AU2020102262A4

AU2020102262A4 - Use of ratiometric fluorescent probe in measurement of peroxynitrite anion

Info

Publication number: AU2020102262A4
Application number: AU2020102262A
Authority: AU
Inventors: Luo BAO; Keyin Liu; Changwei ZHANG
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2020-04-26
Filing date: 2020-09-15
Publication date: 2020-10-29
Anticipated expiration: 2028-09-15
Also published as: CN111423423A; CN111423423B

Abstract

The present invention discloses use of a ratiometric fluorescent probe in measurement of ONOO-, where the ratiometric fluorescent probe has a structural formula of OHOOH 0 5 CS-NA . When the fluorescent probe is not reacted with a ONOO-, a carboxyl group in the molecule forms an intramolecular spiro ring which damages a conjugation structure of a fluorophore and results in fluorescence quenching. When the fluorescent probe is reacted with an ONOO-, the spiro ring opens and the conjugation structure is recovered, showing relatively strong fluorescence under light. The o preparation method of the fluorescent probe in the present invention is simple and has a high productivity, thus it is suitable for promotion and application at a large scale. The fluorescent probe of the present invention can be used to recognize and measure ONOO- in water system or organic system with high selectivity. Measurement with a ratiometric fluorescent probe uses a ratio of two fluorescence signal intensities as an output signal where background 15 interference can be eliminated and interference from other factors can be reduced. Thus, sensitivity and accuracy can be improved in measurement. 16573718_1 (GHMatters) P114466.AU 1/3 § Psj diI I4 4I I4

Description

1/3

§

Psj diI I4 4I

I4

USE OF RATIOMETRIC FLUORESCENT PROBE IN MEASUREMENT OF PEROXYNITRITE ANION

TECHNICAL FIELD The present invention belongs to the technical field of fluorescent probes, and particularly relates to use of a ratiometric fluorescent probe in measurement of peroxynitrite anion (ONOO-).

BACKGROUND o ONOO- is regarded as one of the most important anions and is generated by reaction of excess amount of nitric oxide (NO) with oxygen free radical (02-). It is both a strong oxidant and a nitrating agent which can react with macromolecular substances such as nucleic acid, protein and lipid in a human cell. Thus, it can disturb metabolism in a cell and consume a lot of energy and thus causes a series of biological and pathological processes such as tumor, arteriosclerosis, inflammation in a body and retinal damage. In recent years, there are a variety of researches for ONOO- measurement in medical field. However, measurement of ONOO- is inconvenient as it is strongly oxidative and thus is very unstable in vivo. Therefore, it is very important to develop a novel and effective technique for measurement of ONOO-. With development of novel small-molecule fluorescent probes, fluorescent probes are o generally more selective and less invasive in measurement of these peroxides compared with many other methods. The fluorescent probes are more convenient in measurement of biologically relevant analytes in cells. In order to investigate the pathophysiology of in vivo ONOO-, it is crucial to develop an effective imaging tool to monitor ONOO- in a brain. Fluorescence imaging technology based on an active sensing probe has become an important means in studies of biological species in biological systems due to its advantages such as high sensitivity, high selectivity, real-time measurement and non-invasion. Some fluorescent probes have been reported to be used in in vivo cell or tissue imaging, but there is no in vivo imaging method for use in a brain including an epilepsy brain. In recent years, measurement with fluorescent probes, as an excellent measurement technique, has attracted increasing concerns due to its high selectivity, high sensitivity and real-time imaging. It is widely used in measurement of various substances with advantages such as simple operation and desired reproducibility. It can be conveniently used in in situ,

16573718_1 (GHMatters) P114466.AU real time and non-invasive measurement of biomolecules and tracking of biomolecules and biological processes of the biomolecules. Generally, measurement with a fluorescent probe depends on increase or decrease of fluorescence intensity. Thus, an output signal can be affected by factors such as concentration of a probe, efficiency of a device and environment. In contrast, ratiometric fluorescent probe uses a ratio of two fluorescence signal intensities as an output signal where these factors can be eliminated based on fluorescence intensities at two different wavelengths. There are few fluorescent probes for measurement of ONOO-. Most reported probes can be interfered by other active oxygen (e.g. hydrogen peroxide). These probes can be interfered o by background to a relatively large extent in measurement for organisms. Thus, it is very important to develop a novel fluorescent probe for measurement of ONOO-.

SUMMARY To overcome the defect in the prior art that a fluorescent probe for measurement of ONOO- has low sensitivity and large background interference, the present invention provides use of a ratiometric fluorescent probe in measurement of ONOO-. It can be found through measurement by fluorescence spectrophotometer that, the fluorescent probe is highly sensitive in measurement of ONOO- with an obvious change in appearance and is convenient for identification. o The present invention provides use of a ratiometric fluorescent probe in measurement of ONOO-, where the fluorescent probe has a molecular formula of C5 2 HoN 7 06 and a structural formula of CS-NA of: OOH

OOH N'N N CS-NA

Further, the use is in a system including water system, organic solvent system or organism system.

16573718_1 (GHMatters) P114466.AU

Further, the fluorescent probe is prepared by a method as follows: step (1) adding a compound 1 and propargylamine to ethanol, heating to allow reaction, pouring a reaction solution to ice water after completion of the reaction, extracting with dichloromethane (DCM) and purifying by column chromatography to obtain a compound 2; step (2) dissolving the compound 2 and N-(2-aminoethyl)morpholine in ethylene glycol monomethyl ether, heating to allow reaction, washing a reaction solution with a dilute hydrochloric acid solution and purifying by column chromatography to obtain a compound 3; step (3) adding a compound 4 and cyclohexanone to sulfuric acid, heating to allow reaction, pouring a reaction solution to ice water, adding perchloric acid dropwise to o precipitate a solid, recrystallizing the solid with ethanol to obtain a compound 5; step (4) adding the compound 5 and p-azidobenzaldehyde to glacial acetic acid, heating to allow reaction, washing a reaction solution with a saturated sodium bicarbonate solution and purifying by column chromatography to obtain a compound 6; step (5) adding the compound 3 and the compound 6 to chloroform, adding triethylamine dropwise to allow reaction at room temperature with cuprous bromide as catalyst, and purifying by column chromatography to obtain the compound CS-NA; where, a synthetic route is as follows:

0 o aH2N 0NH2 "

/ N HN

1 1

0 OH OCH ~\#M/

4 5

H OOH CS-NA

16573718_1I(GHMatters) P114466.AU

Further, in step (1), a molar ratio of the compound I to propargylamine is 1:1.5; in step (2), a molar ratio of the compound 2 to N-(2-aminoethyl)morpholine is 1:1.2; in step (3), a molar ratio of the compound 4 to cyclohexanone is 1:3; in step (4), a molar ratio of the compound 5 to p-azidobenzaldehyde is 1:1.5; in step (5), a molar ratio of the compound 3 the compound 6 : cuprous bromide: triethylamine is 1:1.1:3:2. Further, in step (1), the heating is carried out at 80°C for 12 h; in step (2), the heating is carried out at 110°C for 12 h; in step (3), the heating is carried out at 90°C for 12 h; in step (4), the heating is carried out at 90°C for 12 h; in step (5), the heating is carried out for 12 h. Further, in step (1), the purifying by column chromatography is carried out by removing a o solvent by rotary evaporation, dissolving a solid with DCM, using a mixed solvent of DCM and methanol in a volume ratio of 5:1 for separation with column chromatography; in step (2), the purifying by column chromatography is carried out by removing an aqueous phase after extraction with DCM, removing a solvent by rotary evaporation, dissolving a solid with a small amount of DCM, using a mixed solvent of DCM and methanol in a volume ratio of 20:1 for separation with column chromatography; in step (4), the purifying by column chromatography is carried out by removing an aqueous phase after extraction with DCM, removing a solvent by rotary evaporation, dissolving a solid with a small amount of DCM, using a mixed solvent of DCM and methanol in a volume ratio of 5:1 for separation with column chromatography; in step (5), the purifying by column chromatography is carried out by removing a solvent by rotary evaporation, dissolving a solid with DCM, using a mixed solvent of DCM and methanol in a volume ratio of 10:1 for separation with column chromatography. Further, in step (3), a mass fraction of the sulfuric acid is 36%, and a mass fraction of the perchloric acid is 70%. The fluorescent probe of the present invention measures ONOO- based on change of fluorescence and color. When the fluorescent probe is not reacted with a ONOO-, a carboxyl group in the molecule forms an intramolecular spiro ring which damages a conjugation structure of a fluorophore and results in fluorescence quenching. When the fluorescent probe is reacted with a ONOO-, the spiro ring opens and the conjugation structure is recovered, showing relatively strong fluorescence under light. The fluorescent probe is able to recognize and measure ONOO- in a water system or an organic solvent system with high selectivity. The probe itself shows very weak fluorescence at 490 nm, and a solution thereof in water or organic solvent is light yellow which color disappears after the reaction of the probe with a

16573718_1 (GHMatters) P114466.AU

ONOO-. Beneficial Effects: 1. The fluorescent probe for measurement of ONOO- has a high selectivity. It can be found through measurement by fluorescence spectrophotometer that, the fluorescent probe is highly sensitive in measurement of ONOO- with an obvious change in appearance and is convenient for identification. 2. The preparation method of the fluorescent probe for measurement of ONOO- is simple and has a high productivity, thus it is suitable for promotion and application at a large scale. 3. The present invention has high selectivity in measurement for organisms. Measurement o with a ratiometric fluorescent probe uses a ratio of two fluorescence signal intensities as an output signal where background interference can be eliminated and interference from other factors can be reduced. Thus, sensitivity and accuracy can be improved in measurement.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a mass spectrum of CS-NA synthesized in Example 1 of the present invention; FIG. 2 shows fluorescence intensity of a fluorescent probe in response to different concentrations of ONOO- (pH=7.4); FIG. 3 shows the relationship between ratio of fluorescence intensities at excitation o wavelengths of 420 nm and 580 nm (1490/165o) and concentration of ONOO- (pH=7.4); FIG. 4 shows the effect of a fluorescent probe at different concentrations on cell viability with zero ONOO-; and FIG. 5 shows comparison of fluorescence intensity of fluorescent probe with different small biological molecules where 1-14 represent Glu, Cys, S2032,2-, SO32-, SO4 2 -, HS032 N02-, H 2 0 2 , Vc-, Zn2+, Fe 3+, Mg2+ and ONOO-.

DETAILED DESCRIPTION The technical solution of the present invention is clearly and completely described hereinafter for better understanding by those of ordinary skill in the art where other examples obtained by those of ordinary skill in the art based on the examples of the present application without creative efforts should fall within the protection scope of the present application. Example 1 (1) 0.276 g compound1 (6-Bromo-1H,3H-benzo[de]isotryptamine-1,3-dione) and 0.08

16573718_1 (GHMatters) P114466.AU g propargylamine were added to 20 ml ethanol for reaction at 80°C for 12 h monitored by TLC. After completion of the reaction, the reaction solution was subjected to rotary evaporation to remove the solvent. The solid was dissolved with DCM and subjected to column chromatography for separation with a mixed solvent of DCM and methanol in a volume ratio of 5:1 to obtain a compound 2; (2) 0.156 g compound 2 and 0.078 g N-(2-aminoethyl)morpholine were dissolved in 8 ml ethylene glycol monomethyl ether, and heated to allow reaction at 110°C for 12 h monitored by TLC. The reaction solution was washed with dilute hydrochloric acid having a mass fraction of 10%. The reaction solution after completion of the reaction was extracted o with DCM and the aqueous phase was removed. Rotary evaporation was carried out to remove the solvent. The solid was dissolved with a small amount of DCM and subjected to column chromatography for separation with a mixed solvent of DCM and methanol in a volume ratio of 20:1. 0.13 g compound 3 was obtained with a yield of 72%. IH NMR (400 MHz, CDC3) 6 8.53 (d, J= 7.3 Hz, 1H), 8.40 (d, J= 8.4 Hz, 1H), 8.02 (s, 1H), 7.57 (t, J= 7.4 Hz, 1H), 6.57 (d, J= 8.1 Hz, 1H), 6.24 (s, 1H), 4.85 (d, J= 2.2 Hz, 2H), 3.69 (s, 4H), 3.31 (d, J= 13.5 Hz, 2H), 2.74 (s, 2H), 2.47 (s, 3H), 2.17 - 1.95 (m, 1H), 1.14 (s, 1H)."C NMR (100 MHz, CDCl 3) 6 164.02, 163.24, 149.73, 135.02, 131.57, 129.95, 126.42, 124.89, 120.48, 116.00, 104.61, 90.27, 88.51, 79.17, 69.92, 67.04, 59.80, 55.99, 53.13, 38.85, 29.09. Results were calculated with HRMS (ESI). For C 2 1H 21 N 3 0 3 [M] ':363.1583. found: 364.1649; o (3) 0.269 g compound 4 ((4-(diethylamino)-2-hydroxyphenyl)(phenyl)methanone) and 0.294 g cyclohexanone were added to 20 ml concentrated sulfuric acid having a mass fraction of 360% for reaction at 90°C for 12 h. The reaction solution was poured to ice water, and 10 ml perchloric acid having a mass fraction of 70% was added dropwise to precipitate a solid. The solid was recrystallized with ethanol to obtain a compound 5; (4) 0.188 g compound 5 and 0.22 g p-azidobenzaldehyde were added to 10 ml glacial acetic acid for reaction using a reflux condensation method at 90°C for 12 h monitored by TLC. After completion of the reaction, the solution was extracted with DCM and the aqueous phase was removed. Rotary evaporation was carried out to remove the solvent. The solid was dissolved with a small amount of DCM and subjected to column chromatography for separation with a mixed solvent of DCM and methanol in a volume ratio of 5:1. 0.17 g compound 6 was obtained with a yield of 67%. 1 3C NMR (101 MHz, CDCl 3) 6 212.74, 175.28, 169.90, 166.60, 161.79, 155.90, 151.84, 150.43, 143.60, 138.67, 134.40, 134.00,

16573718_1 (GHMatters) P114466.AU

131.02, 130.57, 130.55, 129.35, 129.30, 128.65, 123.65, 123.64, 122.58, 118.85, 101.15, 97.17, 47.75, 44.51, 29.72, 27.17, 23.14, 22.29, 12.47; (5) 0.072 g compound 3 and 0.11 g compound 6 were added to 10 ml chloroform. 3-4 drops of triethylamine was added dropwise to allow Click reaction with 0.086 g cuprous bromide as catalyst to prepare the probe of CS-NA. The reaction was monitored by TLC. After completion of the reaction, the solution was subjected to rotary evaporation to remove the solvent. The solid was dissolved with DCM and subjected to column chromatography for separation with a mixed solvent of DCM and methanol in a volume ratio of 10:1 to obtain the compound as shown by CS-NA. The mass spectrum of the CS-NA was shown in FIG. 1. 1H NMR (400 MHz, MeOD) 6 8.30 (s, 1H), 7.99 (s, 1H), 7.75 - 7.65 (m, 1H), 7.52 (d, J= 6.5 Hz, 1H), 7.34 (d, J= 7.2 Hz, 3H), 7.21 (d, J= 31.1 Hz, 3H), 6.93 (d, J= 9.3 Hz, 1H), 6.69 (s, 1H), 6.31 (s, 1H), 6.21 (d, J= 14.2 Hz, 1H), 4.14 (d, J= 7.2 Hz, 3H), 3.57 (d, J= 7.0 Hz, 3H), 2.84 (s, 3H), 2.16 (d, J= 20.5 Hz, 1H), 1.84 - 1.44 (m, 4H), 1.38 - 1.22 (m, 18H), 1.18 - 0.73 (m, 13 4H). C NMR (101 MHz, CDCl3) 5 170.06, 167.37, 164.48, 163.78, 156.16, 152.53, 152.19, 149.67, 149.40, 145.00, 137.68, 137.22, 135.56, 134.88, 134.49, 131.76, 131.50, 131.48, 130.59, 130.56, 129.95, 129.26, 128.52, 128.33, 128.14, 127.63, 126.34, 124.96, 124.83, 123.79, 123.47, 122.93, 121.35, 120.46, 120.23, 110.04, 108.84, 104.54, 97.31, 66.93, 55.92, 53.07, 44.52, 41.05, 38.93, 35.02, 29.67, 29.26, 27.16, 23.09, 22.30, 12.51. Results were calculated with HRMS (ESI). For C 2H 5 oN 7 0 6 [M] :868.3817. Found: 868.3798. o A synthetic route was as follows:

0 ON 0 O NH2 ON 0 H 2 NWN

- HN IN

1 2 3

O 0 O 0 O OH COOH '/\N COOH 3

4 5 6 N3

16573718_1 (GHMatters) P114466.AU

0 N 0 COOH COOH

NNN _)ON H

N N3 NN O (H

3 6 CS-NA

Example 2 Titration experiment with the fluorescent probe and ONOO 1 mM fluorescent probe at an initial concentration was added to PBS buffer (pH=7.4) to achieve a concentration of 10 M in the solution. A different amount of fluorescent probe at an initial concentration of 1.00 mM was added to obtain a series of solutions having 0.5 [M, 1 ptM, 2 M, 3 M, 4 M and 5 M ONOO- respectively, with a solution having no ONOO- as control. The solutions were allowed to stand still for 0.5 h for sufficient reaction of ONOO with the fluorescent probe. A fluorescence spectrometer was used to obtain fluorescence spectra at different concentrations of ONOO-. The excitation wavelengths were 420 nm and 580 nm, the emission wavelengths and detection wavelengths were 490 nm and 660 nm. Results were shown in FIG. 2 where it can be seen that, as the concentration of ONOO- was increasing, the fluorescence intensity at 490 nm was gradually increased while the fluorescence intensity at 660 nm was gradually decreased. A working curve was obtained with x-axis of concentration of ONOO- and y-axis of 1490/1660, ratio of fluorescence intensities. The concentration of ONOO- linearly correlated with the ratio of 1490/1660with a correlation coefficient of 0.99742 as shown in FIG. 3. Thus, the fluorescent probe can result in a desired linear ratio relationship and have a relatively desired effect in eliminating background interference in measurement for organism. Example 3 (1) MTT assay for cell viability with the fluorescent probe Hela cells were cultivated in an incubator at 37°C for 24 h, and then transferred to a 96-well plate for incubation with the fluorescent probe at different concentrations for 12 h. Subsequently, 10 L MTT was added to each well for further incubation for 4 h followed by addition of DMSO (100 [L). Herm Fisher Scientific Reader was used to measure the number of cells in each well. Cytotoxicity was shown in FIG. 4. Thus, the fluorescent probe of the present invention had low cytotoxicity.

16573718_1 (GHMatters) P114466.AU

(2) Test of selectivity of the fluorescent probe in measurement of ONOO Excess amounts of other bioactive small molecules were added to a solution under the same testing conditions to obtain fluorescence spectra. Results were shown in FIG. 5. It can be seen from FIG. 5 that, 1-14 represented the bioactive small molecules of Glu, Cys, S2032-, 2-,

S032-, S042-, HS0 32-, NO2-, H 2 0 2 , Vc-, Zn2+, Fe3+, Mg2+ and ONOO-. For measurement at 490 nm, only ONOO- had significantly increased fluorescence intensity, other bioactive small molecules had no interference in measurement results. Thus, the fluorescent probe had relatively high selectivity in measurement of ONOO-. It is to be understood that, if any prior art publication is referred to herein, such reference o does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

16573718_1 (GHMatters) P114466.AU

Claims

Claims

1. Use of a ratiometric fluorescent probe in measurement of peroxynitrite anion (ON00), wherein the fluorescent probe has a molecular formulaof C 52 H 5 N 7 06* and a structural formula of CS-NA of:

I OOH

N N

N'N 0

CS-NA

2. The use according to claim 1, wherein the use is in a system comprising water system, organic solvent system or organism system.

3. The use according to claim 1, wherein the fluorescent probe is prepared by the following method: step (1) adding a compound 1 and propargylamine to ethanol, heating to allow reaction, pouring a reaction solution to ice water after completion of the reaction, extracting with dichloromethane (DCM) and purifying by column chromatography to obtain a compound 2; step (2) dissolving the compound 2 and N-(2-aminoethyl)morpholine in ethylene glycol monomethyl ether, heating to allow reaction, washing a reaction solution with a dilute hydrochloric acid solution and purifying by column chromatography to obtain a compound 3; step (3) adding a compound 4 and cyclohexanone to sulfuric acid, heating to allow reaction, pouring a reaction solution to ice water, adding perchloric acid to precipitate a solid, recrystallizing the solid with ethanol to obtain a compound 5; step (4) adding the compound 5 and p-azidobenzaldehyde to glacial acetic acid, heating to allow reaction, washing a reaction solution with a saturated sodium bicarbonate solution and purifying by column chromatography to obtain a compound 6; step (5) adding the compound 3 and the compound 6 to chloroform, adding triethylamine to allow reaction at room temperature with cuprous bromide as catalyst, and purifying by

16573718_1 (GHMatters) P114466.AU column chromatography to obtain the fluorescent probe CS-NA; wherein, a synthetic route is as follows:

0 NN

0 H2Ni r tt 1:.:3

0 N -C0 OOH -- NN

5 . The use according to claim 3, wherein in step (1), th hel ati is carred coud t 80Cto

1-2h; ine 2the~rhating is carried;out at110°C3fora12o;lin stepo(3) the heatin is carie

out aTh 0°C fordn 2 h;i , hri in step (), the heating is carried out at 90°C for 1 ;i tp() h

wherein in step (1), the purifying by column chromatography is carried out by removing a solvent by rotary evaporation, dissolving a solid with DCM, using a mixed solvent of DCM and methanol in a volume ratio of 5:1 for separation with column chromatography; in step (2),

16573718_1 (GHMatters) P114466.AU the purifying by column chromatography is carried out by removing an aqueous phase after extraction with DCM, removing a solvent by rotary evaporation, dissolving a solid with a small amount of DCM, using a mixed solvent of DCM and methanol in a volume ratio of 20:1 for separation with column chromatography; in step (4), the purifying by column chromatography is carried out by removing an aqueous phase after extraction with DCM, removing a solvent by rotary evaporation, dissolving a solid with a small amount of DCM, using a mixed solvent of DCM and methanol in a volume ratio of 5:1 for separation with column chromatography; in step (5), the purifying by column chromatography is carried out by removing a solvent by rotary evaporation, dissolving a solid with DCM, using a mixed o solvent of DCM and methanol in a volume ratio of 10:1 for separation with column chromatography; wherein in step (3), a mass fraction of the sulfuric acid is 36% , and a mass fraction of the perchloric acid is 70%.

16573718_1 (GHMatters) P114466.AU