A highly water-soluble and stable chemical sensor for cysteine

Abstract

The present invention relates to chemical probes for the improved detection of cysteine ​​in test samples, preferably aqueous test samples, and respective uses and kits.

Description

[Technical Field]

[0001] The present invention relates to chemical probes for the improved detection of cysteine ​​in test samples, preferably aqueous test samples, as well as respective uses and kits. [Background technology]

[0002] Background of the Invention Cysteine ​​(Cys) is important in biosynthesis, detoxification, and metabolism. Elevated levels of total cysteine ​​can predict cardiovascular disease and metabolic syndrome. Cysteine ​​deficiency is known to be one of the consequences of aging. Selective detection of Cys relative to the structurally similar homocysteine ​​(Hcy) or glutathione (GSH) remains a significant challenge. Although there are many methods for detecting Cys, photoluminescence (PL) and electrochemiluminescence (ECL) techniques are well suited for clinical diagnostics and analytical techniques due to their high sensitivity.

[0003] Trisulfide formation in recombinant monoclonal antibodies is a source of heterogeneity that needs to be controlled for consistent product quality. Ryll et al. (Kshirsagar, R.; McElearney, K.; Gilbert, A.; Sinacore, M.; Ryll, T. Biotechnol. Bioeng. 2012, 109, 2523) have shown that the L-cysteine ​​(Cys) concentration in the feed medium directly correlates with the trisulfide level in the product (IgG1 mAb). Therefore, a controlled Cys feeding strategy is required to reduce trisulfide formation to an acceptable level.

[0004] To date, the detection of Cys has attracted much attention for various biochemical applications. Numerous methods have been developed to detect Cys, including fluorometry, potentiometry, electrochemical voltammetry, and HPLC coupled with Ellman's reagent or fluorescence. These methods require complex instrumentation, involve laborious laboratory procedures, or have low throughput.

[0005] Kim and Hong (Photoluminescence and Electrochemiluminescence Dual-Signaling Sensors for Selective Detection of Cysteine ​​Based on Iridium(III) Complexes. ACS Omega 2019, 4, 7, 12616-12625) report a PL and ECL dual-channel sensor using a cyclometallated iridium(III) complex for the discrimination of Cys from Hcy and GSH.

[0006] UV-vis spectroscopy offers a fast and simple measurement procedure, so automated analyzers, e.g., Cedex, are often used to quantify essential metabolites in bioprocesses. (registered trademark) Photometric assays using Bio HT (Roche Diagnostics, Penzberg, Germany) are used. However, only a few candidates are available. (registered trademark) Bio HT Analyzer (Cedex (registered trademark) "), the analytical device only offers a limited set of wavelengths: 340, 378, 409, 480, 512, 520, 552, 583, 629, 652, 659, and 800 nm. Additionally, Cedex (registered trademark) An ideal probe for the system must include high sensitivity, rapid response, water solubility and stability, and ease of use.

[0007] To date, most indicators for Cys are based on the strong nucleophilicity of the thiol group. Various mechanisms, including Michael addition and cleavage reactions, have been used. The sensing strategy based on acrylate groups appears promising because it allows for the discrimination of Cys from other amino acids and thiols (Han, Q.; Shi, Z.; Tang, X.; Yang, L.; Mou, Z.; Li, J.; Shi, J.; Chen, C.; Liu, W.; Yang, H.; Liu, W. Organic & Biomolecular Chemistry 2014, 12, 5023). The sensing mechanism is shown in Figure 1. This strategy involves the conjugate addition of Cys to an acrylate to generate a thioester, followed by intramolecular cyclization. The acrylate moiety as a thiol-activated site undergoes rapid cyclization only with Cys, since the reaction rate strongly depends on the ring size of the resulting lactam. After the masking acrylate group is removed, the conjugated π-electron system of the chromophore is restored, which enables the colorimetric response.

[0008] Unfortunately, only a few acrylate-based probes are available from Cedex (registered trademark) They are characterized by an intense colorimetric response at the wavelengths required for their use in the above. Their chromophores are based on xanthene, merocyanine, heptamethine, and fluorescein. The fact that most of the reported acrylates have been applied in organic solvent-water mixtures is obviously necessitated by their poor solubility in aqueous media. However, the water solubility of the probes is confirmed by Cedex. (registered trademark) This is essential for the above applications because the instrument parts are unstable to organic solvents. Also, the assay solution must be kept in a constant state for a reasonable length of time. (registered trademark) The stability of existing probes needs to be assessed to ensure they can be stored in a stable state.

[0009] In view of these and other drawbacks, it is an object of the present invention to provide a Cys probe that can be used in aqueous solution and that provides a satisfactory colorimetric response at one of the required wavelengths. Other objects and advantages will become apparent to those skilled in the art upon review of this specification of the present invention. Summary of the Invention

[0010] In a first aspect of the present invention, the above object is to provide a compound of formula (I): [ka] (In the formula, R 1 and R 2 is R 3 , OR 3 , S.R. 3 , SO3 - , SO3-R 3 are independently selected from R 3 is C1~C 18 alkyl, and polyethylene glycol (PEG) residues; Acc is a compound of formula II [ka] wherein X is selected from -N(CH3)-, -S-, -Se-, -O-, and -C(CH3)2-; Formula III [ka] Formula IV [ka] and Formula V [ka] (wherein in each of Formulas II-V, the aromatic ring may contain one, two, or three SO3 - may be substituted with a group, R 4 is C1~C 18Alkyl, C1-C6 cycloalkyl, and (CH2) m -SO3 - and m is an integer selected from 1 to 18. selected from the group selected from n is selected from 1, 2 and 3 and suitable salts and solvates thereof.

[0011] We synthesized a series of acryloyl esters based on the merocyanine chromophore (Figure 2, see below); probe LZ07 was prepared as a control and for comparison and is known from the literature (Han, Q.; Shi, Z.; Tang, X.; Yang, L.; Mou, Z.; Li, J.; Shi, J.; Chen, C.; Liu, W.; Yang, H.; Liu, W. Organic & Biomolecular Chemistry 2014, 12, 5023). We then investigated the spectral properties, aqueous solubility, and stability of the acryloyl esters and evaluated their response to Cys. The chemical design was carried out using a proprietary method, specifically developed by Cedex. (registered trademark) It has been demonstrated that this method can provide specialized probes for

[0012] Preferred is R 1 and R 2 But R 3 , OR 3 , S.R. 3 , SO3 - , SO3-R 3 are independently selected from R 3 is selected from C1-C6 alkyl and polyethylene glycol (PEG) residues; Acc is a compound of formula II [ka] wherein X is selected from -N(CH3)-, -S-, -Se-, -O-, and -C(CH3)2-, and the aromatic ring is substituted with one, two, or three SO3 - may be substituted with R 4is C1-C6 alkyl, C1-C6 cycloalkyl, and (CH2) m -SO3 - and m is an integer from 1 to 6. and Compounds of formula I according to the present invention, wherein n is 1, and suitable salts and solvates thereof.

[0013] More preferably, R 1 and R 2 But R 3 , OR 3 , S.R. 3 , SO3 - , SO3-R 3 are independently selected from R 3 is selected from C1-C3 alkyl and polyethylene glycol (PEG) residues; Acc is a compound of formula II [ka] (Wherein, X is -C(CH3)2- and the aromatic ring contains 1, 2 or 3 SO3 - R4 may be substituted with a (CH2) group. m -SO3 - and m is an integer from 1 to 6. and Compounds of formula I according to the present invention, wherein n is 1, and suitable salts and solvates thereof.

[0014] More preferred are compounds of the following formulae VI to IX: [ka] and suitable salts and solvates thereof.

[0015] In the context of the present invention, suitable salts are generally those which do not interfere or do not substantially interfere with the solubility of the compounds according to the invention, in particular in aqueous media. Examples are salts of Group I elements (Li + , Na + , K. + , Cs+ , Rb + ), ammonium ion (NH4 + ), nitrate ions (NO3 - ) and Cl - , Br - , or I - or sulfates.

[0016] A further aspect of the present invention then relates to a process for preparing a compound according to formula I according to the present invention, comprising: a) Formula VI [ka] (In the formula, R 1 and R 2 is as defined above, and n is 1 or 2) The compound Formula II [ka] a compound of, or Formula III [ka] a compound of, or Formula IV [ka] or a compound of formula V [ka] Compounds of (wherein in each of Formulas II-V, the aromatic ring may contain one, two, or three SO3 - may be substituted with R 4 is C1~C 18 Alkyl, C1-C6 cycloalkyl, and (CH2) m -SO3 - and m is an integer from 1 to 18. to form a compound of formula VIII [ka] (In the formula, R 1 , R 2 and Acc is as defined above, and n is 1 or 2. obtaining a compound of formula (I), b) reacting the compound of formula VIII with suitable acryloyl chloride Suitable conditions for carrying out the above-mentioned methods are known to those skilled in the art and are exemplarily disclosed in the examples and schemes below.

[0017] Yet another aspect of the present invention then relates to a method for detecting cysteine ​​in a test sample, comprising the steps of: a) measuring the UV / Vis absorbance of a solution of a compound defined according to the present invention in a suitable solvent before and after contacting it with a test sample expected to contain cysteine, b) determining the difference in absorbance by comparison of the UV / Vis spectra measured in step a), and c) detecting cysteine ​​in the test sample based on the difference in absorbance determined in step b). [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows a scheme of the mechanism of reaction of acryloyl esters with Cys (R—OH=merocyanine). [Figure 2] FIG. 2 shows the structure of the probes synthesized in the context of the present invention. [Figure 3] Figure 3 shows the results of fertilization in feed medium (DMT118F.01 without Cys). SR MF70 in DMSO / water (1:1). R1: 100 mM K-PO4. DETAILED DESCRIPTION OF THE INVENTION

[0019] Test samples according to the present invention can include any sample that contains cysteine ​​or is expected to contain cysteine. Examples are the detection of biogenic thiols in, for example, plasma, in samples obtained from patients, total proteins in different types of cell lines, in tissue samples, cell lysates, serum, saliva or urine, in antibody samples, and in samples used in biotechnology applications. Preferred are Cedex (registered trademark) An aqueous biological sample is analyzed in the system.

[0020] Spectra recorded in the presence of Cys confirmed the colorimetric response through cleavage of the acryloyl ester. The synthesized probes showed a significant bathochromic shift into the green and yellow range of the visible spectrum (Table 2). Preferred is the method according to the present invention, in which UV / Vis absorbance is measured at discrete wavelengths within the range of 200 nm to 1000 nm. Furthermore, their spectral profiles were analyzed by Cedex. (registered trademark) The wavelength requirements (340, 378, 409, 480, 512, 520, 552, 583, 629, 652, 659 and 800 nm) for Cys sensing applications in the system were advantageously met.

[0021] More preferred is a process according to the invention, wherein the solvent is an aqueous solvent.

[0022] In a preferred embodiment of the present invention, the method of the present invention is one in which the difference in absorbance is determined by visual inspection, such as a color change, e.g., a significant bathochromic shift into the green and yellow range of the visible spectrum. For example, in a slightly different approach, Hai-Feng Yin et al. ("Simple probe with visible color change for selective detection of cysteine," Spectroscopy Letters, (2020) DOI: 10.1080 / 00387010.2020.1821063) synthesized a fluorescent probe capable of selectively detecting cysteine. Upon addition of cysteine, the probe solution exhibited a noticeable color change from pale yellow to orange, as seen with the naked eye.

[0023] The determination of the difference in absorbance is (registered trademark) The method according to the invention is by Bio HT (Roche Diagnostics, Penzberg, Germany).

[0024] Yet another aspect of the present invention then relates to a kit for detecting cysteine ​​in a test sample, comprising a vial or container containing a predetermined amount of a compound according to the present invention, together with instructions for using said kit. Examples of materials that may be included include, for example, standards, a probe according to the present invention, and a buffer.

[0025] Yet another aspect of the present invention then relates to the use of a compound according to the present invention, or a kit according to the present invention, for detecting cysteine ​​in a test sample, preferably an aqueous test sample as disclosed herein.

[0026] The present invention is further described, but not limited, in the following examples and with reference to the figures, in which: For purposes of the present invention, all references cited herein are incorporated by reference in their entirety. [Example]

[0027] A series of acryloyl esters based on the merocyanine chromophore were synthesized (Figure 2) and compared with the known probe LZ07 from the literature (Han, Q.; Shi, Z.; Tang, X.; Yang, L.; Mou, Z.; Li, J.; Shi, J.; Chen, C.; Liu, W.; Yang, H.; Liu, W. Organic & Biomolecular Chemistry 2014, 12, 5023). The spectral properties, aqueous solubility, and stability were studied, and the response to Cys was evaluated. The chemical design according to the present invention was carried out by Cedex. (registered trademark) The present inventors have demonstrated that the present invention provides specialized probes for the detection of HIV-1.

[0028] The following is a brief summary of the current state of the art regarding known Cys probes and their properties. [Table 1-1] [Table 1-2] [Table 1-3]

[0029] Experimental procedure material and method Reagents and solvents were purchased from Sigma-Aldrich at the highest commercial quality and used without further purification. CHROMASOLV solvent was used as eluent in HPLC. Yields were determined chromatographically (HPLC-MS) and spectroscopically ( 1 H NMR) refers to homogeneous material. Counterion anions are omitted for clarity.

[0030] Analytical HPLC-MS (ESI-MS) The purity of the compounds was determined with the aid of an HPLC-MS instrument from Waters (Milford, USA) containing the following components: a 2695 Separation module, a 2696 photodiode array, and a Waters Micromass ZQ (ESCI ionization mode) detector. Data acquisition was performed by MassLynx (V4.1) software.

[0031] Column:YMC-Triart C18 3μM(4.6×150mm) / Product Nr.TA12S03-1546WT. Flow rate: 0.7mL / min. Phase A: Triethylammonium acetate (TEAAc) buffer (10 mM, pH 7.0) in deionized water. Phase B: MeCN. Gradient 80:5-80B (7 min); 80-80B (2 min); 80-5B (0.5 min); 5-5B (2.5 min). Gradient 100:5-100B (7 min); 100-100B (2 min); 100-5B (0.5 min); 5-5B (2.5 min).

[0032] NMR NMR spectra were recorded on Bruker Avance (500 and 600 MHz) and Agilent 400 MR DD2 (400 MHz) instruments and were corrected using residual non-deuterated solvent as an internal reference. 1 The following abbreviations were used to describe NMR peak multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad.

[0033] HRMS For HRMS (high-resolution mass spectra), samples were dissolved in MeCN and analyzed by direct flow injection (injection volume = 5 μL) electrospray ionization time-of-flight (ESI-TOF) mass spectrometry in positive ion mode on a Waters Q-ToF Premier instrument.

[0034] General Procedure I Preparation of merocyanine dyes A mixture of the respective aldehyde (1 equiv.) and indolium salt (1 equiv.) in ethanol was refluxed under Ar in the presence of piperidine (0.1–2 equiv.) for 1–16 h. After the reaction mixture was slowly cooled to room temperature, the solvent was removed in vacuo, and the residue was purified by reverse-phase column chromatography (C-18, TEAB buffer (10 mM, pH 7.4) / MeCN or HO (0.1% TFA) / MeCN).

[0035] General Procedure II Preparation of acryloyl esters Acryloyl chloride (4–5 equiv.) was added to a mixture of the respective merocyanine dye (1 equiv.) and EtN (4–5 equiv.) in dry DCM at 0 °C under Ar. After stirring at 0 °C for 1 h, the reaction was quenched by adding aqueous NH4Cl (0.1 M), and the organic material was extracted twice with DCM. The combined extracts were washed with NH4Cl (0.1 M), dried over Na2SO4, and concentrated in vacuo. The residue was purified by reverse-phase column chromatography (C-18, HO / MeCN).

[0036] General Procedure III Preparation of acryloyl esters Acryloyl chloride (4–5 equiv.) was added to a mixture of the respective merocyanine dye (1 equiv.) and EtN (4–5 equiv.) in dry DCM at 0 °C under Ar. After stirring at 0 °C for 1 h, the reaction was quenched by adding aqueous NH Cl (0.1 M), and the water-soluble product was extracted twice with HO. The combined extracts were washed with DCM, concentrated in vacuo (20 mbar, 20 °C), and then purified by reverse-phase column chromatography (C-18, HO / MeCN).

[0037] Spectroscopic Materials and Methods Absorption spectra were recorded on a Cary 50 UV-vis spectrometer from Varian. All measurements were performed in 1 cm UV-vis disposable cuvettes (BRAND semi-micro) and air-equilibrated solutions at 25±0.1°C. A total assay volume of 1.0 mL was used for each measurement. UV-vis scan spectra were recorded using the following parameters: averaging time 0.05 s; data interval 1 nm; scan speed 1200 nm / min; baseline correction was performed.

[0038] Solutions were prepared in 1.5 mL vials (Eppendorf® Microtubes 3810X) using a Vortex Mixer.

[0039] Stock solutions of the compounds to be assayed (2–5 mM) were prepared in HO-DMSO (1:1), stored at −20 °C, and diluted to 1.0 mM with buffer before use. L-Cys stock solution (20.0 mM) was freshly prepared in buffer before measurements. HEPES buffer (25 mM, pH 7.4) was used for all measurements.

[0040] 18MΩ cm obtained using a Millipore purification system -1 All aqueous solutions were made up in deionized water (MQ water) with a resistivity of 0.1% or higher. 2

[0041] Extinction coefficient For measurements, 1000 μL of buffer and 1-50 μL of probe (1.0 mM) were mixed and then transferred to a cuvette. Absorbance spectra (250-800 nm) were measured against a buffer blank. Extinction coefficients were calculated from the slope of the probe concentration vs. absorbance plot using at least six concentrations of each compound using MS Excel software (Microsoft).

[0042] Similarly, ε was obtained from a solution of the probe reacted with excess Cys (100 μM). CysThe reaction was carried out at 37°C (incubation time 15 min). A blank reaction was carried out without adding Cys.

[0043] Stability assessment For the measurements, 1000 μL of buffer solution and 16 μL of probe (1.0 mM) were mixed and then incubated at +4°C and +37°C for 5 h. The resulting mixture was transferred to a cuvette, and the absorbance (250-800 nm) was measured. Each measurement was performed in triplicate.

[0044] Solubility evaluation In experiments, 5–10 mg of dried material was suspended in 250–500 μL of HO at room temperature. The resulting suspension was centrifuged (16,000 rcf) for 10 min at room temperature. The UV–vis of the supernatant was recorded in buffer (25 mM HEPES pH 7.4) at room temperature. Each measurement was performed in triplicate. The pellet was dried in vacuum for 16 h and then weighed.

[0045] The final products were obtained in a two-step synthesis (condensation and acrylation; Schemes 1-3) as described above. The overall yields were 21-59%, except for 6% in the case of MF65. The products were analyzed by HPLC-MS. 1 H and 13 It was characterized by C NMR and UV-Vis. [ka]

[0046] Scheme 1. Reagents and conditions: (i) piperidine (catalyst), EtOH, reflux, Ar; (ii) EtN (4 equiv), DCM, 0 °C, t<2 h, Ar. [ka]

[0047] Scheme 2. Reagents and conditions: (i) piperidine (catalyst), EtOH, reflux, Ar; (ii) EtN (4 equiv), DCM, 0 °C, t<2 h, Ar. [ka]

[0048] Scheme 3. Reagents and conditions: (i) piperidine (catalyst), EtOH, reflux, Ar; (ii) EtN (4 equiv), DCM, 0 °C, t<2 h, Ar. UV-vis and Cys responses

[0049] The resulting dyes were evaluated for UV-visibility. Selected substituents on the benzene ring helped increase the UV-visibility for the initial merocyanine dyes. The most striking results were observed for the preferred intermediate compounds MF56, MF57, and MF66. [Table 2]

[0050] Spectra recorded in the presence of Cys confirmed the colorimetric response through cleavage of the acryloyl ester. The synthesized probes exhibited a significant bathochromic shift into the green and yellow range of the visible spectrum (Table 2). Furthermore, their spectral profiles were compared with those of Cedex. (registered trademark) The wavelength requirements (340, 378, 409, 480, 512, 520, 552, 583, 629, 652, 659 and 800 nm) for Cys sensing applications in the system were advantageously met.

[0051] stability Stability is another important parameter for the evaluation of colorimetric probes for biological assays. Probes for commercial applications need to be able to be stored at 4°C for several months. Additionally, stability is measured by Cedex (registered trademark) It must be tested under the assay conditions (37°C) of the Bio HT analyzer.

[0052] We qualitatively examined the stability of the probes in buffer (10 mM HEPES pH 7.4) at 4°C and 37°C to simulate typical storage and assay conditions. Solutions of each probe were monitored by UV-vis for 5 h. The results of all spectroscopic evaluations are summarized in Table 3. [Table 4]

[0053] solubility

[0054] The aqueous solubility of the probe is another important characteristic for the performance of a compound in biological assays. We used UV-vis to determine the solubility of the probes of the present invention in water. First, a supersaturated mixture of each probe was prepared. The mixture was centrifuged, and the concentration of the probe in the supernatant was then determined by UV-vis. To further verify the results, the concentration was calculated from the weight of the isolated pellet. As can be seen in Table 4, the results determined by both approaches were within the same range and followed the same trend. The preferred probe MF70, which contains two sulfo groups, is highly water-soluble compared to its analog LZ05 and MF59, which contains only one sulfo group. The solubility of MF70 is also superior to that of the literature-known compound LZ07. [Table 5]

[0055] Cedex (registered trademark) Performance of the MF70 probe in Bio HT The rapid kinetic profile, stability and low background signal obtained are the hallmarks of Cedex (registered trademark)This prompted further use of MF70 for Cys sensing in BioHT. The reagent solution was treated with various Cys concentrations (0.5-7.6 mM) in feed medium used for monoclonal antibody production. As shown in Figure 3, progressively enhanced absorbance was observed with increasing amounts of Cys. Under these conditions, reliable responses were obtained over a 1-week period (Table 5), with a corresponding detection limit of 3.6 μM Cys and a blank limit of 2.2 μM Cys.

[0056] In conclusion, the probes according to the present invention, and preferably probe MF70, meet the requirements for commercial assays. The merocyanine dye scaffold ensures a bright chromogenic signal. The methyl and sulfonic acid groups at the ortho positions appear to ensure hydrolytic stability and aqueous solubility, respectively.

Claims

1. Formula I 【Chemistry 1】 (In the formula, R １ and R ２ is R ３ , O-R ３ , S.R. ３ , S.O. ３ － , S.O. ３ -R ３ and R ３ is C １ ~C １８ alkyl, and polyethylene glycol (PEG) residues; Acc is a compound of formula II 【Chemistry 2】 (Wherein, X is —N(CH ３ )-, -S-, -Se-, -O-, and -C(CH ３ ) ２ - selected from), Formula III 【Transformation 3】 Formula IV 【Chemistry 4】 and Formula V 【Transformation 5】 wherein in each of Formulas II-V, the aromatic ring contains one, two, or three SO ３ － may be substituted with a group, R ４ is C １ ~C １８ Alkyl, C １ ~C ６ Cycloalkyl, and (CH ２ ) ｍ -SO ３ － and m is an integer selected from 1 to 18. selected from the group selected from n is selected from 1, 2 and 3. and suitable salts and solvates thereof.

2. R １ and R ２ But, R ３ , O-R ３ , S.R. ３ , S.O. ３ － , S.O. ３ -R ３ and R ３ But C １ ~C ６ alkyl, and polyethylene glycol (PEG) residues; Acc is a compound of Formula II 【Transformation 6】 (Wherein, X is —N(CH ３ )-, -S-, -Se-, -O-, and -C(CH ３ ) ２ -, wherein the aromatic ring contains 1, 2 or 3 SO ３ － may be substituted with a group, R ４ is C １ ~C ６ Alkyl, C １ ~C ６ Cycloalkyl, and (CH ２ ) ｍ -SO ３ － and m is an integer from 1 to 6. and 2. A compound of formula I according to claim 1, wherein n is 1, and suitable salts and solvates thereof.

3. R １ and R ２ But, R ３ , O-R ３ , S.R. ３ , S.O. ３ － , S.O. ３ -R ３ and R ３ But C １ ~C ３ alkyl, and polyethylene glycol (PEG) residues; Acc is a compound of Formula II 【Transformation 7】 (Wherein, X is —C(CH ３ ) ２ -, and the aromatic ring contains 1, 2 or 3 SO ３ － may be substituted with a group, R ４ (CH ２ ) ｍ -SO ３ － and m is an integer from 1 to 6. and 2. A compound of formula I according to claim 1, wherein n is 1, and suitable salts and solvates thereof.

4. Formulas VI to IX below 【Transformation 8】 and suitable salts and solvates thereof.

5. 10. A process for preparing a compound of formula I according to claim 1, comprising: a) Formula VI 【Chemistry 9】 (In the formula, R １ and R ２ is as defined in claim 1 and n is 1 or 2, Formula II 【Chemistry 10】 a compound of, or Formula III 【Chemistry 11】 a compound of, or Formula IV 【Chemistry 12】 a compound of, or Formula V 【Chemistry 13】 Compounds of wherein in each of Formulas II-V, the aromatic ring contains one, two, or three SO ３ － may be substituted with a group, R ４ is C １ ~C １８ Alkyl, C １ ~C ６ Cycloalkyl, and (CH ２ ) ｍ -SO ３ － and m is an integer from 1 to 18. to form a compound of formula VIII 【Chemistry 14】 (In the formula, R １ , R ２ and Acc is as defined in claim 1, and n is 1 or 2. obtaining a compound of formula (I), b) suitably reacting said compound of formula VIII with acryloyl chloride A method comprising:

6. 1. A method for detecting cysteine ​​in a test sample, comprising: a) measuring the UV / Vis absorbance of a solution of a compound according to any one of claims 1 to 4 in a suitable solvent before and after contacting it with a test sample suspected of containing cysteine; b) determining the difference in absorbance by comparing the UV / Vis spectra measured in step a); and c) detecting cysteine ​​in said test sample based on said difference in absorbance determined in step b). A method comprising:

7. 7. The method of claim 6, wherein the UV / Vis absorbance is measured at discrete wavelengths in the range of 200 nm to 1000 nm.

8. 8. The method of claim 7, wherein the wavelength is selected from the group consisting of 340, 378, 409, 480, 512, 520, 552, 583, 629, 659, and 800 nm.

9. The method according to any one of claims 6 to 8, wherein the solvent is an aqueous solvent.

10. The method of any one of claims 6 to 9, wherein said determining said difference in absorbance is by visual inspection of a color change.

11. 10. A kit for detecting cysteine ​​in a test sample, comprising a vial or container containing a predetermined amount of a compound according to any one of claims 1 to 4, together with instructions for using said kit.

12. Use of a compound according to any one of claims 1 to 4 or a kit according to claim 11 for detecting cysteine ​​in a test sample.

13. The use of claim 12, wherein the test sample is an aqueous test sample.

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