KR20120061576A - Coumarin-based compound having cysteine selectivity, preparation method of the same, chemodosimeter using the same - Google Patents

Coumarin-based compound having cysteine selectivity, preparation method of the same, chemodosimeter using the same Download PDF

Info

Publication number
KR20120061576A
KR20120061576A KR1020100122923A KR20100122923A KR20120061576A KR 20120061576 A KR20120061576 A KR 20120061576A KR 1020100122923 A KR1020100122923 A KR 1020100122923A KR 20100122923 A KR20100122923 A KR 20100122923A KR 20120061576 A KR20120061576 A KR 20120061576A
Authority
KR
South Korea
Prior art keywords
cysteine
compound
coumarin
formula
present
Prior art date
Application number
KR1020100122923A
Other languages
Korean (ko)
Other versions
KR101337434B1 (en
Inventor
김종승
Original Assignee
고려대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 고려대학교 산학협력단 filed Critical 고려대학교 산학협력단
Priority to KR20100122923A priority Critical patent/KR101337434B1/en
Publication of KR20120061576A publication Critical patent/KR20120061576A/en
Application granted granted Critical
Publication of KR101337434B1 publication Critical patent/KR101337434B1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/16—Peri-condensed systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Hematology (AREA)
  • General Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)

Abstract

The present invention relates to a coumarin derivative of formula (1) having a cysteine selectivity, a method for preparing the same, and a cysteine detection system using the same, wherein the coumarin derivative represented by formula (1) according to the present invention is structurally similar in biomolecules. Cysteine can be selectively detected even when homocysteine and glutathione are mixed.

Figure pat00010
(One)

Description

Coumarin derivatives having cysteine selectivity, preparation methods thereof, and cysteine detection systems using the same {COUMARIN-BASED COMPOUND HAVING CYSTEINE SELECTIVITY, PREPARATION METHOD OF THE SAME, CHEMODOSIMETER USING THE SAME}

The present invention relates to a coumarin derivative having cysteine selectivity, a method for preparing the same, a method for detecting cysteine and a chemosimeter using the same, and more particularly, a coumarin derivative having excellent cysteine selectivity in vivo, a method for preparing the same It relates to a cysteine detection system.

Thiols play an important role in cellular antioxidant defense systems. Of the biomolecules containing thiols, cysteine (Cys) is of particular interest. Cysteine, the major amino acid of proteins, plays an important role in maintaining biological redox homeostasis through equilibrium at a given potential between reduced free thiol (RSH) and oxidized disulfide (RSSR). In addition, abnormal levels of cysteine are known to be associated with human diseases such as low growth, liver damage, skin disorders, Alzheimer's disease, cardiovascular disease and coronary heart disease.

Fluorescent probes have inherent advantages over other types of probes such as high sensitivity, specificity, simplified implementation and real-time monitoring capability due to fast response time. Therefore, considerable efforts have been made to develop fluorescent probes, particularly fluorescent probes for detecting thiol systems comprising cysteines in biomolecules.

However, most of these efforts have been difficult to apply due to their interference with other biological analytes, low water solubility, narrow pH range and slow response under physiological conditions. In particular, the distinction of the thiol groups of cysteine from two other important thiols, homocysteine (Hcy) and glutathione (GSH) in biological systems, has been a difficult task in the field of chemical quantitative thiol detection.

Recently, ketocoumarin-based thiol detection probes have been reported that feature 1,4-addition of thiols to α, β-unsaturated ketones based on intramolecular charge transfer (ICT). However, there is a need for development of probes capable of selectively detecting cysteine through electrostatic or steric adjustment in any biological system.

The problem to be solved by the present invention is to provide a coumarin derivative that can effectively detect cysteine in vivo, its preparation method, a cysteine detection method and a cysteine detection system using the same.

The present invention provides coumarin derivatives of formula (1) having cysteine selectivity:

Figure pat00001
(One)

The present invention also provides a process for preparing coumarin derivatives of formula (1) according to Scheme 1 below:

Scheme 1

Figure pat00002

In the above Reaction Scheme 1,

(Iii) CH 2 (COOC 2 H 5 ) 2 , HCl / AcOH; (Ii) is POCl 3 , DMF; And (iii) are reaction conditions of diethyl malonate and DCM.

In another aspect of the present invention, a method of selectively detecting cysteine using a coumarin derivative of formula (1) is provided. This detection can be carried out using the fluorescence and absorption spectrum change by the binding of the coumarin derivative of the formula (1) and cysteine. In particular, cysteine detection using the coumarin derivative of the formula (1) according to the present invention can be carried out in an aqueous solution, it is possible to detect in vivo.

On the other hand, as another aspect of the present invention, a cysteine detection system using a coumarin derivative of the formula (1), specifically a chemo dosimeter may be provided.

Since the coumarin derivative represented by the formula (1) according to the present invention has excellent cysteine selectivity, cysteine can be effectively detected in an aqueous solution such as in vivo. In particular, even when structurally similar homocysteine and glutathione are mixed in a living body, cysteine can be selectively detected, which is very useful as a chemodometer.

1 is a molecular structure showing a fluorescence change when the coumarin derivative according to the present invention reacts with cysteine.
2 is a graph showing (a) UV-Vis spectrum and (b) fluorescence spectrum of coumarin derivatives according to one embodiment of the present invention.
Figure 3 is a molecular model showing the structure of the first excited state of coumarin derivatives, coumarin derivatives-cysteine and coumarin derivatives as one embodiment of the present invention.
Figure 4 is a molecular model showing the HOMO and LUMO of coumarin derivatives and coumarin derivative-cysteine as one embodiment of the present invention.
5 is a graph showing fluorescence changes according to reaction of coumarin derivatives with Cys, Hcy and GSH as one embodiment of the present invention.
6 is a graph showing fluorescence spectra of coumarin derivatives in the presence of various amino acids, metals, ROS and glucose as one embodiment of the present invention.
7 is a diagram showing a fluorescence-enhancement factor (FEF) of coumarin derivatives in the presence of various amino acids, metals, ROS and glucose as one embodiment of the present invention.
8 is a graph showing chromatograms of coumarin derivatives in the presence of Cys, Hcy and GSH as one embodiment of the present invention.
9 is an image showing the results of confocal microscopy analysis of HepG2 cells treated with coumarin derivatives as one embodiment of the present invention.
10 is a graph showing chromatograms of coumarin derivatives in the presence of a metabolite sample as one embodiment of the invention.
11 is a graph showing UV-Vis (10 μM) spectra of coumarin derivatives as one embodiment of the present invention.
FIG. 12 shows fluorescence intensities of coumarin derivatives, coumarin derivatives + cysteine (cysteine, Cys), coumarin derivatives + homocysteine (homocycteine, Hcy) and coumarin derivatives + glutathione (GSH) as one embodiment of the present invention. It is a graph.
FIG. 13 is a graph showing UV-Vis (10 μM) spectrum and fluorescence intensity of cysteine-added coumarin derivatives as one embodiment of the present invention.
14 is a graph showing a correlation between fluorescence intensity and cysteine concentration as one embodiment of the present invention.
15 is a graph showing a standard response of a fluorescence signal to a change in cysteine concentration as one embodiment of the present invention.
FIG. 16 is a graph showing fluorescence intensity versus pH of a coumarin derivative including cysteine and a coumarin derivative not containing cysteine as one embodiment of the present invention.
17 is a photograph showing fluorescence using a UV lamp of a coumarin derivative and a coumarin derivative in which Cys, Hcy, GSH or Cys + Hcy + GSH is combined as one embodiment of the present invention.
18 is a graph showing the absorbance against time of a coumarin derivative in the presence of cysteine as one embodiment of the present invention.
19 is a graph showing ln2 / t1 versus cysteine concentration as one embodiment of the present invention.
20 is a diagram showing molecular orbitals of coumarin derivatives and coumarin derivatives-cysteine as one embodiment of the present invention.
FIG. 21 is a graph showing LC-MS spectra of (a) coumarin derivatives, (b) coumarin derivatives + Cys, (c) coumarin derivatives + Hcy, and (d) coumarin derivatives + GSH as one embodiment of the present invention.
22 is a graph showing chromatograms of coumarin derivatives to which a protein sample is added as one embodiment of the present invention.
Figure 23 is a graph showing the FAB-MS results of coumarin derivatives according to one embodiment of the present invention.
24 is a graph showing FAB-MS results of coumarin derivative-cysteine according to one embodiment of the present invention.
25 is a graph showing 1 H-NMR results of coumarin derivatives according to one embodiment of the present invention.
26 is a graph showing 13 C-NMR results of a coumarin derivative according to one embodiment of the present invention.
27 is a graph showing HSQC results of coumarin derivatives according to one embodiment of the present invention.
28 is a graph showing 1 H NMR change when cysteine is added to a coumarin derivative as one embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples and drawings.

Coumarin derivatives according to the present invention are represented by the following formula (1).

Figure pat00003
(One)

The coumarin derivative represented by Formula (1) according to the present invention may exhibit strong fluorescence by binding to cysteine in a biomolecule, and thus has the characteristic of effectively detecting cysteine.

The coumarin derivative represented by the formula (1) according to the present invention can be prepared by the condensation reaction of coumarin aldehyde and diethylmalonate. More specifically, the coumarin derivative represented by Formula (1) may be prepared by the following Scheme 1.

Scheme 1

Figure pat00004

In the above Reaction Scheme 1,

(Iii) CH 2 (COOC 2 H 5 ) 2 , HCl / AcOH; (Ii) is POCl 3 , DMF; And (iii) are reaction conditions of diethyl malonate and DCM.

The present invention also provides a method for selectively detecting cysteine in a biomolecule using a coumarin derivative represented by the formula (1), and a chemodometer using the same.

The coumarin derivative represented by the formula (1) according to the present invention can effectively detect cysteine even when structurally similar homocysteine and glutathione are mixed in a biomolecule. In addition, from the TDDFT calculation, it can be seen that the fluorescence amplification for the cysteine reaction of the coumarin derivative represented by the formula (1) according to the present invention is due to ICT blocking.

In addition, the coumarin derivative represented by the formula (1) according to the present invention can be more usefully used as an effective cysteine detection probe by a confocal laser scanning microscope.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided by way of example only to more easily understand the present invention, and the scope of the present invention should not be construed as being limited thereto.

Synthetic example Coumarin derivatives (1)

All fluorescence and UV / Vis absorption spectra were measured using Shimadzu RF-5301PC and Shinco S-3100 spectrophotometer, respectively. NMR and mass spectra were measured using a Varian instrument (400 MHz) and JMS-700 MStation mass spectrometer, respectively. Infrared spectra were measured from KBr windows using a Bomen MB-104 spectrometer. All analytes were purchased from Aldrich, solvent was purchased from Duksan Pure Chemical Co., Ltd., and CH 3 CN for spectral detection was used with HPLC reagent.

 Scheme 1

Figure pat00005

In the above Reaction Scheme 1,

(Iii) CH 2 (COOC 2 H 5 ) 2 , HCl / AcOH; (Ii) is POCl 3 , DMF; And (iii) are reaction conditions of diethyl malonate and DCM.

Coumarinaldehyde (Coumarinaldehyde, 135 mg, 0.5 mmol) and diethyl malonate (diethyl malonate, 96 mg, 0.6 mmol) were dissolved in 5 mL of DCM, and then piperidine (25 μL, 0.27 mmol) was added to the solution. The reaction mixture was stirred at room temperature for 5 hours. The mixture was extracted with DCM and dried over anhydrous magnesium sulfate. Thereafter, the dried reagent was filtered and the organic solution was evaporated under reduced pressure. The residue was purified by column chromatography on silica (n-hexane / EtOAc, 1: 1 v / v) to give compound 1 as a red solid.

Mp: 178-180 ° C .; R f = 0.3;

1 H NMR (400 MHz, CDCl 3 ): δ 7.79 (s, 1H), 7.68 (s, 1H), 6.82 (s, 1H), 4.39-4.33 (q, J = 7.19 Hz, 2H), 4.31-4.25 (q, J = 7.19 Hz, 2H), 3.36-3.29 (q, J = 6.01 Hz, 4H), 2.87-2.82 (t, J = 6.33 Hz, 2H), 2.76-2.71 (t, J = 6.33 Hz, 2H), 2.00-1.92 (m, 4H), 1.35-1.30 (m, 6H);

13 C NMR (100 MHz, CDCl 3 ): 166.8, 164.5, 160.9, 152.0, 147.7, 143.9, 136.7, 126.3, 124.0, 119.2, 111.6, 108.2, 106.0, 64.5, 61.3, 50.1, 49.7, 27.3, 21.1, 20.1 , 20.0, 14.1, 14.0;

IR (film): ν max 2928, 2851, 1717, 1619, 1587, 1557, 1520, 1446, 1365, 1344, 1286, 1247, 1201, 1067, 1034, 858, 759 cm −1 ;

FAB-MS calc. for C 23 H 22 N 2 O 2 S [M + H] + 412.17, found 411.5.

Example  1: Cysteine Detection Experiment

Fluorescence-activated cysteine detection coumarin derivatives and their crystal structures are shown in FIG. 1.

As shown in FIG. 1, cysteine may react with β-carbon of α, β-unsaturated diester and 1,4-addition of Michael type to generate Compound 1-Cys. Detailed NMR spectra including 2D Heteronuclear Single Quantum Coherence (HSQC) for Compound 1 and Compound 1-Cys products are shown in FIGS. 27 and 28 below. The quantum signals of H 1 -H 3 of Compound 1 were found to be 7.65, 7.75 and 6.99 ppm, respectively. Thereafter, 1 H-NMR spectrum of Compound 1 was observed by adding cysteine at room temperature with D 2 O / CD 3 CN (1: 1). When cysteine was added, vinyl protons (H 1 ) disappeared at 7.65 ppm and four new peaks appeared at 4.35, 4.27, 4.26 and 4.23 ppm, indicating the re- or β-carbon reactivity of the compound 1 of the cysteine thiol group The si-face may be due to the presence of two diastereoisomers derived from nucleophilic attack.

During the 1,4-addition reaction of compound 1, the conjugation of the coumarin ring system to the α, β-unsaturated carbonyl group is impaired, resulting in changes in UV / Vis and fluorescence (see FIG. 2). In fact, the addition of cysteine to Compound 1 results in a 55 nm blue shift with a maximum absorption spectrum of λ ab = 426 nm, a color change from dark orange to green, with a maximum fluorescence of λ em = 499 nm (see Table 1). In contrast, Compound 1 exhibits non-fluorescence due to intactolecular charge transfer (ICT) from the fluorescent receptor (coumarin) near the conjugated diester (see Table 1).

In order to understand the mechanism of fluorescence enhancement during the formation of compound 1-cysteine products, density functional theory (DFT) calculations were performed with a 6-31G * base set using a Gaussian 03 program. The optimized structure of compound 1 and compound 1-cysteine is shown in FIG. 3.

Compound 1 comprises a bridge (-C = C-) conjugated between coumarin and diester groups, while compound 1-cysteine includes a saturated bridge (-C-C-). One of the two ester groups of compound 1 is located on the same plane as the coumarin ring system differently from compound 1-cysteine. As shown in FIG. 1, the calculated structure of Compound 1 is in good agreement with the experimental crystal structure. This structural difference expects a strong ICT process in Compound 1 and a weak ICT process in Compound 1-cysteine.

In addition, as shown in FIG. 3, the optimized structure for the first excited state of compound 1 exhibits a planar geometry with a coumarin plane larger than compound 1. Therefore, it is estimated that ICT is prominent in an excited state, and it suppresses fluorescence emission from compound 1.

(A) UV-Vis (10 μM) and (b) fluorescence spectra of cysteine at various concentrations in Compound 1 (5 μM) in 10% aqueous solution of CH 3 CN (pH 7.4, 10 mM PBS buffer) are shown in FIG. 2. . The optimized structure of Compound 1, Compound 1-Cys, and the first excited state of Compound 1 is shown in Figure 3 below.

Details relating to fluorescence enhancement in the formation of Compound 1-Cys can be obtained from time-dependent density functional theory (TDDFT) calculations. The calculated wavelength of the excited state of Compound 1-Cys was 355 nm, and 56 nm of blue shifted from Compound 1 (411 nm). The blue shift value was in good agreement with the experimentally observed blue shift (55 nm).

HOMO and LUMO of Compound 1 and Compound 1-Cys are shown in FIG. 4. The HOMO to LUMO transition contributed 100 and 97.4% to the excited states of Compound 1 and Compound 1-Cys, respectively. Thus, LUMO exhibits an electron density distribution when exclusively excited. From LUMO, ICT is carried out via bridges conjugated between coumarin and diesters in compound 1, and is inhibited mainly due to beaconjugated bridges in compound 1-Cys. These results strongly support the conjecture that the strong fluorescence emission from Compound 1-Cys is mainly due to the relatively weakened ICT process compared to Compound 1.

Comparison of the reaction of Compound 1 with Cys, Hcy and GSH was performed by monitoring the fluorescence change of the reaction mixture in aqueous solution (10 mM PBS buffer, pH 7.4, 10% CH 3 CN), which is shown in FIG. 5 below.

Fluorescence intensity over time in the mixture of Compound 1 and Cys showed the result of a single exponential function. Hcy and GSH also increased the fluorescence intensity, but the rate was very low. Indeed, kinetic analysis based on a single exponential decay model showed that the reaction of Compound 1 and Cys was 5.7 times faster (see FIGS. 18, 19 and Table 2). These results indicate that Compound 1 is selective for cysteine by covering a biologically important thiol and thus can be used as a selective sensor of cysteine in biological systems including cells.

The fluorescence intensity was proportional to the proportional constant R = 0.99557 depending on the amount of cysteine added as μM level (see FIG. 14), and the detection limit was reduced to 6.47 × 10 −7 M (see FIG. 15). Nevertheless, as shown in FIG. 16, the fluorescent reaction of Compound 1 with cysteine was pH independent at pH 6-11. These results indicate that the fluorescent reaction of Compound 1 can be usefully used for the selective and sensitive detection of cysteine in biological systems.

Fluorescence spectra of compound 1 (5 μM) in 10% aqueous solution of CH 3 CN (pH 7.4, 10 mM PBS buffer) in the presence of various amino acids, metal ions, ROS and glucose are shown in FIG. 6.

Amino acid (Hcy, GSH, Ala, Arg, Asn, Asp, Gln, Gluc, Glu, Gly, His, Ile, Leu, Lys, Met, Phe in 10% aqueous solution of CH 3 CN (pH 7.4, 10 mM PBS buffer) , Pro, Ser, Tau, Thr, Trp, Tyr, Val), metal ions (K + , Ca 2 + , Mg 2 + , Na + , Zn 2 + , Fe 2 + , Fe 3 + ), ROS (H 2 Fluorescence-enhancement factor (FEF) for cysteine of compound 1 (5 μM) in the presence of O 2 , NADH) and glucose is shown in FIG. 7.

For further study of the selective cysteine detectability in biological media, fluorescence spectral changes from the reaction of Compound 1 with various biological analytes were observed in aqueous solution (10 mM PBS buffer, pH 7.4, 10% CH 3 CN). Compound 1 contains other amino acids (Ala, Arg, Asn, Asp, Gln, Gluc, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Tau, Thr, Trp, Tyr, Val), biological Common metal ions (K +, Ca 2 +, Mg 2 +, Na +, Zn 2 +, Fe 2 +, Fe 3 +), ROS in cysteine than (H 2 O 2, NADH), and glucose stronger fluorescence intensity Appear (see FIGS. 6 and 11). In contrast, the nucleophilic thiols of Hcy and GSH showed relatively minor changes in fluorescence intensity.

As described above, it can be seen that the coumarin derivative represented by the formula (1) according to the present invention shows surprising selectivity to cysteine.

Regarding the cysteine selectivity of compound 1, there is a strong H-bond between zwitterionic NH and coumarin carbonyl oxygen atom under neutral pH, which leads to the Hcy reaction of the Michael reaction of compound 1 with cysteine. And faster than GSH (see FIG. 1). In addition, cysteine may have an improved selectivity for cysteine of Compound 1 due to its steric effect, which is relatively less steric hindrance than Hcy and GSH.

An LC / MS spectrometer was used to observe the selectivity of the cysteine of Compound 1 over Hcy and GSH. The chromatogram of Compound 1 (10 μM) under addition of Cys, Hcy and GSH in 10% aqueous CH 3 CN solution (pH 7.4, 10 mM PBS buffer) is shown in FIG. 8.

Confocal microscopic analysis of HepG2 cells treated with Compound 1 is shown in FIG. 9. 9 shows N-ethylmaleimide (NEM) dependent fluorescence changes in the human hepatoma line, a HepG2 cell line. Fluorescence intensity decreased with the addition of NEM, meaning that cysteine is responsible for fluorescence enhancement in cells by compound 1 because NEM is a species reactive to thiols inside cells.

For LC-MS spectroscopic studies, a mixture of cytosolic proteins and metabolites was obtained from cell lysis, small metabolites were removed using dialysis, and acetone precipitation was used. Macromolecules were removed. Upon reaction with compound 1, LC-MS analysis was performed and the results of the LC-MS profiles are shown in FIG. 10 below. Although GSH was the most thiol species in cells, the main fluorescent product was Cys. Therefore, it can be seen that the coumarin derivative represented by the formula (1) according to the present invention can be very useful for the detection of cysteine in a biological system.

The chromatogram of Compound 1 (10 μM) with the addition of a metabolite sample in 10% aqueous CH 3 CN solution (pH 7.4, 10 mM PBS buffer) is shown in FIG. 10. Figure 10 (a) shows the total ion chromatogram in the cation mode, (b) shows the chromatogram monitoring the selected ions.

UV-Vis of compound 1 in 10% aqueous CH 3 CN solution (pH 7.4, 10 mM PBS buffer) after 5 minutes of 440 nm excitation in the presence of various amino acids, metals, ROS, glucose (100 equiv each) (10 μM) spectrum is shown in FIG. 11.

For concentrations of Compound 1 (5 μM), Compound 1 + cysteine (cysteine, Cys), Compound 1 + homocysteine (Hcy) and Compound 1 + glutathione (GSH) in an excited state of 440 nm (λ em = 499 nm) Fluorescence intensity is shown in FIG. 12.

The UV-Vis (10 μM) spectrum and fluorescence intensity of the compound 1 to which cysteine was added in 10% aqueous solution of CH 3 CN (pH 7.4, 10 mM PBS buffer) are shown in FIG. 13, and the correlation between fluorescence intensity and cysteine concentration was shown in FIG. 13. Is shown in FIG. 14. In addition, the standard response of the fluorescence signal to the change in cysteine concentration is shown in FIG. 15, and the compound 1 containing cysteine and compound 1 not containing cysteine in 10% aqueous solution of CH 3 CN Fluorescence intensity against pH is shown in FIG. 16.

The photophysical results (absorption spectra, emission spectra, relative quantum efficiencies, etc.) of Compound 1 combined with various amino acids such as Compound 1 and Cysteine are shown in Table 1 below.

compound Absorption spectrum
(λ max , nm) Emission spectrum
(λ max , nm) Relative quantum efficiency
(Φ f ) Compound 1 481 559 0.0056 Compound 1-Cys 426 499 0.3268 Compound 1-Hcy 481 500 0.0823 Compound 1-GSH 481 501 0.0464 Compound 1
All other analytes 481 558 0.0082

In Table 1, the relative quantum efficiency (Φ f ) was based on 0.85, which is a value of fluorescein in 0.1N NaOH. In addition, all other analytes include amino acids (Ala, Arg, Asn, Asp, Gln, Gluc, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Tau, Thr, Trp, Tyr and Val), refers to a metal (K +, Ca 2 +, Mg 2 +, Na +, Zn 2 +, Fe 2 + and Fe 3 +), ROS (H 2 O 2, NADH) , and glucose.

Fluorescence using a UV lamp of Compound 1 in which Cys, Hcy, GSH or Cys + Hcy + GSH is combined with Compound 1 (5 μM) in a 10% aqueous solution of CH 3 CN (pH 7.4, 10 mM PBS buffer) is shown in FIG. 17. It was.

The absorbance over time of compound 1 (10 μM) in the presence of cysteine in 10% aqueous solution of CH 3 CN (pH 7.4, PBS buffer) is shown in FIG. 18. In FIG. 18, the results of the dynamic analysis are shown as the first attenuation model.

Ln2 / t1 for cysteine concentration is shown in FIG. 19. The lifetime of the compound in the presence of each cysteine is represented by t1 and kinetic experiments were performed in 10% aqueous CH 3 CN solution (pH 7.4, PBS buffer).

Kinetic parameters for the reaction of compound 1 with Cys, Hcy and GSH are shown in Table 2 below.

Speed constant
(rate constant, / Msec) Error 1-Cys 1.95 0.13 1-Hcy 0.327 0.024 1-GSH 0.342 0.013

Excitation characteristics and λ max experimental values calculated by TDDFT (time-dependent density functional theory) are shown in Table 3 below.

Theoretical value
(λ max , nm) Excitation Experimental value
(λ max , nm) Compound 1 411 HOMO → LUMO (100%) 481 Compound 1-Cys 355 HOMO-1 → LUMO + 2 (2.6%)
HOMO → LUMO (97.4%) 426

The molecular orbitals of Compound 1 and Compound 1-cysteine are shown in FIG. 20.

LC-MS spectra of (a) Compound 1, (b) Compound 1 + Cys, (c) Compound 1 + Hcy, and (d) Compound 1 + GSH in cationic mode are shown in FIG. 21.

The chromatogram of Compound 1 (10 μΜ) with protein samples added in 10% aqueous CH 3 CN solution (pH 7.4, 10 mM PBS buffer) is shown in FIG. 22. Figure 22 (a) shows the total ion chromatogram of the protein in the cation mode, (b) is a chromatogram monitoring each selected ion.

The FAB-MS results of the compound 1 are shown in FIG. 23, and the FAB-MS results of the compound 1-cysteine are shown in FIG. 24.

Also, CDCl 3 within the compound showed the 1 H NMR spectrum (400MHz) of 1 (10mM) in Figure 25, showed a 13 C NMR spectrum (100MHz) in CDCl 3 in compound 1 in Figure 26, CDCl 3 in compound 1 The HSQC spectrum of (100MHz) is shown in FIG. 27.

In addition, 1 H NMR change when cysteine was added at various concentrations to Compound 1 (5 mM) in D 2 O / CD 3 CN (1: 1) at room temperature is shown in FIG. 28.

In conclusion, the coumarin derivative represented by formula (1) according to the present invention can detect cysteine more effectively and selectively than homocysteine and glutathione which are structurally similar in biomolecules.

From the TDDFT calculation, it can be seen that the fluorescence amplification for the cysteine reaction of the coumarin derivative represented by the formula (1) according to the present invention is due to ICT blocking.

Therefore, the coumarin derivative represented by the formula (1) according to the present invention can be more usefully used as an effective cysteine detection probe by a confocal laser scanning microscope.

Claims (8)

Coumarin derivatives of formula (1) having cysteine selectivity:
Figure pat00006
(One) To prepare a coumarin derivative of formula (1) according to (Scheme 1):
[Reaction Scheme 1]
Figure pat00007

Where
(Iii) CH 2 (COOC 2 H 5 ) 2 , HCl / AcOH; (Ii) is POCl 3 , DMF; And (iii) is diethyl malonate, DCM. A method for selectively detecting cysteine using a coumarin derivative of formula (1)
Figure pat00008
(One) The method of claim 3,
A method for selectively detecting cysteine characterized by using a fluorescence and absorption spectrum change by combining a coumarin derivative of the formula (1) with cysteine. The method of claim 3,
A method for selectively detecting cysteine, wherein cysteine detection using the coumarin derivative of the formula (1) is performed in an aqueous solution. The method of claim 3,
A method for selectively detecting cysteine, wherein cysteine detection using the coumarin derivative of the formula (1) is performed in vivo. Cysteine detection system using the coumarin derivative of formula (1).
Figure pat00009
(One) The method of claim 7, wherein
A cysteine detection system, characterized in that it is a fluorescent chemosimeter.
KR20100122923A 2010-12-03 2010-12-03 Coumarin-based compound having cysteine selectivity, preparation method of the same, chemodosimeter using the same KR101337434B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR20100122923A KR101337434B1 (en) 2010-12-03 2010-12-03 Coumarin-based compound having cysteine selectivity, preparation method of the same, chemodosimeter using the same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR20100122923A KR101337434B1 (en) 2010-12-03 2010-12-03 Coumarin-based compound having cysteine selectivity, preparation method of the same, chemodosimeter using the same

Publications (2)

Publication Number Publication Date
KR20120061576A true KR20120061576A (en) 2012-06-13
KR101337434B1 KR101337434B1 (en) 2013-12-06

Family

ID=46612101

Family Applications (1)

Application Number Title Priority Date Filing Date
KR20100122923A KR101337434B1 (en) 2010-12-03 2010-12-03 Coumarin-based compound having cysteine selectivity, preparation method of the same, chemodosimeter using the same

Country Status (1)

Country Link
KR (1) KR101337434B1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101255261B1 (en) * 2011-10-25 2013-04-16 고려대학교 산학협력단 Cysteine-targeting chemodosimetric fluorescent probe, method for preparing the same, and method for bioimaging of cysteine using the same
CN108727326A (en) * 2018-07-06 2018-11-02 广西师范学院 Identify fluorescence probe and preparation method and the application of cysteine and glutathione
CN110894193A (en) * 2018-09-13 2020-03-20 南京大学 Synthesis of novel fluorescent probe and application of novel fluorescent probe in cysteine detection

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101862581B1 (en) 2014-11-19 2018-07-13 메타볼랩(주) Real-time Imaging Sensor for Measuring Cellular Thiol Levels
KR102249148B1 (en) 2019-12-27 2021-05-10 영남대학교 산학협력단 Electrochemically reduced graphene oxide modified with cupper and phenanthroline derivatives for cysteine detection and its applications to sensor-electrodes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4929452B2 (en) * 2006-02-06 2012-05-09 国立大学法人 東京大学 New coumarin derivatives

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101255261B1 (en) * 2011-10-25 2013-04-16 고려대학교 산학협력단 Cysteine-targeting chemodosimetric fluorescent probe, method for preparing the same, and method for bioimaging of cysteine using the same
CN108727326A (en) * 2018-07-06 2018-11-02 广西师范学院 Identify fluorescence probe and preparation method and the application of cysteine and glutathione
CN108727326B (en) * 2018-07-06 2021-12-21 南宁师范大学 Fluorescent probe for identifying cysteine and glutathione and preparation method and application thereof
CN110894193A (en) * 2018-09-13 2020-03-20 南京大学 Synthesis of novel fluorescent probe and application of novel fluorescent probe in cysteine detection
CN110894193B (en) * 2018-09-13 2022-04-22 南京大学 Synthesis of novel fluorescent probe and application of novel fluorescent probe in cysteine detection

Also Published As

Publication number Publication date
KR101337434B1 (en) 2013-12-06

Similar Documents

Publication Publication Date Title
Sheng et al. A highly selective ESIPT-based fluorescent probe with a large Stokes shift for the turn-on detection of cysteine and its application in living cells
Hirayama et al. Chemical tools for detecting Fe ions
Xu et al. Ultrasensitive near-infrared fluorescence-enhanced probe for discriminative detection of GSH and Cys from different emission channels
Wang et al. An iminofluorescein-Cu2+ ensemble probe for selective detection of thiols
Noh et al. Solvent-dependent selective fluorescence assay of aluminum and gallium ions using julolidine-based probe
Huo et al. A turn-on green fluorescent thiol probe based on the 1, 2-addition reaction and its application for bioimaging
Wang et al. A fluorescent sensor bearing nitroolefin moiety for the detection of thiols and its biological imaging
Qiu et al. A selective and sensitive fluorescent probe for homocysteine and its application in living cells
Liu et al. A triphenylamine as a fluorophore and maleimide as a bonding group selective turn-on fluorescent imaging probe for thiols
Wang et al. Thiazolyl substituted NBD as fluorescent probe for the detection of homocysteine and cysteine
Han et al. A colorimetric and ratiometric fluorescent probe for distinguishing cysteine from biothiols in water and living cells
KR101337434B1 (en) Coumarin-based compound having cysteine selectivity, preparation method of the same, chemodosimeter using the same
Yang et al. BODIPY-based fluorescent probe for cysteine detection and its applications in food analysis, test strips and biological imaging
Wei et al. A TICT based two-photon fluorescent probe for cysteine and homocysteine in living cells
Meng et al. A ratiometric two-photon fluorescent probe for cysteine and homocysteine in living cells
Hou et al. A colorimetric and red emissive fluorescent probe for cysteine and its application in bioimaging
Liu et al. A squaraine-based red emission off–on chemosensor for biothiols and its application in living cells imaging
Kang et al. A red emitting fluorescent probe based on TICT for selective detection and imaging of HSA
Liu et al. Development of a mitochondria targetable ratiometric time-gated luminescence probe for biothiols based on lanthanide complexes
Ma et al. Dual-functional probes for sequential thiol and redox homeostasis sensing in live cells
KR101686886B1 (en) Compounds and fluorescent probes for selectively detecting cysteine/homocysteine based on fluorescein structure
Zhang et al. Two-photon small molecular fluorogenic probe visualizing biothiols and sulfides in living cells, mice brain slices and zebrafish
KR101872155B1 (en) Two-photon fluorescent probe for detection of Cysteine
Wang et al. A novel reaction-based fluorescent turn-on probe for biothiols and its application in cell imaging
Long et al. An ultralow concentration of two-photon fluorescent probe for rapid and selective detection of lysosomal cysteine in living cells

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E902 Notification of reason for refusal
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20160928

Year of fee payment: 4