AU2020102202A4

AU2020102202A4 - Ratiometric fluorescent probe for ph measurment and preparation method and use thereof

Info

Publication number: AU2020102202A4
Application number: AU2020102202A
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: 2020-09-10
Publication date: 2020-10-22
Anticipated expiration: 2028-09-10
Also published as: CN111518089B; CN111518089A

Abstract

The present invention discloses a ratiometric fluorescent probe for pH measurement and preparation method and use thereof. The ratiometric fluorescent probe has a structural formula of OOH 00 CS-NA . The preparation method for the fluorescent probe 5 of 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 has very weak fluorescence under neutral conditions and a solution thereof in water or organic solvent is light yellow in color. As the solution is increasingly acidic, the color fades gradually and turns into blue-green, and the solution shows increasing fluorescence. The fluorescent probe of the present invention can measure a pH in a range 10 of pH 2-7 and can result in a desired linear relationship at pH 2-4. Measurement with the probe can eliminate background interference effectively with increased sensitivity and accuracy. 1/2 j 415 2246 20 S72788 814 *7M 58 10714694 =!091 15260126 1 .7312 FIG. 1 O.3 pH=2 0.2 cu C ~0.1 400 So 60 70 800 Wavelength/m FIG. 2

Description

1/2

j 4152246

20 S72788

814 *7M 58 10714694 =!091 15260126 1 .7312

FIG. 1

O.3

pH=2 0.2 cu

C

~0.1

400 So 60 70 800 Wavelength/m FIG. 2

RATIOMETRIC FLUORESCENT PROBE FOR PH MEASURMENT AND PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD The present invention belongs to the technical field of fluorescent probes, and particularly relates to a ratiometric fluorescent probe for pH measurement and a preparation method and use thereof. BACKGROUND The reference to prior art in the background is not and should not be taken as an acknowledgment or any form of suggestion that the referenced prior art forms part of the common general knowledge in Australia or in any other country. Concentration of in vivo H' indicating a pH value plays an important role in maintaining internal environment of an organism. For example, H' regulates a series of cell physiological activities such as cell growth, apoptosis and autophagy. PH value as an important parameter in cell metabolism plays a key role in maintaining normal physiological processes such as cell growth, proliferation, apoptosis and signaling. In a eukaryotic cell, pH value varies among organelles. For example, pH in lysosome or nucleus is 4.0-6.0 which is slightly acidic while pH in mitochondria is about 8.0 which is slightly alkaline. Methods for pH measurement mainly include colorimetric analysis, electrochemical method and the like. Compared with these methods, measurement with a fluorescence microscope is superior in performances since a proper fluorescent probe can mark a pH value at a micron level. Real time dynamic pH changes in a living cell can be shown under a fluorescence microscope in situ. In order to achieve fluorescence imaging of pH in a cell, a variety of fluorescent probes are constructed with different responsive sites and different mechanisms of fluorescence. In recent years, measurement with a fluorescent probe as an excellent detection technique has attracted increasing concerns due to high selectivity, high sensitivity and real time imaging thereof and is widely used for measurement of various substances. 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 ratio of two fluorescence signal intensities as an output signal where these factors can be eliminated based on fluorescence intensities at two different wavelengths. Fluorescent probe is an effective means for pH measurement and is widely used in fields such as industry, environment and medical test. Compared with an electrochemical method, a method with a fluorescent probe can have a better effect in eliminating background interference and relatively large improvement in sensitivity. However, current measurement of pH with a fluorescent probe is only used in a relatively limited range in the field of life science. Most reported probes can result in a relatively desired linear relationship under slightly acidic conditions. But these probes are not suitable for many highly acidic cases such as stomach, ribozyme and lysosome due to insufficient sensitivity. Moreover, these fluorescent probes may be affected by background interference to a relatively large extent in measurement for organisms which greatly limits applications thereof. Therefore, it is very important to develop a ratiometric fluorescent probe for pH measurement which has a wider range of applications. SUMMARY Aspects of the present invention provide a ratiometric fluorescent probe for pH measurement, and a preparation method and use thereof. In embodiments, the fluorescent probe of the present invention can result in a desired linear relationship at pH 2-4 with high sensitivity and is easy to be identified. In embodiments, the preparation method of the present invention may be a simple process with high productivity and thus is suitable for promotion and application at a large scale. Aspects or embodiments of the present invention may overcome a defect in the prior art wherein a fluorescent probe for pH measurement has low sensitivity under highly acidic conditions and large background interference. Embodiments of one aspect of the present invention provide: A ratiometric fluorescent probe for pH measurement, having a molecular formula of C52H5oN70O'and a structural formula of CS-NA of:

OOH N

0

CS-NA

Embodiments of another aspect of the present invention provide a preparation method for the ratiometric fluorescent probe for pH measurement includes the following steps: 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 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 for the ratiometric fluorescent probe for pH measurement is as follows:

NH ON N HN N

H-N Br IN

13

45

H HNN

~Ec2 NH

3 G CS-NA

Preferably, in step (1), a molar ratio of the compound 1 to the propargylamine is 1:1.5; in step (2), a molar ratio of the compound 2 to the N-(2-aminoethyl)morpholine is 1:1.2; in step (3), a molar ratio of the compound 4 to the cyclohexanone is 1:3; in step (4), a molar ratio of the compound 5 to the p-azidobenzaldehyde is 1:1.5; in step (5), a molar ratio of the compound 3 : the compound 6 : the cuprous bromide : the triethylamine is 1:1.1:3:2. Preferably, 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. Preferably, 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), 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. Preferably, in step (3), a mass fraction of the sulfuric acid is 36%, and a mass fraction of the perchloric acid is 70%. In the present invention, the ratiometric fluorescent probe for pH measurement may be used to measure pH in different systems. Preferably, the systems include water systems, organic solvent systems or organism systems. The fluorescent probe of the present invention measures pH based on a PET mechanism. When the fluorescent probe is not binding H+ as a solution has a neutral pH, an electron at a highest energy level in a morpholine ring of the probe can be transferred to an empty electronic orbit in a fluorophore at an excited state and results in fluorescence quenching. When a morpholine ring binds to H' where electron donation from the morpholine ring is reduced and the PET process is inhibited, a photoexcited electron in the fluorophore can directly transit back to its original ground state orbit and thus increases fluorescence emission from the fluorophore. In particular, the fluorescent probe has very weak fluorescence under neutral conditions and a solution thereof in water or organic solvent is light yellow in color. As the solution is increasingly acidic, the color fades gradually and turns into blue-green, and the solution shows increasing fluorescence. Thus, pH can be measured based on change of fluorescence and color. The fluorescent probe can be used for measurement of pH between 2 and 7. When pH is 7, the probe generates weak fluorescence at 525 nm, and when the pH is getting increasingly acidic, the color of a solution of the probe gradually turns from light yellow to blue-green and the solution shows increasing fluorescence at 525 nm. Beneficial Effects: (1) The fluorescent probe for pH measurement of the present invention is highly sensitive with an obvious change in appearance and is convenient for identification in pH measurement. (2) The preparation method of the fluorescent probe for pH measurement of the present invention is simple and has a high productivity, thus it is suitable for promotion and application at a large scale. (3) The present invention has a high selectivity in measurement for organisms where the ratiometric fluorescent probe can eliminate background interference effectively. Moreover, when pH is 2-4, the fluorescent probe can result in a desired linear relationship which widens its use in highly acidic environments with reduced interference from other factors and increased sensitivity and accuracy in measurement. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a mass spectrum of CS-NA; FIG.2 is an ultraviolet (UV) absorption spectrum of the fluorescent probe in Example 2 at 300-800 nm; FIG. 3 is a fluorescence spectrum of the fluorescent probe in Example 2 at an excitation wavelength of 420 nm under different pH conditions; and FIG. 4 shows linear fitting of ratio of fluorescence intensities at excitation wavelengths of 420 nm and 580 nm of the fluorescent probe in response to pH under different pH conditions in Example 2. 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 compound 1 (6-Bromo-1H,3H-benzo[de]isotryptamine-1,3-dione) and 0.08 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 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 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%. 1H NMR (400 MHz, CDCl3) 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).1 3C NMR (100 MHz, CDCl3) 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 C21H21N303 [M] :363.1583. found:

364.1649; (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 36% 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 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 colunm 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 3 C NMR (101 MHz, CDC3) 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, 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 colunm 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, 13 4H), 1.38 - 1.22 (m, 18H), 1.18 - 0.73 (m, 4H). C NMR (101 MHz, CDCl3) 6 170.06, 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 C2HoN70 [M] *: 868.3817. Found: 868.3798. A synthetic route was as follows:

0 0 0 NH 2 O 0 OOO 0 H 2N N N

N

1 2 3 N

O OH COOH O a N3 COOH

N NN N

4 5 6 N N-N N0 __

I COOH COOH N 0 N 01

N N O SHN I

NO 3N O NH

3 6 CS-NA

Example 2 s (1) Titration experiment with the fluorescent probe and H' Solutions with pH gradients of 2, 3, 3.5, 4, 5, 6 and 7 were prepared with PBS solution and

hydrochloric acid. 1 mM fluorescent probe at an initial concentration was added to achieve a concentration of 10 pM in each of the solutions. The solutions with different pH gradients were allowed to stand still for 0.5 h for sufficient reaction with the fluorescent probes. A UV spectrophotometer was used to obtain UV absorption spectra in the range of 300-800 nm. Results were shown in FIG. 2 where the absorption wavelengths were 425 nm and 575nm

respectively. The results provided preliminary experimental reference for titration experiment for fluorescence spectrum.

(2) Titration experiment with the fluorescent probe and H' Solutions with pH gradients of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 and 7 were prepared with PBS solution and hydrochloric acid. 1 mM fluorescent probe at an initial concentration was added to achieve a concentration of 10 pM in each of the solutions. The solutions with different pH gradients were allowed to stand still for 0.5 h for sufficient reaction with the fluorescent probes. A fluorescence spectrometer was used to obtain fluorescence spectra under different pH conditions. The excitation wavelengths were 420 nm and 580 nm while the emission wavelengths and detection wavelengths were 525 nm and 675 nm. Results were shown in FIG. 3 where it can be seen that, as the pH was increasing, the fluorescence intensity at 525 nm or 675 nm was gradually decreased.

A working curve was obtained with x-axis of pH and y-axis of 1525/1675, ratio of fluorescence intensities. The pH linearly correlated with 1525/1675in the range of pH 2.00-4.00 with a correlation coefficient of 0.9932 as shown in FIG. 4. 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 organisms. In this specification, the terms "comprises", "comprising", "includes", "including", and similar terms are intended to mean a non-exclusive inclusion, such that a product or method that comprises or includes a list of elements need not have those elements solely, and may well have other elements not listed.

Claims

What is claimed is: 1. A ratiometric fluorescent probe for pH measurement, wherein the probe has a molecular formula of C52H5oN706*and a structural formula of CS-NA of:

1OOH

- N

0

CS-NA
2. A preparation method for the ratiometric fluorescent probe for pH measurement according to claim 1, comprising the following steps: 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 1 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 column chromatography to obtain the fluorescent probe CS-NA; wherein a synthetic route for the ratiometric fluorescent probe for pH measurement is as follows:

K? 0N0 -NH 2 // 0 N C , -N0

HN Br

1 2

C*4

4 5 6

0 N H H

HN N

3 G CS-NA
3. The preparation method according to claim 2, wherein in step (1), a molar ratio of the compound 1 to the propargylamine is 1:1.5; in step (2), a molar ratio of the compound 2 to the N-(2-aminoethyl)morpholine is 1:1.2; in step (3), a molar ratio of the compound 4 to the cyclohexanone is 1:3; in step (4), a molar ratio of the compound 5 to the p-azidobenzaldehyde is 1:1.5; in step (5), a molar ratio of the compound 3 : the compound 6 : the cuprous bromide : the triethylamine is 1:1.1:3:2.
4. The preparation method according to claim 2, wherein 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.
5. The preparation method according to claim 2, 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), 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; wherein in step (3), a mass fraction of the sulfuric acid is 36%, and a mass fraction of the perchloric acid is 70%.