CN110776460B

CN110776460B - Fluorescent compound for LED visible light induced release of nitric oxide and preparation and application thereof

Info

Publication number: CN110776460B
Application number: CN201910641530.7A
Authority: CN
Inventors: 朱勍; 蒋建泽; 刘江
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2019-07-16
Filing date: 2019-07-16
Publication date: 2021-05-04
Anticipated expiration: 2039-07-16
Also published as: CN110776460A

Abstract

The invention relates to a LED, a fluorescent compound (I) capable of releasing nitric oxide under the induction of visible light, and a preparation method and application thereof. The compound (I) can sufficiently release nitric oxide under the induction of LED yellow light, and reaches the concentration of the nitric oxide released by similar compounds under the induction of ultraviolet; meanwhile, the fluorescence response induced by LED yellow light (18W) in the PBS solution is higher than that induced by common ultraviolet light (8W), so that the method is better applied to cell biological imaging; according to the invention, the LED yellow light source is used as a trigger light source for nitric oxide release, and the two-photon dye is used as a compound fluorescent parent, so that the phototoxicity is greatly reduced during application, and the damage to organisms and cells is reduced; due to the good light stability and thermal stability of the conjugated structure containing the naphthalimide, the compound (I) can be irradiated in organisms for a long time and can continuously release nitric oxide for a long time, and can be applied to inhibiting the proliferation of cancer cells.

Description

Fluorescent compound for LED visible light induced release of nitric oxide and preparation and application thereof

(I) technical field

The invention relates to a fluorescent compound capable of releasing nitric oxide under the induction of LED visible light, and a preparation method and application thereof.

(II) background of the invention

Nitric Oxide (NO) is considered to be an important gaseous signaling molecule that plays an important role in a variety of physiological and pathological processes, and is involved in the regulation of angiogenesis, blood flow and vascular function. In recent years, NO has not only found its important role in various biological functions, but also has potential anticancer activity, and the level of NO in tumors and its microenvironment can directly affect the response of cancer cells to excessive amounts of NO (more than several hundred nanomolar) can destroy DNA or mitochondria through apoptosis mechanisms, inhibit key metabolic pathways, block growth or completely kill cancer cells. NO may also exert a local cytotoxic effect on infectious microorganisms; can also be used as free radical scavenger to reduce free radical mediated oxidation process. Therefore, the carbon monoxide release molecule which constructs rapid and accurate space-time control and has the fluorescence response function and cancer cytotoxicity has important application value.

Several classes of nitric oxide releasing molecules with different release mechanisms and kinetics have been developed so far, but real-time monitoring and control of the released concentration are difficult, and heavy metal toxicity is present in part. Compared with the prior art, the light-induced on/off triggered nitric oxide releasing molecules have the advantages of being relatively mild, and the existing light-induced released fluorescent molecules are induced by an ultraviolet light source, a pulse light source and a halogen lamp light source and have the defects of high phototoxicity, high cost and inconvenience in use.

Disclosure of the invention

The invention aims to provide a fluorescent compound for inducing the release of nitric oxide by LED visible light, and a preparation method and application thereof.

The technical scheme adopted by the invention is as follows:

an LED visible light-induced nitric oxide-releasing fluorescent compound has a structure shown in formula (I):

compared with a compound with a similar effect, the compound (I) provided by the invention firstly uses the fluorescent molecules released by the nitric oxide induced by the LED yellow light source, has obvious fluorescent response, small phototoxicity and high light stability, has the performance of releasing the nitric oxide by long-time illumination, can be applied to inhibiting cell proliferation, and simultaneously meets the requirements of monitoring the nitric oxide release and biological cell imaging.

The invention also relates to a method for preparing the fluorescent compound, which comprises the following steps: under the conditions of ice bath at 0 ℃ and nitrogen protection, adding a compound shown as a formula (II) dissolved in anhydrous tetrahydrofuran into an anhydrous tetrahydrofuran solution added with NaH, continuously reacting for 1-2 hours at a low temperature, adding a compound shown as a formula (III) dissolved in anhydrous tetrahydrofuran, continuously reacting for 5-6 hours at room temperature, and separating and purifying to obtain a compound shown as a formula (I) after the reaction is finished;

preferably, the amount ratio of the compound (II), the compound (III) and the NaH substance is 1:0.7: 3. The volume of the anhydrous tetrahydrofuran is 20mL/mmol based on the amount of the compound substance shown in the formula (II).

Specifically, the separation and purification method comprises the following steps: removing the solvent from the reaction solution by rotary evaporation under reduced pressure, and separating the concentrate by a silica gel column, wherein the reaction solution is prepared by the following steps of: and (3) taking a mixed solution with the methanol volume ratio of 20:1 as an eluent, collecting a target component, and drying to obtain the fluorescent compound shown in the formula (I) (a nuclear magnetic hydrogen spectrum is shown in figure 1a, and a mass spectrum is shown in figure 1 b).

The formula (II) of the invention is a disclosed compound, and the preparation method thereof can be referred to as the literature: [ T, Suzuki, O.Nagae, Y.Kato, H.Nakagawa, K.Fukuhara, and N.Miyata, Photoinked Nitric Oxide Release from Nitrobenzene Derivatives, J.Am. chem.Soc., 127(2005)1720 11726 ]

The formula (III) of the invention is a disclosed compound, and the preparation method thereof can be referred to as the literature: [ H, Park, S. -K.Chang, signalling of water content in organic solvents by solvatochromism of ahydroxynapthalimide-based merocyanine dye, DyesPigm,122(2015)324-

The invention also relates to application of the fluorescent compound in preparing a fluorescent probe.

In particular, the fluorescent probe is used for photoinduced monitoring of nitric oxide release.

The synthesis route of the nitric oxide releasing fluorescent compound of formula (I) is as follows:

the invention simultaneously prepares other two new nitric oxide releasing fluorescent molecules:

detection experiments prove that the o-trifluoromethyl nitrobenzene light release groups in the compound (A) (the mass spectrogram is shown in figure 3a) and the compound (B) (the mass spectrogram is shown in figure 3B) cannot effectively release nitric oxide under an LED visible light source. The cy-7 parent of the compound (A) is easy to crack under the illumination condition due to a huge conjugated structure, and has no application capability; the parent of the naphthalimide of the compound (B) meets the requirement of long-time illumination, can effectively release nitric oxide under the condition of ultraviolet illumination, but has insignificant effects of fluorescence quenching and photoinduced turn-on based on photoinduced electron transfer. In contrast, the fluorescent compound (I) takes the naphthalimide with good light stability as a parent body, and is connected with the o-dimethyl nitrobenzene in a conjugated manner, so that the fluorescent turn-on performance after the nitric oxide is released by good fluorescent quenching and light induction is generated.

In the present invention, the 2, 6-dimethylnitrobenzene structure of compound (I) acts as a nitric oxide donor, and the nitro group is unstably perpendicular to the plane of the benzene ring due to the large steric hindrance caused by the methyl group ortho to the nitro group. Under the irradiation condition of an 18W yellow LED lamp source, the naphthalimide fluorescent parent absorbs irradiation light energy and transmits the irradiation light energy to a donor, NO molecules are easily released, and the 2, 6-dimethyl nitrobenzene structure is converted into a 2, 6-dimethyl nitrophenol structure (a high performance liquid chromatogram is shown in figure 2a, and a mass spectrogram is shown in figure 2 b). At this time, the fluorescent compound (I) fluoresces turn-on due to an intramolecular charge transfer effect.

The fluorescence detection method of the nitric oxide releasing fluorescent compound I comprises the following steps: the solution of compound (I) was illuminated under 18W yellow LED lamp with a lamp spacing of 20 cm. After light irradiation, fluorescence response is detected by using a microplate reader, the fluorescence excitation wavelength is set to 410nm, the maximum fluorescence emission wavelength is 520nm, and the illumination time fluorescence spectrum and the concentration gradient fluorescence spectrum of the compound I are obtained.

In particular, the fluorescent probe can be used for fluorescent confocal cell imaging. The specific method can be as follows: the compound (I) is used as a fluorescent probe, and is incubated with HeLa cells in a culture solution for 30 minutes, and fluorescence imaging is carried out under a confocal microscope after the illumination of an LED visible light source.

The invention also relates to application of the fluorescent compound in preparing a medicament for inhibiting cancer cell proliferation. The compound (I) and cancer cells are incubated for 1 hour, the lamp source is irradiated for a certain time and then is continuously cultured for 12 hours, and the survival rate of the cells is measured by an MTT method, so that the compound (I) has obvious inhibition effect on the cancer cells.

Preferably, the cancer cell is a Hela cell.

The invention has the following beneficial effects: the compound (I) can sufficiently release nitric oxide under the induction of LED yellow light, and reaches the concentration of the nitric oxide released by similar compounds under the induction of ultraviolet; meanwhile, in the PBS solution, the fluorescence response induced by the yellow light of the LED is higher than that induced by common ultraviolet (365nm/8W), so that the fluorescent response is better applied to cell biological imaging; according to the invention, an LED yellow light source (18W) is used as a trigger light source for releasing nitric oxide, and a two-photon dye is used as a compound fluorescent parent, so that the phototoxicity is greatly reduced during application, and the damage to organisms and cells is reduced; due to the good light stability and thermal stability of the conjugated structure containing the naphthalimide, the compound (I) can be irradiated in organisms for a long time and can continuously release nitric oxide for a long time, and can be applied to inhibiting the proliferation of cancer cells.

(IV) description of the drawings

FIG. 1a is a nuclear magnetic hydrogen spectrum of compound (I) in the present invention.

FIG. 1b is a mass spectrum of compound (I) according to the present invention.

FIG. 2a is a high performance liquid chromatogram of a solution of compound (I) in the present invention after light irradiation.

FIG. 2b is a mass spectrum of the photoproduct of compound (I) according to the present invention.

FIG. 3a is a mass spectrum of Compound (A) in the present invention.

FIG. 3B is a mass spectrum of the compound (B) of the present invention.

FIG. 4 is a graph showing the change of fluorescence intensity with time of illumination of compound (I) of the present invention in a 1% dimethylsulfoxide PBS solution at a concentration of 50. mu.M, with an 18W yellow LED lamp, the excitation wavelength being 410nm and the emission wavelength being 520 nm.

FIG. 5 shows the fluorescence intensity as a function of the concentration of compound (I) of the present invention in 1% dimethylsulfoxide in PBS for 30 minutes under irradiation with a lamp source, the excitation wavelength being 410nm and the emission wavelength being 520 nm.

FIG. 6 shows the change of fluorescence intensity of compound (I) in 1% dimethylsulfoxide in PBS at a concentration of 100. mu.M after 2 hours of irradiation with different light sources, with an excitation wavelength of 410nm and an emission wavelength of 520 nm.

FIG. 7a is the concentration of nitric oxide released from compound (I) in 1% DMSO-PBS at 50 μ M for different periods of time under illumination with a 18W yellow LED light source; FIG. 7b is a standard curve.

FIG. 8a is the concentration of nitric oxide released from the probe (I) in 1% dimethylsulfoxide (PBS) solution within 1 hour of illumination by the lamp at concentrations of compound (I) of 2. mu.M, 5. mu.M, 10. mu.M, 50. mu.M, and 100. mu.M; fig. 8b is a standard curve.

FIG. 9 shows the cell viability after the HeLa cells in the culture medium were irradiated with 18W yellow LED light for 10 minutes, 30 minutes, and 60 minutes and further cultured for 12 hours.

FIG. 10 shows the cell viability of compound (I) at concentrations of 2. mu.M, 5. mu.M, 10. mu.M, 50. mu.M and 100. mu.M after culturing in Hela cell culture medium for 30 minutes without exposure to light, respectively, and further for 12 hours.

FIG. 11 is a confocal fluorescent image of compound (I) in HeLa cells irradiated with light from the lamp for different periods of time. 1) 2), 3) the light irradiation time was 0 minute, 4), 5), 6) the light irradiation time was 30 minutes, 7), 8), 9) the light irradiation time was 60 minutes.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1: synthesis of Compound (I)

In a 50mL round-bottom flask, 100mg of Compound 6 (0.33mmol) was dissolved in 1mL of anhydrous tetrahydrofuran at 0 ℃ under nitrogen protection, and 40mg of NaH (60% content, 1.00mmol of NaH) dissolved in 3mL of anhydrous tetrahydrofuran was added and stirred for 1.5 hours. 70mg of Compound 8(0.23mmol) was dissolved in 3mL of anhydrous tetrahydrofuran and added to the reaction mixture, and the reaction was carried out at ordinary temperature for 6 hours. The TLC plate reaction was completed, the solvent was removed by rotary evaporation of the mixture under reduced pressure, and the concentrate was subjected to silica gel column separation (dichloromethane: methanol 20:1, v/v) to obtain 63.5mg of a red solid in 62% yield. The nuclear magnetic hydrogen spectrum is shown in figure 1a, and the mass spectrum is shown in figure 1 b.

1H NMR(600MHz,DMSO)δ8.67(d,J＝7.7Hz,2H),8.42(s, 1H),7.81–7.75(m,1H),7.58(s,2H),7.37(d,J＝16.4Hz,1H),4.03 (d,J＝6.0Hz,2H),2.30(s,6H),1.61(s,2H),1.35(dd,J＝14.7,7.4 Hz,2H),1.23(t,J＝7.0Hz,2H),0.93(t,J＝7.4Hz,3H). C₂₆H₂₄N₂O₅[M-H]444.17,found 443.48.

Example 2:

compound (I) 4.45mg of Compound (I) was accurately weighed, 10mM probe stock solution was prepared using dimethyl sulfoxide, 5. mu.L of the solution was pipetted into 995. mu. LPBS solution (pH7.2, 50mmol/L) at a concentration of 50. mu.M, and the fluorescence was measured at 37 ℃ for 3 minutes, 10 minutes, 30 minutes, 1 hour, 1.5 hours, and 2 hours using 18w yellow LED light, at an excitation wavelength of 410nm and an emission wavelength of 520nm, as shown in FIG. 4. The HPLC diagram of the light product is shown in FIG. 2a, and the mass spectrum diagram is shown in FIG. 2 b. C₂₆H₂₅NO₄[M-H]414.18, found 414.35.

Experiments prove that in PBS solution, under the condition that the emission wavelength is 520nm, the fluorescence intensity of the compound (I) due to illumination turn-on increases along with the increase of illumination time, and the fluorescence is enhanced by 60 times after 2 hours. The LC-MS spectrum simultaneously proves the generation of the photoproduct compound II in the solution and the correctness of the mechanism.

Example 3:

the pipette was pipetted 10. mu.L of the compound (I) stock solution (10mM) into 990. mu.L of PBS solution (pH7.2, 50mmol/L) at a compound (I) concentration of 100. mu.M, diluted sequentially to different concentrations (0. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 50. mu.M), irradiated with 18w yellow LED light for 30 minutes, and the fluorescence was measured at 37 ℃ under excitation at 410 nm. The emission wavelength was 520nm and the fluorescence spectrum is shown in FIG. 5.

Experiments prove that in a PBS (phosphate buffer solution), under the condition that the emission wavelength is 520nm, the fluorescence intensity of the fluorescent compound (I) is gradually enhanced along with the increase of the concentration of the compound (I) after the fluorescent compound (I) is irradiated for 30 minutes, and a certain linear relation is shown.

Example 4:

the pipette gun pipetted 10. mu.L of the compound (I) stock solution (10mM) to 990. mu.L of LPBS solution (pH7.2, 50mmol/L) at a probe concentration of 100. mu.M. Respectively irradiating under 18W yellow LED light source, ultraviolet 365nm light source (8W) and natural light for 2 hr, and measuring fluorescence value at 37 deg.C with excitation wavelength of 410nm and emission wavelength of 520nm under dark condition as control, wherein the fluorescence spectrogram is shown in FIG. 6.

Experiments prove that in the PBS solution, the compound (I) has good fluorescent response to the irradiation of an 18W LED yellow lamp source, and is superior to a common ultraviolet lamp source.

Example 5:

formulation of 0 to 100. mu.M gradient NaNO₂After adding Griess reagent, the absorbance at 540nm was measured to obtain the NO concentration standard curve (FIG. 7 b). The pipette gun pipetted 5. mu.L of the compound (I) stock solution (10mM) to 995. mu.L of PBS solution (pH7.2, 50mmol/L) at a compound (I) concentration of 50. mu.M. Set 4 sets of replicates and irradiate with 18W yellow LED lamp for 10 min, 20 min, 30min, 40 min, and 50 μ L of each sample was added to a 96 well plate. 50 μ L of Griess I and II solutions were added to each well, incubated for 10 minutes, and then the absorbance at 540nm was measured with a microplate reader to obtain the NO release concentration according to a standard curve (see FIG. 7 a).

Experiments prove that the compound (I) can effectively release nitric oxide under 18W yellow LED light, the release concentration is increased along with the illumination time, and the release duration is more than 1 hour.

Example 6:

a gradient NaNO2 solution of 0 to 100. mu.M was prepared, and the absorbance at 540nm was measured after adding Griess reagent to obtain a standard NO concentration curve (FIG. 8 b). The pipette gun pipetted 10. mu.L of the compound (I) stock solution (10mM) into 990. mu.L of PBS solution (pH7.2, 50 mmol/L). The dilution was sequentially carried out at different concentrations (0. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 50. mu.M), and 4 sets of replicates were set. After 1 hour of illumination with 18W yellow LED light, 50. mu.L of each sample was added to a clear 96-well plate. 50 μ L of Griess I and II solutions were added to each well, incubated for 10 minutes, and then the absorbance at 540nm was measured with a microplate reader, and the NO release concentration was obtained according to a standard curve (see FIG. 8 a).

Experiments prove that the probes (I) with different concentrations can effectively release nitric oxide under 18W yellow LED light, the release concentration is increased along with the concentration of the probes, and the release concentration of 100 mu M of the compound (I) nitric oxide can reach 10 mu M.

Example 7:

hela cells were cultured in a transparent 96-well plate at a constant temperature of 37 ℃ under 5% CO2, the plate was covered with the Hela cells, and then irradiated under an 18W yellow LED light source for 10 minutes, 30 minutes, and 60 minutes, and all the cells were set in 5 groups in parallel with each other as a control. And continuing to culture at constant temperature for 12 hours, sucking out the culture solution, adding fresh culture solution, adding 10 mu L (5mg/mL) of MTT PBS solution, continuing to culture for 4 hours, stopping culturing, sucking out the culture solution, adding 150 mu L of DMSO into each hole, detecting an absorption value at 490nm by using a microplate reader, setting the OD value of light-shielding culture to be 1, and calculating the survival rate of the cells. (see FIG. 9).

Experiments prove that the LED visible light source used in the invention has low phototoxicity, and the influence of irradiation for 60 minutes on the normal proliferation of cell growth does not exceed 10% of the total number of normal cultured cells.

Example 8:

hela cells were plated in clear 96-well plates at 37 ℃ with 5% CO₂The culture was carried out at a constant temperature, and after the well plate was covered, 1. mu.L of compound I at different concentrations was added to the culture solution to give final concentrations of 0. mu.M, 10. mu.M, 30. mu.M, and 50. mu.M. Culturing at 37 deg.C under 5% CO2 at constant temperature, incubating for 1 hr, irradiating under 18W yellow LED lamp for 30min, and setting 5 groups at all concentrations as control. Culturing is continued for 12 hours, the culture solution is aspirated and fresh culture solution is added, 10 mu L (5mg/mL) of MTT PBS solution is added, the culturing is stopped after continuing to culture for 4 hours, 150 mu L of DMSO is added to each well after the culture solution is aspirated, the absorbance at 490nm is detected by a microplate reader, the OD value of the light-shielded culture without the compound (I) is 1, and the cell survival rate is calculated. (see FIG. 10).

Experiments prove that after illumination, the compound (I) releases nitric oxide with a concentration exceeding the inhibition concentration, and the average inhibition rate reaches 49.1% when the compound (I) is at a concentration of 50 mu M in the co-culture with cells.

Example 9:

HeLa cells were found to be approximately 3 x 10⁵The cells were inoculated and cultured in 3 confocal plates respectively, and cultured in DMEM at 37 deg.C and 5% CO₂The incubation was performed at constant temperature under the conditions, after 24 hours of incubation, DMEM medium was removed, PBS 7.2 buffer was washed 3 times, and 1. mu.L of compound (I) in DMSO was added to 999. mu.L of fresh DMEM medium to a final concentration of 25. mu.M. After mixing uniformly, the mixture was added to the cells in a confocal dish and incubated at 37 ℃ for 15 minutes. The light was irradiated with a yellow LED light source for 0min, 30min and 60 min, respectively. And continuing to culture for 2 hours at constant temperature, removing DMEM culture solution, washing 3 times by PBS 7.2 buffer solution, and performing fluorescence imaging by using a fluorescence confocal microscope, wherein the emission wavelength is 520 nm. (see FIG. 11). 1) And 2) and 3) culturing in a dark place; 4) 5) and 6) culturing after illumination for 30 minutes; 7) 8), 9) was incubated under light for 60 minutes. Wherein 1), 4) and 7) are bright field images; 2) 5), 8) fluorescence imaging; 3) 6), 9) are superimposed images.

Experiments prove that as the illumination time increases, the fluorescent signal in the cells is gradually enhanced, and compared with the cells cultured in the dark, the morphology of part of the cells is changed. The compound (I) has the function of intracellular fluorescence imaging, and the released nitric oxide has destructive effect on the growth of cancer cells.

Claims

1. An LED visible light-induced nitric oxide-releasing fluorescent compound has a structure shown in formula (I):

。

2. a method of making the fluorescent compound of claim 1, the method comprising: under the conditions of ice bath at 0 ℃ and nitrogen protection, adding a compound shown as a formula (II) dissolved in anhydrous tetrahydrofuran into an anhydrous tetrahydrofuran solution added with NaH, continuously reacting for 1-2 hours, adding a compound shown as a formula (III) dissolved in anhydrous tetrahydrofuran, continuously reacting for 5-6 hours at room temperature, and separating and purifying after the reaction is finished to obtain a compound shown as a formula (I);

。

3. the method according to claim 2, wherein the amount of the compound of formula (II), the amount of the compound of formula (III) and the amount of NaH is 1:0.7:3, and the volume of the anhydrous tetrahydrofuran is 20mL/mmol based on the amount of the compound of formula (II).

4. The method according to claim 2, wherein the separation and purification method comprises: removing the solvent from the reaction solution by rotary evaporation under reduced pressure, and separating the concentrate by a silica gel column, wherein the reaction solution is prepared by the following steps of: and (3) taking a mixed solution with the methanol volume ratio of 20:1 as an eluent, collecting a target component, and drying to obtain the fluorescent compound shown in the formula (I).

5. Use of the fluorescent compound of claim 1 in the preparation of a fluorescent probe for LED light-induced monitoring of nitric oxide release.

6. Use of the fluorescent compound of claim 1 in the manufacture of a medicament for inhibiting proliferation of Hela cells.