High water solubility and stability chemical sensor for cysteine

Abstract

This invention relates to an improved chemical probe for the detection of cysteine ​​in test samples, preferably aqueous test samples, and the corresponding uses and kits.

Description

[0001] This invention relates to an improved chemical probe for the detection of cysteine ​​in test samples, preferably aqueous test samples, and the corresponding uses and kits. Background Technology

[0002] Cysteine ​​(Cys) plays a crucial role in biosynthesis, detoxification, and metabolism. Elevated total homocysteine ​​levels can predict cardiovascular disease and metabolic syndrome. Cysteine ​​deficiency is known to be a consequence of aging. Selectively detecting Cys on structurally similar homocysteine ​​(Hcy) or glutathione (GSH) remains a significant challenge. Although numerous methods exist for Cys detection, photoluminescence (PL) and electrochemiluminescence (ECL) techniques are well-suited for clinical diagnostics and analytical applications due to their high sensitivity.

[0003] Trisulfide formation in recombinant monoclonal antibodies is a source of heterogeneity and needs to be controlled to ensure product quality consistency. Ryll et al. (Kshirsagar, R.; McElearney, K.; Gilbert, A.; Sinacore, M.; Ryll, T. Biotechnol. Bioeng. 2012, 109, 2523) have shown a direct correlation between L-cysteine ​​(Cys) concentration in the feed medium and trisulfide levels in the product (IgG1 mAb). Therefore, a controlled Cys feed strategy is needed to reduce trisulfide formation to acceptable levels.

[0004] To date, the detection of Cys has attracted widespread attention in various biochemical applications. Many methods have been developed for the detection of Cys, such as fluorescence assays combined with Ellman's reagents or with fluorescence, potentiometry, electrochemical voltammetry, and HPLC. These methods require complex instrumentation, involve cumbersome laboratory procedures, or have low throughput.

[0005] Kim and Hong (in: Photoluminescence and Electrochemiluminescence Dual-Signaling Sensors for Selective Detection of Cysteine ​​Based on Iridium(III) Complexes. ACS Omega 2019, 4, 7, 12616-12625) reported a dual-channel PL and ECL sensor using cyclometalated iridium(III) complexes to distinguish Cys from Hcy and GSH.

[0006] UV-vis spectroscopy offers a rapid and simple measurement procedure. Therefore, to quantify essential metabolites in biological processes, automated analyzers such as the Cedex Bio HT (Roche Diagnostics, Penzberg, Germany) are required for photometric determination. However, only a limited number of candidates can be used for colorimetric Cys detection on the Cedex Bio HT analyzer (“Cedex”), and this analyzer only provides a limited set of wavelengths: 340, 378, 409, 480, 512, 520, 552, 583, 629, 652, 659, and 800 nm. Furthermore, ideal probes for the Cedex system must include high sensitivity, rapid response, water solubility and stability, and ease of use.

[0007] To date, most indicators of Cys have been based on the strong nucleophilicity of thiol groups. Various mechanisms have been employed, including Michael addition and cleavage reactions. Sensing strategies based on acrylate groups appear promising because they allow for the differentiation 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). Figure 1 The sensing mechanism is presented. This strategy involves the conjugated addition of Cys to acrylate to form a thioester, followed by intramolecular cyclization. The acrylate moiety, acting as the thiol activation site, undergoes rapid cyclization only with Cys, as the reaction rate largely depends on the ring size of the resulting lactam. Removal of the masking acrylate group restores the conjugated π-electron system of the chromophore, thus achieving a colorimetric response.

[0008] Disadvantageously, only a few acrylate-based probes exhibit strong colorimetric responses at the desired wavelengths for use on the Cedex. Their chromophores are based on xanthracene, benzocyanine, heptamethrin, and fluorescein. The fact that most reported acrylate probes are used in organic solvent-water mixtures is clearly necessary due to their poor solubility in aqueous media. However, for applications on the Cedex, the probes must be water-soluble because parts of the instrument are unstable in organic solvents. Furthermore, the stability of existing probes must be evaluated to ensure that the assay solution can be stored in the Cedex for a reasonably long period.

[0009] In view of these and other disadvantages, one object of the present invention is to provide a Cys probe that can be used in aqueous solutions and provides sufficient colorimetric response at one of the desired wavelengths. Other objects and advantages will become apparent to those skilled in the art upon studying the present description of the invention.

[0010] In a first aspect of the invention, the above-mentioned objective is achieved by a compound according to formula (I).

[0011]

[0012] in

[0013] R 1 and R 2 Independently selected from R 3 OR 3 SR 3 SO3 - SO3-R 3 , where R 3 Selected from C1-C 18 Alkyl groups and polyethylene glycol (PEG) residues,

[0014] Acc is selected from the group that is selected from Equation II.

[0015]

[0016] Where X is selected from -N(CH3)-, -S-, -Se-, -O-, and -C(CH3)2-.

[0017] Formula III

[0018]

[0019] Formula IV

[0020] ,and

[0021] Formula V

[0022]

[0023] In each of formulas II to V, the aromatic ring is optionally enclosed by one, two, or three SO3 rings. - Group substitution,

[0024] R 4 Selected from C1-C 18 Alkyl, C1-C6 cycloalkyl, and (CH2) m -SO3 - The group, where m is an integer selected from 1 to 18, and n is selected from 1, 2, and 3, as well as its suitable salts and solvates.

[0025] The inventors synthesized a series of acryloyl esters based on anthocyanin chromophores ( Figure 2(See below); Probe LZ07 was prepared as a control and for comparison, and this probe 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). The inventors then investigated the spectral properties, water solubility, and stability of the acryloyl ester and evaluated its response to Cys. It can be demonstrated that the chemical design provides a dedicated probe, particularly for Cedex.

[0026] Preferably, it is a compound of formula I according to the invention, wherein R 1 and R 2 Independently selected from R 3 OR 3 SR 3 SO3 - SO3-R 3 , where R 3 Selected from C1-C6 alkyl groups and polyethylene glycol (PEG) residues.

[0027] Acc is Equation II

[0028]

[0029] Where X is selected from -N(CH3)-, -S-, -Se-, -O-, and -C(CH3)2-, and optionally the aromatic ring has 1, 2, or 3 SO3 groups. - Group substitution, R 4 Selected from C1-C6 alkyl, C1-C6 cycloalkyl, and (CH2) m -SO3 - , where m is an integer from 1 to 6 and n is 1, along with its suitable salt and solvate.

[0030] Further preferred are compounds of formula I according to the present invention, wherein R 1 and R 2 Independently selected from R 3 OR 3 SR 3 SO3 - SO3-R 3 , where R 3 Selected from C1-C3 alkyl groups and polyethylene glycol (PEG) residues.

[0031] Acc is Equation II

[0032]

[0033] Where X is -C(CH3)2-, and optionally the aromatic ring has 1, 2 or 3 SO3 groups. - Group substitution, R4 is (CH2). m -SO3 - , where m is an integer from 1 to 6 and n is 1, along with its suitable salt and solvate.

[0034] Further preferred are compounds of formula I according to the invention, based on formulas VI to IX below.

[0035]

[0036] And its suitable salts and solvates.

[0037] In the context of this invention, a suitable salt is generally one that does not interfere with or substantially does not interfere with the solubility of the compounds according to the invention, particularly in aqueous media. An example is a salt containing a Group I element (Li). + Na + K + Cs + 、Rb + ), ammonium ions (NH4) + ), nitrate ions (NO3) - ), containing Cl - ,Br - Or I - Salts, or sulfates.

[0038] Another aspect of the present invention relates to a method for preparing a compound of formula I according to the present invention, the method comprising the following steps:

[0039] a) Make compound of formula VI

[0040]

[0041] Where R 1 and R 2 As defined above, and n is 1 or 2, and the compound of formula II

[0042]

[0043] Or with compounds of formula III

[0044]

[0045] Or with compound of formula IV

[0046]

[0047] Or with compound of formula V

[0048]

[0049] In each of formulas II to V, optionally, the aromatic ring passes through 1, 2, or 3 SO3 rings. - Group substitution, R 4 Selected from C1-C 18 Alkyl, C1-C6 cycloalkyl, and (CH2) m -SO3 - , and where m is an integer from 1 to 18, react appropriately to obtain compound of formula VIII.

[0050]

[0051] Where R 1 R 2 And Acc as defined above, and n is 1 or 2, and b) react the compound of formula VIII with acryloyl chloride appropriately. Suitable conditions for carrying out the above methods are known to those skilled in the art and are exemplary disclosed in the examples and schemes below.

[0052] Another aspect of the invention relates to a method for detecting cysteine ​​in a test sample, the method comprising the steps of: a) measuring the UV / Vis absorbance of a solution of a compound as defined according to the invention in a suitable solvent before and after contact with a test sample expected to contain cysteine; b) determining the difference in absorbance by comparing 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).

[0053] The test samples according to the invention can include any sample that contains or is intended to contain cysteine. Examples include, for instance, detecting biothiols in plasma, samples obtained from patients, total protein in different cell lines, tissue samples, cell lysates, serum, saliva or urine, antibody samples, and samples used in biotechnological applications. Preferably, it is an aqueous biological sample to be analyzed in the Cedex system.

[0054] The spectra recorded in the presence of Cys confirmed the colorimetric response through the cleavage of acryloyl esters. The synthesized probes showed a significant redshift into the green and yellow ranges of the visible spectrum (Table 2). Preferably, the method according to the invention is used, wherein UV / Vis absorbance is measured at discrete wavelengths in the range of 200 nm to 1000 nm. Furthermore, their spectral distribution advantageously meets the wavelength requirements (340, 378, 409, 480, 512, 520, 552, 583, 629, 652, 659, and 800 nm) for Cys sensing applications in the Cedex system.

[0055] More preferably, the method according to the present invention is an aqueous solvent.

[0056] In a preferred aspect of the invention, according to the method of the invention, the determination of the difference in absorbance is performed by visual inspection of color changes, such as a significant redshift into the green and yellow ranges of the visible spectrum. As an example, using a slightly different method, Hai-Feng Yin et al. (in: simple probe with visible color change for selective detection of cysteine, Spectroscopy Letters, (2020) DOI: 10.1080 / 00387010.2020.1821063) synthesized a fluorescent probe that selectively detects cysteine. Upon addition of cysteine, a distinct color change from pale yellow to orange was observed in the probe solution by the naked eye.

[0057] According to the method of the present invention, the determination of the difference in absorbance is performed by Cedex Bio HT (Roche Diagnostics, Penzberg, Germany).

[0058] Another aspect of the invention relates to a kit for detecting cysteine ​​in a test sample, the kit comprising a vial or container and a manual for using the kit, the vial or container comprising a predetermined amount of a compound according to the invention. Examples of the included materials are, for example, standards, probes according to the invention, and buffer solutions.

[0059] Another aspect of the invention relates to the use of compounds according to the invention, or kits according to the invention, for detecting cysteine ​​in test samples, preferably aqueous test samples as disclosed herein.

[0060] The present invention will now be further described in the following examples, and with reference to the accompanying drawings, but is not limited thereto. For the purposes of this invention, all references cited herein are incorporated herein by reference in their entirety.

[0061] Figure 1 The reaction mechanism diagram of acryloyl ester with Cys(R-OH = cyanine) is shown.

[0062] Figure 2 The structure of the probe synthesized in the context of this invention is shown.

[0063] Figure 3 The calibration results are shown in the feed medium (DMT118F.01 without Cys). SR: MF70 in DMSO / water (1:1). R1: 100 mM K-PO4.

[0064] Example

[0065] A series of acryloyl esters based on anthocyanin chromophores were synthesized. Figure 2 The probe was designed and compared with the known probe LZ07 in 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). Spectroscopic properties, water solubility, and stability were investigated, and the response to Cys was evaluated. The inventors demonstrate that the chemical design according to the invention provides a dedicated probe for Cedex.

[0066] The following is a brief summary of the latest techniques regarding known Cys probes and their properties:

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Experimental Procedure

[0074] Materials and methods

[0075] Reagents and solvents were purchased from Sigma-Aldrich at the highest commercial quality and were ready for use without further purification. CHROMASOLV solvent was used as the eluent in HPLC. Unless otherwise stated, yields refer to chromatographic (HPLC-MS) and spectroscopic (HPLC-MS) results. 1 (H NMR) homogeneous material. For clarity, counter anions have been omitted.

[0076] Analytical HPLC-MS (ESI-MS)

[0077] The purity of the compounds was determined using an HPLC-MS system from Waters (Milford, USA), which includes a 2695 separation module, a 2696 photodiode array, and a Waters Micromass ZQ (ESCI ionization mode) detector. Data acquisition was performed using MassLynx (V4.1) software.

[0078] Column: YMC-Triart C18 3μM (4.6x150mm) / Product Nr.TA12S03-1546WT.

[0079] Flow rate: 0.7 mL / min.

[0080] Phase A: Triethylammonium acetate (TEAAc) buffer in deionized water (10 mM, pH 7.0).

[0081] Phase B: MeCN.

[0082] Gradient 80: 5-80B (7 min); 80-80B (2 min); 80-5B (0.5 min); 5-5B (2.5 min).

[0083] Gradient 100: 5-100B (7 min); 100-100B (2 min); 100-5B (0.5 min); 5-5B (2.5 min).

[0084] NMR

[0085] NMR spectra were recorded on a Bruker Avance (500 and 600 MHz) and an Agilent 400MR DD2 (400 MHz) instrument and calibrated using residual non-deuterated solvent as an internal reference. 1 The following abbreviations are used to explain NMR peak multiplicity: s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, br = broad peak.

[0086] HRMS

[0087] For HRMS (High Resolution Mass Spectrometry), the sample was dissolved in MeCN and analyzed in positive ion mode by direct current injection (injection volume = 5 μL) electrospray ionization time-of-flight (ESI-TOF) mass spectrometry on a Waters Q-ToF Premier instrument.

[0088] General Procedure I

[0089] Preparation of cyanine dyes

[0090] A mixture of the corresponding aldehyde (1 equivalent) and indole salt (1 equivalent) in ethanol was refluxed under Ar for 1 to 16 h in the presence of piperidine (0.1 to 2 equivalents). The reaction mixture was allowed to cool slowly to room temperature, the solvent was removed under vacuum, and the residue was purified by reversed-phase column chromatography (C-18, TEAB buffer (10 mM, pH 7.4) / MeCN or H2O (0.1% TFA) / MeCN).

[0091] General Procedure II

[0092] Preparation of Acrylamide

[0093] Acryloyl chloride (4 to 5 equivalents) was added to a mixture of the corresponding cyanine dye (1 equivalent) and Et3N (4 to 5 equivalents) in dry DCM at 0 °C and Ar. After stirring at 0 °C for 1 h, the reaction was quenched by adding aqueous NH4Cl (0.1 M), and the organic matter was extracted twice with DCM. The combined extracts were washed with NH4Cl (0.1 M), dried over Na2SO4, and concentrated under vacuum. The residues were purified by reversed-phase column chromatography (C-18, H2O / MeCN).

[0094] General Procedure III

[0095] Preparation of Acrylamide

[0096] Acryloyl chloride (4 to 5 equivalents) was added to a mixture of the corresponding cyanine dye (1 equivalent) and Et3N (4 to 5 equivalents) in dry DCM at 0 °C and Ar. After stirring at 0 °C for 1 h, the reaction was quenched by adding aqueous NH4Cl (0.1 M), and the water-soluble product was extracted twice with H2O. The combined extracts were washed with DCM, concentrated under vacuum (20 mbar, 20 °C), and then purified by reversed-phase column chromatography (C-18, H2O / MeCN).

[0097] Spectroscopic Materials and Methods

[0098] Absorption spectra were recorded on a Varian Cary 50 UV-vis spectrometer. All measurements were performed in 1 cm UV-vis disposable cuvettes (BRAND semi-micro) and air-equilibrated solution at 25 ± 0.1 °C. A total measurement 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 rate 1200 nm / min; and baseline correction.

[0099] Use a vortex mixer in a 1.5 mL bottle ( The solution was prepared in a microtube (3810X).

[0100] Stock solutions (2 to 5 mM) of the compounds to be measured were prepared in H2O-DMSO (1:1), stored at -20°C, and diluted to 1.0 mM with buffer before use. L-Cys stock solutions (20.0 mM) were freshly prepared in buffer before measurement. All measurements were performed using HEPES buffer (25 mM, pH 7.4).

[0101] All aqueous solutions have a resistivity ≥18 MΩcm -1 It is made from deionized water and obtained using the Millipore purification system (MQ-water). 2

[0102] Extinction coefficient

[0103] For measurement, 1000 μL of buffer and 1 to 50 μL of probe (1.0 mM) were mixed and then transferred to a cuvette. The absorbance spectrum (250 to 800 nm) was measured against the buffer blank. The extinction coefficient was calculated from the slope of the probe concentration versus absorbance plot using MS Excel software (Microsoft) with at least six concentrations of each compound.

[0104] In the same manner, ε was determined from the solution of the probe reacted with excess Cys (100 μM). Cys The reaction was performed at 37°C (incubation time 15 min). A blank reaction was performed without the addition of Cys.

[0105] Stability assessment

[0106] For the measurements, 1000 μL of buffer 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 was measured (250 to 800 nm). Each measurement was performed in triplicate.

[0107] Solubility assessment

[0108] In the experiment, 5 to 10 mg of dried material was suspended in 250 to 500 μL of H₂O at RT. The resulting suspension was centrifuged at RT (16000 rcf) for 10 min. The UV-vis of the supernatant was recorded at RT in buffer (25 mM HEPES, pH 7.4). Each measurement was performed in triplicate. The precipitate was dried under vacuum for 16 h and then weighed.

[0109] As described above, the final product was obtained through a two-step synthesis (condensation and acrylic acidification; schemes 1 to 3). Except for a 6% yield of MF65, the overall yield ranged from 21% to 59%. The product was analyzed by HPLC-MS, 1 H and 13The product was characterized by C NMR and UV-Vis.

[0110]

[0111] Scheme 1. Reagents and conditions: (i) piperidine (catalyst), EtOH, reflux, Ar; (ii) Et3N (4 equivalents), DCM, 0℃, t < 2h, Ar.

[0112]

[0113] Scheme 2. Reagents and conditions: (i) piperidine (catalyst), EtOH, reflux, Ar; (ii) Et3N (4 equivalents), DCM, 0℃, t < 2h, Ar.

[0114]

[0115] Scheme 3. Reagents and conditions: (i) piperidine (catalyst), EtOH, reflux, Ar; (ii) Et3N (4 equivalents), DCM, 0℃, t < 2h, Ar.

[0116] UV-vis and Cys response

[0117] The UV-vis of the obtained dyes were evaluated. Selected substituents on the benzene ring contribute to improving the value of the initial cyanine dye. The most significant results were observed for the preferred intermediate compounds MF56, MF57, and MF66.

[0118] Table 1. Spectral properties of the dyes according to the present invention. *

[0119]

[0120] *UV-vis spectra were recorded in an aqueous buffer (25 mM HEPES, pH 7.4). Probe concentrations ranged from 1 to 24 vM. At least six concentrations of each compound were used in the experiments.

[0121] The experiment was conducted in the same buffer solution at pH 8.0.

[0122] The spectra recorded in the presence of Cys confirmed the colorimetric response through the cleavage of acryloyl esters. The synthesized probes showed a significant redshift into the green and yellow ranges of the visible spectrum (Table 2). Furthermore, their spectral distribution favorably met the wavelength requirements (340, 378, 409, 480, 512, 520, 552, 583, 629, 652, 659, and 800 nm) for Cys sensing applications in the Cedex system.

[0123] Table 2. Spectral properties and Cys response screening of compounds according to the present invention. *

[0124]

[0125] *UV-vis spectra were recorded before and after adding excess Cys (100 pM) to an aqueous buffer (25 mM HEPES, pH 7.4). Probe concentrations ranged from 1 to 24 μM. Reactions with Cys were performed at 37 °C (incubation time 15 min). SNR was obtained from the reaction with Cys (ε... Cys The values ​​obtained after the reaction and the blank measurement values ​​(ε) 空白 (Estimated.) At least six concentrations of each compound were used in the experiment.

[0126] The experiment was conducted in the same buffer solution at pH 8.0.

[0127] stability

[0128] Stability is another important parameter for evaluating colorimetric probes used in bioassays. Probes for commercial applications must be able to be stored at 4°C for several months. Furthermore, stability must be checked under the assay conditions (37°C) of a Cedex Bio HT analyzer.

[0129] The inventors qualitatively examined the stability of the probes in buffer solutions (10 mM HEPES, pH 7.4) at 4°C and 37°C to simulate typical storage and assay conditions. The solutions of each probe were monitored by UV-vis for 5 hours. The results of all spectroscopic evaluations are summarized in Table 3.

[0130] Table 3. *Stability of the compounds of the present invention

[0131]

[0132] * Experiments were performed in an aqueous buffer (25 mM HEPES pH 7.4) at 4 °C and 37 °C (concentration 15.6 μM (incubation time 5 h)). The amount of hydrolysis probe was determined using ε values ​​derived from anthocyanin analogs (Table 1). Each measurement was performed in triplicate.

[0133] The amount of hydrolysis probe was determined using the ε value obtained from the Cys response screening (Table 2).

[0134] solubility

[0135] The water solubility of probes is another important property of compound performance in bioassays. The inventors used UV-vis to determine the water solubility of the probes according to the invention. First, a supersaturated mixture of each probe was prepared. The mixture was centrifuged, and then the concentration of the probe in the supernatant was examined by UV-vis. To further verify the results, the concentration was calculated from the weight of the separated particles. As shown in Table 4, the results determined by the two methods are in the same range and follow the same trend. The preferred probe MF70 contains two sulfonyl groups, making it highly water-soluble compared to its analogs LZ05 and MF59, which contain only one sulfonyl group. Moreover, MF70 exhibits superior solubility compared to the known compound LZ07 in the literature.

[0136] Table 4. *Solubility.

[0137]

[0138] *In the experiments, 5 to 10 mg of dried material was suspended in 250 to 500 μL of H₂O at RT. The UV-vis of the supernatant was recorded at RT in buffer (25 mM HEPES, pH 7.4). Each measurement was performed in triplicate. Probes derived from the synthesis were used.

[0139] Performance of probe MF70 in Cedex Bio HT

[0140] The obtained rapid kinetic curves, stability, and low background signal encourage further Cys sensing using MF70 in CedexBio HT. The reagent solution, used for monoclonal antibody production, was treated in the feed medium with various Cys concentrations (0.5 to 7.6 mM). Figure 3 As shown, the absorbance gradually increased with increasing Cys concentration. Under these conditions, a reliable response was obtained within one week (Table 5), with a detection limit of 3.6 μM Cys and a blank limit of 2.2 μM Cys.

[0141] Table 5. Spike recoveries in feed culture medium (DMT118F.01 containing Cys).

[0142]

[0143] In summary, the probe according to the present invention, preferably probe MF70, meets the requirements for commercial assays. The cyanine dye scaffold ensures a bright colorimetric signal. The ortho-methyl and sulfonic acid groups appear to ensure stability against hydrolysis and water solubility, respectively.

Claims

1. Compounds selected from formulas VI to IX and MF70: And its appropriate salt.

2. A method for detecting cysteine ​​in a test sample, the method comprising the following steps: a) Measure the UV / Vis absorbance of a solution of the compound as defined in claim 1 in a suitable solvent before and after contact with a test sample expected to contain cysteine, and b) Determine the difference in absorbance by comparing the UV / Vis spectra measured in step a), and c) Detect cysteine ​​in the test sample based on the difference in absorbance determined in step b).

3. The method of claim 2, wherein the UV / Vis absorbance is measured at discrete wavelengths in the range of 200 nm to 1000 nm.

4. The method according to claim 3, wherein the wavelength is selected from the group consisting of 340, 378, 409, 480, 512, 520, 552, 583, 629, 659 and 800 nm.

5. The method according to any one of claims 2 to 4, wherein the solvent is an aqueous solvent.

6. The method according to any one of claims 2 to 4, wherein the determination of the difference in absorbance is performed by visual inspection of color changes.

7. The method according to any one of claims 2 to 4, wherein the determination of the difference in absorbance is performed by Cedex.

8. A kit for detecting cysteine ​​in a test sample, the kit comprising a container and a manual for using the kit, the container comprising a predetermined amount of the compound according to claim 1.

9. The kit of claim 8, wherein the kit comprises a vial and a manual for using the kit, the vial comprising a predetermined amount of the compound of claim 1.

10. Use of the compound of claim 1 or the kit of claim 8 for detecting cysteine ​​in a test sample, wherein said use is not for disease diagnosis purposes.

11. Use of the compound of claim 1 or the kit of claim 8 for the detection of cysteine ​​in aqueous test samples, wherein said use is not for disease diagnosis purposes.

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