleton 6 (C-SO₂Me) showing ultrahigh react activity. The probe C-SO₂Me could quickly react with low concentration of biological thiols in a complete aqueous solution, showing the turn on fluorescent enhancement signals and high selectivity undisturbed by other endogenous biomolecules. To validate the practical sensing and blocking ability of the probe in a living system, we have constructed different oxidative stress models to observe the thiol changes, including a direct oxidative model and neurodegenerative disease model. The fluorescence imaging results in living cells and in vivo sufficiently demonstrated that the probe is a powerful tool for revealing the roles of biological thiols in redox regulation by monitoring their fluctuations.

RESULTS AND DISCUSSION

With these methylsulfonyl compounds in hand, we firstly investigated their response ability toward biological thiols. After screening via spectral studies, the fluorescent probe C-SO₂Me was picked up from the library for detecting small molecule biological thiols, showing high sensitivity and selectivity. In Figure , we have detailedly studied the dose- and time-dependent fluorescent response of the probe C-SO₂Me with the bio-thiols, including GSH, Cys, and Hcy. The results confirmed that the probe C-SO₂Me could quickly react with thiols and reach up to saturation within 120 s at μM level in completed aqueous solution (10 mM PBS buffer, pH = 7.40). The changes in fluorescent emission may be attributed to the intramolecular charge transfer (ICT) process that the methylsulfonyl moiety (electron withdrawing) has been substituted and transformed into the corresponding sulfide (electron donating), and the reaction mechanism was confirmed by HRMS spectra (Figure ). The response rate order obtained from Figure  is that GSH ﹥ Cys ﹥ Hcy, which is accordance with our previous results of the probe C-SOMe and may relate to their different pK_a values. The fluorescent response performances were greatly improved compared with the reported work. Then, the selectivity experiments were investigated in Figure ; only biological thiols could cause a remarkably fluorescence enhancement of more than 300-fold and other amino acids and endogenous reactive species did not induce a fluorescence response. All these solid results confirmed that the probe C-SO₂Me could react ultrafastly and specifically with biological thiols with high sensitivity and selectivity in a completed aqueous solution.

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Having confirmed its brilliant performances of sensitivity and selectivity toward thiols in completed aqueous solution, we try to check its practical applications in sensing and blocking biological thiols in a living system. Before this, we selected BSA as an external contrast model to check the ability of the probe C-SO₂Me to react with sulfhydryl group in protein. In Figures , it exhibited excellent time- and dose-dependent fluorescent enhancement. The thiol-blocking agent N-ethylmaleimide (NEM) could suppress the enhancement, which indirectly indicates the selectivity of the probe toward thiols. Then, it was used to label BSA samples with different handling methods and separated on a SDS-Page gel (Figure ). A remarkable fluorescent enhancement was observed when the dithiols in BSA were reduced by dithiothreitol (DTT), confirming that the probe has strong labeling ability of proteins containing sulfhydryl group. Interestingly, whenever BSA was treated with or without SDS (Sodium dodecyl sulfate, a protein denaturation reagent), the results showed very slight fluorescent difference, indicating that our developed agent C-SO₂Me has great potential application in labeling thiols in protein.

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Before imaging in cells, we have carried out cytotoxicity assays in HepG2 cells to assess biocompatibility (Figure ). In Figure , we investigated the time-dependent response of the probe C-SO₂Me with thiols in cells. Strong green fluorescence signals were observed after incubation with the probe within 5 min, and then it remained stable as the time increased, proving that the probe could penetrate into cells and react with thiols quickly and the probe has high optical stability, which is a very vital factor for imaging analysis. To validate the specificity with thiols, the thiol-blocking agent NEM was also used for scavenging them. As shown in Figure , there were almost no fluorescence signals in the cells when pretreated with NEM (100 μM). In addition, we investigated the response ability of the probe to exogenous thiols, including GSH, Cys, and Hcy. In Figure , the fluorescence signal enhancement order remains in accordance with that in aqueous solution, viz. GSH ﹥ Cys ﹥ Hcy. All these results suggested that the probe was an excellent agent that could quickly reacts with biological thiols in moderated conditions and act as a useful tool for sensing and blocking analysis.

The biological thiols play key roles in regulating redox balance through variation of their different sulfide intermediates, including sulfhydryl, disulfide, sulfenic acid, sulfinic acid, sulfonic acid, and so on. Abnormal expression levels of biological thiols occur in the pathological processes such as cancer and neurodegenerative disease. Therefore, real-time monitoring of biological thiol fluctuation is of great importance for elucidating their functions. Relative upregulated and downregulated experiments for an oxidative stress model in living cells were carried out to observe the thiol changes. On the one hand, the fluorescent signal was enhanced when the cells were pretreated with tris(2-carboxyethyl)-phosphine (TCEP), known as a reducing agent to cleave disulfide in a biological system, before incubation with the probe (Figure ). This can be attributed to the major reason that the disulfide in protein were opened to enrich the available sulfhydryl in this targeting process. On the other hand, oxidants could consume sulfhydryl and transfer it to other oxidative intermediates. As shown in Figure , exogenous hydrogen peroxide (H₂O₂) was aforehand incubated with the cells and resulted in the significantly suppressed fluorescence signals, showing very weak green fluorescence. As the concentrations of H₂O₂ increased, the fluorescence intensity weakened gradually (Figure ). Inversely, compared with the fluorescence in Figure , the fluorescent signals in cells could be remarkably recovered when the cells sequentially incubated with H₂O₂ and TCEP (Figure ). However, the fluorescent signals were obviously weaker than that in Figure , suggesting that the content of thiols couldn't reach up to previous level even though using the TCEP to destruct formed disulfide. This may be ascribed to the reason that part of the sulfhydryl group transformed into other intermediates rather than disulfide during the oxidative process. All the above response mechanisms of fluorescent signals in cells can be detailedly summarized in Figure . After validation of its sensing and labeling performance in vitro, we further investigated the conjugation ability of the probe C-SO₂Me with biothiols in vivo. In Figure , green fluorescence was observed and enhanced sharply as the incubation time of the probe increased in living zebra fish. All these solid results firmly confirmed that the probe is a vital sensing and labeling tool for monitoring the fluctuations of biological thiols in redox systems.

Aging and neurodegenerative diseases are closely related to oxidative stress. Parkinson's disease (PD), as one of the typical neurodegenerative diseases, was selected for studying the biological thiol fluctuations. The PD model was commonly constructed by using 6-hydroxydopamine (6-OHDA) which was known as neurotoxin yielding potentially toxic products and reactive oxygen species (Figure ); however, it is not clear. Therefore, we intend to use the probe to monitor biothiol fluctuation, revealing the roles of bithiols in the PD model. In Figure , the fluorescent signals in zebra fish showed a time- and dose-dependent damping. The fluorescence signal gradually dimmed as the incubation time and concentrations of 6-OHDA increased. To further confirm the fluorescent decay caused by 6-OHDA induced oxidative stress, the probe was applied in monitoring biological thiols fluctuations in different upregulated and downregulated zebra fish models. As shown in Figure , the fluorescent signals were greatly inhibited when zebra fish was pretreated with NEM. Oppositely, the fluorescence with a certain enhancement was observed when the zebra fish was pretreated with TCEP. Under oxidative stress induced by exogenous addition of H₂O₂, fluorescent signals decay gradually as their concentration increases (Figure ). All the imaging in zebra fish suggests that the probe has strong ability to react with biological thiols in vivo. The fluorescent changes indirectly indicated that biological thiol would be downregulated in Parkinson's disease.

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CONCLUSION

In summary, we have successfully synthesized a series of compounds containing methylsulfonyl groups in different pull–push conjugated fluorophore systems to improve the reaction ability with thiols. The electron-withdrawing inductive effects were productive for discovering novel sensing and protein labeling strategies. We have also picked up the probe C-SO₂Me to monitor the fluctuations of biological thiols with high reactivity and selectivity in completed aqueous solutions. The imaging in living cells and in vivo demonstrated that the probe has excellent biological compatibility and could quickly sense and conjugate endogenous small molecule thiols and protein thiols. The direct oxidative model and neurodegenerative disease model further validate the practical sensing and blocking ability of the probe. Our studies may enlighten the readers to develop novel agents and strategies for sensing and protein labeling.

ACKNOWLEDGMENTS

This work was financially supported by the Natural Science Foundation of China (22376216, 21778026, 21701074, 21976209 and 22204127), the program of the Youth Innovation Promotion Association, CAS (2019217), Taishan Scholar Project Special Funding (TS20190962), the Shenzhen Science and Technology Program (JCYJ20210324142612032), the Guangdong Basic and Applied Basic Research Foundation (2021A1515110906) and the Natural Science Basic Research Program of Shaanxi (No. 2022JQ-106).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

ETHICS STATEMENT

No animal or human experiments were involved in this study.

References

B. Ezraty, A. Gennaris, F. Barras, J. F. Collet, Nat. Rev. Microbiol. 2017, 15, 385.

Y. Xie, S. Du, Z. Liu, M. Liu, Z. Xu, X. Wang, J. X. Kee, F. Yi, H. Sun, S. Q. Yao, Angew. Chem. Int. Ed. 2022, 61, [eLocator: e202200303].

L. Liu, D. Zhang, M. Johnson, N. K. Devaraj, Nat. Chem. 2022, 14, 1078.

C. Jing, V. W. Cornish, Acc. Chem. Res. 2011, 44, 784.

S.‐Y. Dai, D. Yang, J. Am. Chem. Soc. 2020, 142, [eLocator: 17156].

C. H. Knittel, N. K. Devaraj, Acc. Chem. Res. 2022, 55, 3099.

G. Hu, H. Jia, L. Zhao, D. H. Cho, J. Fang, Chin. Chem. Lett. 2019, 30, 1704.

C. E. Paulsen, K. S. Carroll, Chem. Rev. 2013, 113, 4633.

S. E. Leonard, K. S. Carroll, Curr. Opin. Chem. Biol. 2011, 15, 88.

K. G. Reddie, K. S. Carroll, Curr. Opin. Chem. Biol. 2008, 12, 746.

A. K. East, M. C. Lee, L. P. Smaga, C. Jiang, S. C. Mallojjala, J. S. Hirschi, J. Chan, Org. Lett. 2022, 24, 8509.

J. Liu, W. Zhang, C. Zhou, M. Li, X. Wang, W. Zhang, Z. Liu, L. Wu, T. D. James, P. Li, B. Tang, J. Am. Chem. Soc. 2022, 144, [eLocator: 13586].

Z. Zeng, S. S. Liew, X. Wei, K. Pu, Angew. Chem. Int. Ed. Engl. 2021, 60, [eLocator: 26454].

H. Iwashita, E. Castillo, M. S. Messina, R. A. Swanson, C. J. Chang, Proc. Natl. Acad. Sci. 2021, 118, [eLocator: e2018513118].

X. Wu, J. M. An, J. Shang, E. Huh, S. Qi, E. Lee, H. Li, G. Kim, H. Ma, M. S. Oh, D. Kim, J. Yoon, Chem. Sci. 2020, 11, [eLocator: 11285].

J. Zhang, X. Chai, X. P. He, H. J. Kim, J. Yoon, H. Tian, Chem. Soc. Rev. 2019, 48, 683.

D. Cao, Z. Liu, P. Verwilst, S. Koo, P. Jangjili, J. S. Kim, W. Lin, Chem. Rev. 2019, 119, [eLocator: 10403].

F. Yang, P. Lu, T. B. Ren, X. B. Zhang, L. Yuan, Smart Mol. 2023, 1, [eLocator: e20220002].

J. You, H. Liu, Q. Pan, A. Sun, Z. Zhang, X. Shi, J. Anal. Test. 2023, 7, 69.

Y. Geng, Z. Wang, J. Zhou, M. Zhu, J. Liu, T. D. James, Chem. Soc. Rev. 2023, 52, 3873.

J. Yin, L. Huang, L. Wu, J. Li, T. D. James, W. Lin, Chem. Soc. Rev. 2021, 50, [eLocator: 12098].

S. Wang, W. X. Ren, J. T. Hou, M. Won, J. An, X. Chen, J. Shu, J. S. Kim, Chem. Soc. Rev. 2021, 50, 8887.

L. Gao, W. Wang, X. Wang, F. Yang, L. Xie, J. Shen, M. A. Brimble, Q. Xiao, S. Q. Yao, Chem. Soc. Rev. 2021, 50, 1219.

D. Cheng, W. Xu, X. Gong, L. Yuan, X. B. Zhang, Acc. Chem. Res. 2021, 54, 403.

M. Y. Lucero, J. Chan, Nat. Chem. 2021, 13, 1248.

J. C. Qin, Z. W. Li, Z. H. Fu, Z. H. Zhang, Sens. Actuators B Chem. 2022, 357, [eLocator: 131430].

X. Dong, L. Sun, Z. Zhang, T. Zhu, J. Sun, J. Gao, C. Dong, R. Wang, X. Gu, C. Zhao, Sci. China Chem. 2023, 66, 1869.

L. Zhang, D. Duan, Y. Liu, C. Ge, X. Cui, J. Sun, J. Fang, J. Am. Chem. Soc. 2014, 136, 226.

L. Zhang, S. Peng, J. Sun, J. Yao, J. Kang, Y. Hu, J. Fang, Chem. Sci. 2017, 8, 2966.

L. Zhang, S. Peng, J. Sun, R. Liu, S. Liu, J. Fang, Chem. Commun. 2019, 55, 1502.

L. Zhang, R. Liu, J. Kang, J. Sun, Y. Hou, H. Yu, J. Fang, Sci. Sin. Chim. 2017, 47, 1007.

L. W. Zhang, J. Kang, S. D. Liu, X. Zhang, J. Y. Sun, Y. S. Hu, Y. Yang, L. X. Chen, Chem. Commun. 2020, 56, [eLocator: 11485].

D. Zhang, N. O. Devarie‐Baez, Q. Li, J. R. Lancaster, Jr., M. Xian, Org. Lett. 2012, 14, 3396.

R. Chen, H. Lu, C. Liu, Q. Kong, Bioorg. Med. Chem. Lett. 2015, 25, 59.

P. Zhou, J. Yao, G. Hu, J. Fang, ACS Chem. Biol. 2016, 11, 1098.

K. Ulrich, U. Jakob, Free Radic. Biol. Med. 2019, 140, 14.

G. Teskey, R. Abrahem, R. Cao, K. Gyurjian, H. Islamoglu, M. Lucero, A. Martinez, E. Paredes, O. Salaiz, B. Robinson, V. Venketaraman, Adv. Clin. Chem. 2018, 87, 141.

G. J. McBean, M. Aslan, H. R. Griffiths, R. C. Torrao, Redox Biol. 2015, 5, 186.

J. A. Burns, J. C. Butler, J. Moran, G. M. Whitesides, J. Org. Chem. 2002, 56, 2648.

H. J. Forman, H. Zhang, Nat. Rev. Drug Discov. 2021, 20, 689.

N. Simola, M. Morelli, A. R. Carta, Neurotox. Res. 2007, 11, 151.

S. W. Hsu, P. C. Hsu, W. S. Chang, C. C. Yu, Y. C. Wang, J. S. Yang, F. J. Tsai, K. Y. Chen, C. W. Tsai, D. T. Bau, Environ. Toxicol. 2020, 35, 840.