A novel fluorescent probe for imaging the process of HOCl oxidation and Cys/Hcy reduction in living cells

A new on-off-on fluorescent probe, CMOS, based on coumarin was developed to detect the process of hypochlorous acid (HOCl) oxidative stress and cysteine/homocysteine (Cys/Hcy) reduction. The probe exhibited a fast response, good sensitivity and selectivity. Moreover, it was applied for monitoring the redox process in living cells.

Reactive oxygen species (ROS) are indispensable products and are closely connected to various physiological processes and diseases. 1 For instance, endogenous hypochlorous acid (HOCl) as one of the most important ROS, which is mainly produced from the reaction of hydrogen peroxide with chloride catalyzed by myeloperoxidase (MPO), is a potent weapon against invading pathogens of the immune system. 2,3 However, excess production of HOCl may also give rise to oxidative damage via oxidizing or chlorinating the biomolecules. 4 The imbalance of cellular homostasis will cause a serious pathogenic mechanism in numerous diseases, including neurodegenerative disorders, 5 renal diseases, 6 cardiovascular disease, 7 and even cancer. 8 Fortunately, cells possess an elaborate antioxidant defense system to cope with the oxidative stress. 9 Therefore, it is necessary and urgent to study the redox process between ROS and antioxidants biosystems.
Fluorescence imaging has been regarded as a powerful visual methodology for researching various biological components as its advantages of high sensitivity, good selectivity, little invasiveness and real-time detection. 10,11 To date, amounts of small molecular uorescent probes have been reported for detection and visualization of HOCl in vivo and in vitro. [12][13][14][15][16][17][18][19][20][21][22]29 The designed strategies of HOCl sensitive probes are based on various HOCl-reactive functional groups, such as p-methoxyphenol, 13 p-alkoxyaniline, 14 dibenzoyl-hydrazine, 15 selenide, 16 thioether, 17 oxime, 18 hydrazide, 19 hydrazone. 20 But, many of these probes display a delayed response time and low sensitivity. And, only few uorescent probes can be applied for investigating the changes of intracellular redox status. 21 Besides, it's worth noting that most of the redox uorescent probes rely on the organoselenium compounds. 22 Even though these probes are well applied for detection of cellular redox changes, excessive organic selenium is harmful to organisms and the synthesis of organoselenium compounds is high requirement and costly. Additionally, almost all the reports have only investigated the reduction effects of glutathione (GSH) as an antioxidant in the redox events. While, there are the other two important biothiols, cysteine (Cys) and homocysteine (Hcy), which not only present vital antioxidants, but also are tightly related to a wide variety of pathological effects in biosystem, such as slowed growth, liver damage, skin lesions, 23 cardiovascular, 24 and Alzheimer's diseases. 25 However, the uorescent probes for specially studying internal redox changes between HOCl and Cys/Hcy are rarely reported. In this respect, a novel redox-responsive uorescent probe, CMOS, was designed and synthesized in this work, and we hope that it can be a potential tool for studying their biological relevance in living cells.
Based on literature research, the aldehyde group has excellent selectivity in identication of Cys/Hcy, and the thiol atom in methionine can be easily oxidized to sulfoxide and sulfone by HOCl. 26,27 Considering these two points, we utilized 2-mercaptoethanol to protect the 3-aldehyde of 7-diethylamino-coumarin as the recognition part of HOCl, meaning that two kinds of potential recognition moieties are merged into one site. Fluorescent probe CMOS can be easily synthesized by the acetal reaction in one step (Scheme S1 †). A control molecule CMOS-2 was also prepared by 3-acetyl-7-diethylaminocoumarin (CMAC) similarly. The structure of all these compounds have been convinced by 1 H NMR, 13 C NMR, and HR-MS (see ESI †).
As shown in Scheme 1a, we estimated that both CMOS and CMOS-2 can be rapidly oxidized in the appearance of HOCl. The oxidation product CMCHO of CMOS, which has the aldehyde moiety, can further react with Cys/Hcy to obtain the nal product CMCys and CMHcy, respectively. In contrast, the oxidation product CMAC of CMOS-2 cannot combine with Cys/ Hcy or other biothiols anymore (Scheme 1b).
In order to conrm our design concept, the basic photophysical characteristics of CMOS, CMCHO, CMOS-2 and CMAC were tested (Table S1, Fig. S1 †). Under the excitation wavelength 405 nm, CMOS and CMOS-2 exhibited strong uorescence centred at 480 nm in PBS buffer solution, while the uorescence of CMCHO and CMAC was weak around this band. The emission properties of CMOS and CMCHO were also investigated at the excitation wavelength 448 nm under the same experimental conditions as well (Fig. S2 †). Aer careful consideration, we chose 405 nm as the excitation wavelength in the follow-up experiments in vitro and in vivo.
Next, the sensitivity of CMOS and CMOS-2 to HOCl and Cys/Hcy were investigated. As we expected, both the CMOS and CMOS-2 exhibited good response to HOCl. The uorescence intensity of CMOS and CMOS-2 decreased gradually with addition of NaOCl ( Fig. 1a, S3a †), indicating that the uorescence was switched off obviously in the presence of HOCl. The variation of intensity displayed good linearity with concentration of HOCl in the range of 0-20 mM (R 2 ¼ 0.993, Fig. S4 †), and the detection limit of CMOS to HOCl was calculated to be 21 nM (S/N ¼ 3). Subsequently, when Cys/Hcy was added to the nal solution in Fig. 1a, the uorescence intensity increased gradually within 180 min (Fig. 1b, S5 †). However, the uorescence cannot be recovered by addition thiols to the CMOS-2 solution with excess HOCl (Fig. S3b †). These results indicate that the probe CMOS can response to HOCl and Cys/Hcy in a uorescence on-off-on manner, and can be used for monitoring the redox process with high sensitivity.
To further identify the recognizing mechanism of probe CMOS, high performance liquid chromatography (HPLC) and mass spectral (MS) analysis were used to detect the redox process. Initially, probe CMOS displayed a single peak with a retention time at 3.7 min (Fig. 2a, S6 †) while reference compound CMCHO produced a single peak with a retention time at 2.5 min (Fig. 2b, S7 †). Upon the addition of HOCl to the solution of CMOS, the peak at 3.7 min weakened while 2.5 min and 2.2 min appeared (Fig. 2c). According to corresponding mass spectra, the new main peak at 2.5 min is related to compound CMCHO (Fig. S8 †). The other new peak of 2.2 min corresponds to the compound C3, which can be predicted as an intermediate in the oxidation process (Fig. S8 †). 28 The addition of Cys to the solution of CMCHO also caused a new peak with a retention time at 2.1 min, which has been conrmed to be the thioacetal product CMCys (Fig. S9 †). The possible sensing mechanism is depicted in Fig. S10. † To study the selectivity of CMOS towards HOCl, we performed uorescence response to different reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive sulfur species (RSS). As shown in Fig. 3a, CMOS exhibited signicant change of uorescence intensity only in the presence of HOCl, while other ROS and RNS, such as singlet oxygen ( 1 O 2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO$), superoxide anion (O 2 À ), nitric oxide (NO), tert-butylhydroperoxide (t-BuOOH) and tert-butoxy radical (t-BuOO$) had no obvious uorescence emission changes. Additionally, RSS which are abundant in biological samples, showed no inuence in this process under the identical condition. The detection of reducing process was also investigated. As displayed in Fig. 3b, only cysteine and homocysteine induced excellent uorescence recovery towards other reducing materials, such as RSS and various amino acids. Furthermore, the selectivity of CMOS-2 was also studied in the same condition. As expected, CMOS-2 could selectively detect HOCl, and not alter uorescence intensity under various kinds of biothiols (Fig. S11 †). Therefore, Scheme 1 Proposed reaction mechanism of CMOS and CMOS-2 to HOCl and Cys/Hcy.  our design strategy for the on-off-on probe is conrmed by results obtained above, with which CMOS can be utilized for detecting the redox process between HOCl and Cys/Hcy with high selectivity.
Subsequently, the inuence of pH on probe CMOS was measured. The uorescence intensity of CMOS and CMCHO perform no signicant variances in wide pH ranges (pH ¼ 4-11, Fig. S12a †). Fluorescence intensity changes could be observed immediately when HOCl was added into the solution of probe CMOS, especially in alkaline condition (Fig. 4a). Considering the pK a of HOCl is 7.6, 29 CMOS is responsive to both HOCl and OCl À . Alkaline condition was also benet for the uorescence recovery of CMOS from Cys/Hcy (Fig. S12b †). It is reasonable to consider that thiol atom displays higher nucleophilicity in alkaline condition. From the stop-ow test, the UV-visible absorbance of probe CMOS sharply decreased at the wavelength of 400 nm (Fig. 4b). The response time was within 10 s and the kinetic of the reaction was tted to a single exponential function (k obs ¼ 0.67 s À1 ). The ability of instantaneous response is extremely necessary to intracellular HOCl detection.
With these data in hand, we next applied CMOS for uorescence imaging of the redox changes with HOCl and Cys/Hcy in living cells. Aer incubation with 5 mM CMOS at 37 C for 30 min, intense uorescence was observed of the SKVO-3 cells in the optical window 425-525 nm ( Fig. 5a and d), indicating the probe can easily penetrate into cells. Treating the cells with 100 mM NaOCl led to remarkable uorescence quenching as the probe sensed the HOCl-induced oxidative stress ( Fig. 5b and e). Aer 3 min, the cells were washed with PBS buffer three times, and added 5 mM Cys/Hcy for 1 h, respectively. Then the uorescence was recovered obviously (Fig. 5c and f). Experimental results clearly declare that the probe CMOS was successfully used to detect the process of HOCl oxidative stress and Cys/Hcy reducing repair in living cells.

Conclusions
In this work, a novel on-off-on uorescent probe was reported for highly selective detection HOCl oxidative stress and Cys/Hcy reducing repair in vivo and in vitro. The probe CMOS can be easily synthesized and displayed high sensitivity, fast response, and high selectivity. Cells images indicated that CMOS was capable to sense the redox changes between HOCl and Cys/Hcy. Results show that the probe CMOS would be a potential tool to study the oxidative damage and biothiols repairs in the biology and medical research.

Conflicts of interest
There are no conicts to declare.   research team from School of Basic Medical Sciences, Zhengzhou University, for providing SKVO-3 cells in this work.