HIGHLY WATER-SOLUBLE AND STABLE CHEMOSENSOR FOR CYSTEINE

Abstract

The present invention relates to chemical probes for the improved detection of cysteine in a test sample, preferably an aqueous test sample, as well as respective uses and kits.

Claims

1. A compound according to Formula I ##STR00037## wherein R.sup.1 and R.sup.2 are independently selected from R.sup.3, OR.sup.3, SR.sup.3, SO.sub.3.sup.?, SO.sub.3R.sup.3, wherein R.sup.3 is selected from C.sub.1-C.sub.18 alkyl, and a polyethylene glycol (PEG) residue, Acc is selected from the group selected from Formula II ##STR00038## wherein X is selected from N(CH.sub.3), S, Se, O, and C(CH.sub.3).sub.2, ##STR00039## wherein optionally in each of Formula II to V an aromatic ring is substituted with 1, 2 or 3 SO.sub.3.sup.? groups, R.sup.4 is selected from the group of C.sub.1-C.sub.18 alkyl, C.sub.1-C.sub.6 cycloalkyl, and (CH.sub.2).sub.mSO.sub.3.sup.?, wherein m is an integer selected from 1 to 18, and n is selected from 1, 2 and 3, and suitable salts and solvates thereof.

2. The compound of Formula I according to claim 1, wherein R.sup.1 and R.sup.2 are independently selected from R.sup.3, OR.sup.3, SR.sup.3, SO.sub.3.sup.?, SO.sub.3R.sup.3, wherein R.sup.3 is selected from C.sub.1-C.sub.6 alkyl, and a polyethylene glycol (PEG) residue, Acc is Formula II ##STR00040## wherein X is selected from N(CH.sub.3), S, Se, O, and C(CH.sub.3).sub.2, optionally the aromatic ring is substituted with 1, 2 or 3 SO.sub.3.sup.? groups, R.sup.4 is selected from C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 cycloalkyl, and (CH.sub.2).sub.mSO.sub.3.sup.?, wherein m is an integer from 1 to 6, and n is 1, and suitable salts and solvates thereof.

3. The compound of Formula I according to claim 1, wherein R.sup.1 and R.sup.2 are independently selected from R.sup.3, OR.sup.3, SR.sup.3, SO.sub.3.sup.?, SO.sub.3R.sup.3, wherein R.sup.3 is selected from C.sub.1-C.sub.3 alkyl, and a polyethylene glycol (PEG) residue, Acc is Formula II ##STR00041## wherein X is C(CH.sub.3).sub.2, optionally the aromatic ring is substituted with 1, 2 or 3 SO.sub.3.sup.? groups, R.sup.4 is (CH.sub.2).sub.mSO.sub.3.sup.?, wherein m is an integer from 1 to 6, and n is 1, and suitable salts and solvates thereof.

4. The compound of Formula I according to the following formulae VI to IX ##STR00042## and suitable salts and solvates thereof.

5. A method for preparing a compound according to Formula I according to claim 1, comprising the steps of: a) suitably reacting a compound of Formula VI ##STR00043## wherein R.sup.1 and R.sup.2 are as defined in claim 1, and n is 1 or 2, with a compound of Formula II ##STR00044## or with a compound of formula III ##STR00045## or with a compound of Formula IV ##STR00046## or with a compound of Formula V ##STR00047## wherein in each of Formulae II to V optionally an aromatic ring is substituted with 1, 2 or 3 SO.sub.3.sup.? groups, R.sup.4 is selected from C.sub.1-C.sub.18 alkyl, C.sub.1-C.sub.6 cycloalkyl, and (CH.sub.2).sub.mSO.sub.3.sup.?, and wherein m is an integer from 1 to 18, to obtain a compound of Formula VIII ##STR00048## wherein R.sup.1, R.sup.2 and Acc are as defined in claim 1, and n is 1 or 2, and b) suitably reacting the compound of formula VIII with acryloyl chloride.

6. A method for detecting cysteine in a test sample, comprising the following steps of: a) measuring of UV/Vis absorbance of a solution of a compound as defined in claim 1 in a suitable solvent before and after being contacted with a prospectively cysteine-containing test sample, and b) determining the difference in absorbance by comparison of the UV/Vis spectra as measured in step a), and c) detecting cysteine in said test sample based on said difference in absorbance as determined in step b).

7. The method according to claim 5, wherein the UV/Vis absorbance is measured at discrete wavelengths in the range of from 200 nm to 1000 nm.

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

9. The method according to claim 6, wherein the solvent is an aqueous solvent.

10. The method according to claim 6, wherein said determining the difference in absorbance is by a visual inspection of a color change.

11. The method according to claim 6, wherein said determining the difference in absorbance is by Cedex

12. A kit for detecting cysteine in a test sample, comprising a vial or container comprising a predetermined quantity of a compound according to claim 1, together with a manual for using said kit.

13. (canceled)

Description

[0031] The present invention will now be described further in the following examples, and also with reference to the Figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.

[0032] FIG. 1 shows a scheme of the mechanism of reaction of acryloyl esters with Cys (ROH=merocyanine).

[0033] FIG. 2 shows the structure of probes as synthesized in the context of the present invention.

[0034] FIG. 3 shows the results of calibration in a feed medium (DMT118F.01 w/o Cys). SR: MF70 in DMSO/water (1:1). R1: 100 mM KPO.sub.4.

EXAMPLES

[0035] A series of acryloyl esters based on merocyanine chromophore was synthesized (FIG. 2), and was compared with probe LZ07 as 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). Spectral properties, aqueous solubility and stability were studied, and the response to Cys was evaluated. The inventors demonstrated that the chemical design according to the present invention provides dedicated probes for Cedex.

[0036] The following is a brief summary of the state of the art regarding know Cys-probes and their properties:

TABLE-US-00001 Probe Solvent Spectra Reference [00013] Assay EtOH/H2O (2:8, v/v) solution buffered at pH 7.4 (phosphate buffer, 20 mm) Yang, X.; Guo, Y.; Strongin, R. M. Angew. Chem. Int. Ed. 2011, 50, 10690. [00014] Assay EtOH : HEPES (1:9, pH 7.4, 0.01 M) Probe 775 nm With Cys 515 nm Guo, Z.; Nam, S.; Park, S.; Yoon, J. Chemical Science 2012, 3, 2760. [00015] Assay ethanol- phosphate buffer (20 mM, pH 7.4, 2:8 v/v) With Cys 490 nm Wang, H.; Zhou, G.; Gai, H.; Chen, X. Chem. Commun. 2012, 48, 8341. [00016] Probe 384 nm With Cys 512 nm Han, Q.; et al. Organic & Biomolecular Chemistry 2014, 12, 5023. [00017] Li, H.; Jin, L.; Kan, Y.; Yin, B. Sensors and Actuators B: Chemical 2014, 196, 546. [00018] Probe 387 nm With Cys 566 nm [00019] Probe 302, 358 nm With Cys 398 nm Lee, Y. H.; et al. Chem. Commun. 2015, 51, 14401. [00020] Probe 582 nm With Cys 674 nm Zhang, J.; et al. Anal. Chem. 2015, 87, 4856. [00021] Probe 608, 572 nm With Cys 710, 684, 632 nm Han, C.; et al. ACS Applied Materials & Interfaces 2015, 7, 27968. [00022] Probe 335 nm With Cys 372 nm Niu, W.; et al. Anal. Chem. 2016, 88, 1908. [00023] Probe 381 nm With Cys 557 nm Wang, J.; et al. ACS Sensors 2016. [00024] Stock solution DMSO Assay DMSO- PBS Probe 350 nm With Cys 445 nm Chen, C.; Zhou, L.; Huang, X.; Liu, W. Journal of Materials Chemistry B 2017, 5, 5892. [00025] PBS With Cys 460 nm Dai, X.; Kong, X.; Lin, W. Dyes and Pigments 2017, 142, 306. [00026] Stock solution DMSO Assay PBS buffer solution (10 mM, Probe 423 nm With Cys 335, 584 nm Feng, S.; et al. Dyes and Pigments 2017, 146, 103. pH 7.4, with 10% DMSO, v/v) [00027] Assay DMSO-PBS buffer solution (10.0 mM, pH = 7.4, 3:7 (v/v)) Probe 332 nm With Cys 450 nm, Fu, Z .-H.; et al. Anal. Chem. 2017, 89, 1937. [00028] Assay PBS containing 10% CH3CN Probe 315 nm With Cys 380 nm Kang, Y.-F.; et al. Aust. J. Chem. 2017, 70, 952. [00029] Assay PBS containing 1% DMSO Probe 409 nm With Cys 448 nm Liu, G.; et al. J. Talanta 2017, 170, 406. [00030] Assay phosphate bufferC2H5OH (pH 7.4, v/v, 1:1) Probe 400 nm With Cys 427 nm. Pang, L.; et al. Industrial & Engineering Chemistry Research 2017, 56, 7650. [00031] Assay 50 mM PBS solution (THF/water = 1:9, pH 7.4) Probe 360 nm With Cys 400 nm Shen, Y.; et al. Spectrochim. Acta Mol. Biomol. Spectrosc. 2017, 185, 371. [00032] Assay DMSO: H2O = 4:1 v/v, 10 mM HEPES buffer, pH = 7.4 Probe 386 nm With Cys 527 nm Manna, S.; et al. New J. Chem. 2018, 42, 4951. [00033] PBS buffer (10 mM, pH = 7.4, with 50% of DMSO, v/v) Probe 550 nm With Cys 600 nm Qi, Y.; et al. Anal. Chem. 2018, 90, 1014.

EXPERIMENTAL PROCEDURES

Materials and Methods

[0037] Reagents and solvents were purchased at the highest commercial quality from Sigma-Aldrich and used without further purification. CHROMASOLV solvents were used as eluents in HPLC. Yields refer to chromatographically (HPLC-MS) and spectroscopically (.sup.1H NMR) homogeneous material, unless otherwise stated. Counter anions are omitted for clarity.

Analytical HPLC-MS (ESI-MS)

[0038] The purity of the compounds was determined with the help of an HPLC-MS apparatus from Waters (Milford, USA) containing the following components: 2695 Separation module, 2696 photodiode array and Waters Micromass ZQ (ESCI ionization mode) detectors. Data acquisition was carried out by MassLynx (V4.1) software.

[0039] Column: YMC-Triart C18 3 ?M (4.6?150 mm)/Product Nr.TA12S03-1546WT.

[0040] Flow: 0.7 mL/min.

[0041] Phase A: triethylammonium acetate (TEAAc) buffer (10 mM, pH 7.0) in deionized water.

[0042] Phase B: MeCN.

[0043] Gradient 80: 5-80B (7 min); 80-80B (2 min); 80-5B (0.5 min); 5-5B (2.5 min).

[0044] Gradient 100: 5-100B (7 min); 100-100B (2 min); 100-5B (0.5 min); 5-5B (2.5 min).

NMR

[0045] NMR spectra were recorded on a Bruker Avance (500 and 600 MHz) and an Agilent 400 MR DD2 (400 MHz) instruments and were calibrated using residual non-deuterated solvent as an internal reference..sup.1 The following abbreviations were used to explain NMR peak multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad.

HRMS

[0046] For HRMS (high resolution mass spectra), samples were dissolved in MeCN and analyzed by direct-flow injection (injection volume=5 ?L) electrospray ionization time-of-flight (ESI-TOF) mass spectrometry on a Waters Q-ToF Premier instrument in the positive ion mode.

GENERAL PROCEDURE I

Preparation of Merocyanine Dyes

[0047] A mixture of a respective aldehyde (1 equiv.) and an indolium salt (1 equiv.) in ethanol was refluxed for 1-16 h in the presence of piperidine (0.1-2 equiv.) under Ar. The reaction mixture was allowed to cool slowly to rt, solvent was removed in vacuo and the residue was purified by reversed phase column chromatography (C-18, TEAB buffer (10 mM, pH 7.4)/MeCN or H.sub.2O (0.1% TFA)/MeCN).

GENERAL PROCEDURE II

Preparation of Acryloyl Esters

[0048] Acryloyl chloride (4-5 equiv.) was added to the mixture of a respective merocyanine dye (1 equiv.) and Et.sub.3N (4-5 eq.) in dry DCM at 0? C. under Ar. After 1 h of stirring at 0? C., the reaction was quenched by adding aqueous NH.sub.4Cl (0.1 M), and organic materials were extracted twice with DCM. The combined extracts were washed with NH.sub.4Cl (0.1 M), dried over Na2SO4, and concentrated in vacuo. The residue was purified by reversed phase column chromatography (C-18, H2O/MeCN).

GENERAL PROCEDURE III

Preparation of Acryloyl Esters

[0049] Acryloyl chloride (4-5 equiv.) was added to the mixture of a respective merocyanine dye (1 equiv.) and Et.sub.3N (4-5 eq.) in dry DCM at 0? C. under Ar. After 1 h of stirring at 0? C., the reaction was quenched by adding aqueous NH.sub.4Cl (0.1 M), and water-soluble product was extracted twice with H.sub.2O. The combined extracts were washed with DCM, concentrated in vacuo (20 mbar, 20? C.), then purified by reversed phase column chromatography (C-18, H.sub.2O/MeCN).

SPECTROSCOPIC MATERIALS AND METHODS

[0050] 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 following parameters: average time 0.05 s; data interval 1 nm; scan rate 1200 nm/min; with base line correction.

[0051] Solutions were prepared in 1.5 mL-vials (Eppendorf? microtubes 3810X) using Vortex Mixers.

[0052] Stock solutions of assayed compounds (2-5 mM) were prepared in H.sub.2O-DMSO (1:1), stored at ?20? C., and diluted to 1.0 mM with buffer before use. The L-Cys stock solution (20.0 mM) was freshly prepared in buffer before the measurements. HEPES buffer (25 mM, pH 7.4) was used for all measurements.

[0053] All aqueous solutions were made up in deionized water with resistivity?18 M? cm.sup.?1, obtained using a Millipore purification system (MQ-water)..sup.2

Extinction Coefficients

[0054] For a measurement, 1000 ?L of buffer and 1-50 ?L of probe (1.0 mM) were mixed, and then transferred to a cuvette. Absorbance spectra (250-800 nm) were measured against a blank of the buffer. At least six concentrations of each compound were used to calculate extinction coefficients from the slope of probe concentration vs absorbance plots, using MS Excel software (Microsoft).

[0055] In the same manner, ?.sup.Cys was determined from the solutions of probe reacted with an excess of Cys (100 ?M). The reactions were performed at 37? C. (incubation time 15 min). Blank reactions were run without addition of Cys.

Evaluation of Stability

[0056] For a measurement, 1000 ?L of buffer and 16 ?L of probe (1.0 mM) were mixed, and then incubated at +4? C. and at +37? C. for 5 h. The resulting mixtures were transferred to a cuvette, and the absorbance (250-800 nm) was measured. Each measurement was done in triplicate.

Evaluation of Solubility

[0057] In the experiments, 5-10 mg of dried material was suspended in 250-500 ?L of H2O at RT. The resulting suspension was centrifuged for 10 min at RT (16000 rcf). UV-vis of supernatant were recorded in buffer (25 mM HEPES pH 7.4) at RT. Each measurement was done in triplicate. The pellet was dried in vacuo for 16 h, and then weighed.

[0058] As mentioned above, the final products were obtained in a two-step synthesis (condensation and acrylation; Schemes 1 to 3). Overall yields 21-59%, except for 6% in case of MF65. The products were characterized by HPLC-MS, .sup.1H and .sup.13C NMR, and UV-Vis.

##STR00034##

##STR00035##

##STR00036##

UV-Vis and Cys Response

[0059] UV-vis of dyes as obtained was evaluated. Selected substituents on the benzene ring helped to increase the value for the initial merocyanine dye. Most remarkable results were observed for the preferred intermediate compounds MF56, MF57 and MF66.

TABLE-US-00002 TABLE 1 Spectral properties of dyes according to the present invention.* Entry ?.sub.max (? mM.sup.?1cm.sup.?1) nm LZ04 527 (36.6) (contr.) 527 (45.6)** LZ06 .sup.520 (27-37) I.sup.? not quant. (contr.) .sup.520 (37-53) I.sup.? not quant. MF51 550 (73.9) MF52 529 (70.1) MF53 548 (65.3) MF54 528 (44.9) MF56 582 (109.7) MF57 572 (106.3) MF58 514 (48.1, broad) MF66 556 (122.6) 556 (138.6)** MF67 546 (67.4) 546 (84.9)** MF68 575 (70.0) *UV-vis spectra were recorded in aqueous buffer (25 mM HEPES pH 7.4). The concentration of the probes was 1-24 ?M. At least six concentrations of each compound were used in the experiments. **The experiments were performed in the same buffer at pH 8.0.

[0060] Spectra recorded in the presence of Cys confirmed a colorimetric response through the cleavage of acryloyl ester. Probes as synthesized showed significant bathochromic shifts to the green and yellow range of the visible spectrum (Table 2). Moreover, their spectral profiles were advantageously fulfilling the wavelength requirement for the Cys sensing application in Cedex system (340, 378, 409, 480, 512, 520, 552, 583, 629, 652, 659 and 800 nm).

TABLE-US-00003 TABLE 2 Spectral properties and Cys-response screening of compounds according to the present invention.* ?.sub.max ?.sup.Cys (? mM.sup.?1cm.sup.?1) (?.sup.Cys mM.sup.?1cm.sup.?1) Entry nm nm SNR LZ05 (contr.) 527 (1.3), 389 (17.3) 527 (31.9) 12 LZ07 (contr.) 520 (0.6), 383 (16.9) 520 (31.5) 16 MF59 550 (0.7), 391 (16.7) 550 (17.9) 20 550 (52.9)** 44** MF60 530 (2.1), 409 (14.3) 529 (67.8) 14 MF61 548 (0.4), 397 (20.4) 549 (61.7) 36 MF62 582 (1.5), 419 (8.5), 582 (105.3) 20 287 (7.7) MF63 572 (2.9), 397 (14.0) 572 (93.4) 10 MF64 514 (0.7), 350 (8.0) 514 (32.1) 14 MF65 528 (1.2), 381 (17.7) 528 (44.3) 16 MF70 400 (21.9) 556 (23.6) 118 400 (16.6)* 556 (55.8)** 140** MF71 546 (0.6), 387 (29.1) 547 (33.7) 48 546 (0.8), 385 (25.7)* 547 (69.6)** 53** MF72 396 (14.2) 575 (31.0) 103 *UV-vis spectra were recorded before and after addition of an excess of Cys (100 ?M) in aqueous buffer (25 mM HEPES pH 7.4). The concentration of the probes was 1-24 ?M. The reactions with Cys were performed at 37? C. (incubation time 15 min). SNR was estimated from the values obtained after reaction with Cys (?.sup.Cys) and of blank measurements (?.sup.blank). At least six concentrations of each compound were used in the experiments. **The experiments were performed in the same buffer at pH 8.0.

Stability

[0061] The stability is another important parameter for the evaluation of colorimetric probes for biological assays. The probes for commercial applications have to be storable at 4? C. for several months. In addition, the stability has to be examined under assay conditions of Cedex Bio HT analyzer (37? C.).

[0062] The inventors qualitatively examined the stability of probes in buffer (10 mM HEPES pH 7.4) at 4? C. and 37? C. in order to simulate usual conditions of storage and assays. Solutions of each probe were monitored by UV-vis for 5 h. The results of all spectroscopic evaluations are summarized in Table 3.

TABLE-US-00004 TABLE 3 * Stability of compounds of the present invention Entry Hydrolysis @4? C., % Hydrolysis @37? C., % LZ05 (contr.) 8.4 ? 0.2 36.9 ? 0.4 LZ07 (contr.) 7.3 ? 0.1** 30.1 ? 0.2** MF59 1.2 ? 0.0 4.7 ? 0.1 MF60 9.8 ? 0.3 41.8 ? 0.9 MF61 4.8 ? 0.1 29.1 ? 1.9 MF62 7.6 ? 0.5 40.4 ? 0.6 MF63 9.8 ? 1.5 55.3 ? 0.7 MF64 8.9 ? 0.2 45.2 ? 1.4 MF65 7.2 ? 0.2 28.9 ? 1.4 MF70 0.2 ? 0.0 0.8 ? 0.0 MF71 1.0 ? 0.1 2.3 ? 0.2 MF72 0.8 ? 0.1 2.8 ? 0.1 * The experiments were performed in aqueous buffer (25 mM HEPES pH 7.4) at 4? C. and 37? C., with a concentration of 15.6 ?M (incubation time 5 h). The amount of hydrolyzed probe was determined using ? values derived from merocyanine analogues (Table 1). Each measurement was done in triplicate. **The amount of hydrolyzed probe was determined using ? value derived from Cys response screening (Table 2).

Solubility

[0063] The aqueous solubility of probes is another important property for the performance of compounds in biological assays. The inventors determined the solubility in water of the probes according to the invention using UV-vis. First, oversaturated mixtures of each probe were prepared. The mixtures were centrifuged, and then the concentrations in probes of supernatant were examined by UV-vis. To further validate the results, the concentrations were calculated from the isolated pellet weight. As seen in Table 4, the results as determined with both approaches are in the same range, and followed the same trend. Preferred probe MF70, containing two sulpho-groups, is highly water-soluble in comparison to its analogues LZ05 and MF59 containing only one sulpho-group. Also, the solubility of MF70 is superior to the compound LZ07 known from literature.

TABLE-US-00005 TABLE 4 * Solubility. Entry Solubility (by UV-vis), mM Solubility (by weight), mM LZ05 (contr.) 11.0 ? 1.0 12 LZ07 (contr.) 15.4 ? 1.5 17 MF59 5.8 ? 0.2 2 MF70 ?41 ?47 *In the experiments 5-10 mg of dried material was suspended in 250-500 ?L of H.sub.2O at RT. The UV-vis of supernatants was recorded in buffer (25 mM HEPES pH 7.4) at RT. Each measurement was done in triplicate. Probes were used as obtained from synthesis.

Probe MF70 Performance in Cedex Bio HT

[0064] The rapid kinetic profile, stability and low background signal as obtained were encouraging to further use MF70 for Cys-sensing in Cedex Bio HT. Reagent solution was treated with various Cys-concentrations (0.5-7.6 mM) in a feed medium, which is used for monoclonal antibody production. As shown in FIG. 3, progressively enhanced absorbance was observed with the increasing amount of Cys. Under these conditions, a reliable response was obtained over the period of one week (Table 5), with corresponding detection limit of 3.6 ?M Cys, and limit of blank 2.2 ?M Cys.

TABLE-US-00006 TABLE 1 Spike-recovery in a feed medium (DMT118F.01 with Cys). Spiked Cys, mM n (samples) Found, mM CV, % Recovery, % 0.5 21 0.64 1 96 1.5 21 1.64 1 99 3.0 21 3.03 1 96 4.5 21 4.38 1 94 6.0 21 5.83 1 94

[0065] In conclusion, the probes according to the present invention, and preferably probe MF70 meet the requirements for commercial assays. The merocyanine dye scaffold ensures bright chromogenic signal. Methyl groups in the ortho-position and sulfonic acid groups seem to secure stability against hydrolysis and aqueous solubility, respectively.