阅读说明：本技术 Led可见光诱导释放一氧化氮的荧光化合物及其制备与应用 (Fluorescent compound for LED visible light induced release of nitric oxide and preparation and application thereof ) 是由 朱勍 蒋建泽 刘江 于 2019-07-16 设计创作，主要内容包括：本发明涉及一种LED可见光诱导释放一氧化氮的荧光化合物,(I),及其制备方法与应用。本发明化合物(I)在LED黄光诱导下,能充足释放一氧化氮,达到了类似化合物以紫外诱导释放的一氧化氮浓度；同时在PBS溶液中LED黄光(18W)诱导的荧光响应高于常见紫外(8W)诱导的荧光响应,这更好的应用于细胞生物成像；本发明采用LED黄色光源作为一氧化氮释放的触发光源,双光子染料作为化合物荧光母体,在应用时的光毒性大大减弱,生物和细胞损害减小；由于包含内萘酰亚胺的共轭结构有良好的光稳定性和热稳定性,化合物(I)能够在生物体中长时间进行光照射,并长时间地持续释放一氧化氮,可应用于抑制癌细胞增殖。<Image he="553" wi="700" file="DDA0002132051510000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention relates to a fluorescent compound (I) capable of releasing nitric oxide under the induction of LED 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; since the conjugated structure containing the naphthalimide has good photostability and thermal stability, the compound (I) can be irradiated with light in a living body for a long time,and can release nitric oxide continuously for a long time, and can be used for inhibiting cancer cell proliferation.)

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 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;

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

6. The use according to claim 5, wherein said fluorescent probe is used for LED light-induced monitoring of nitric oxide release.

7. The use of claim 5, wherein said fluorescent probe is used for fluorescence confocal cell imaging.

8. Use of the fluorescent compound of claim 1 in the preparation of a medicament for inhibiting cancer cell proliferation.

9. The use of claim 8, wherein said cancer cell is a Hela cell.

(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 hydro xynaphalimide-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: