CN113831756B - Red fluorescent protein two-photon photosensitive dye and preparation method and application thereof - Google Patents
Red fluorescent protein two-photon photosensitive dye and preparation method and application thereof Download PDFInfo
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- UOOWXWNVPFBLPZ-UHFFFAOYSA-N 5-methylideneimidazol-2-one Chemical compound C=C1NC(=O)N=C1 UOOWXWNVPFBLPZ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a red fluorescent protein two-photon photosensitive dye RFP-Ac and a preparation method and application thereof. The red fluorescent protein photosensitive dye molecule RFP-Ac has the characteristic of two-photon absorption, the Stokes shift exceeds 200nm, the anti-interference capability is strong, the sensitivity is high, the cytotoxicity is low, the biocompatibility is good, the yield of singlet oxygen is high, and the red fluorescent protein photosensitive dye molecule RFP-Ac can be used as a two-photon photosensitive dye and a fluorescent probe with excellent performance and can be widely applied to the aspects of near-infrared band fluorescence sensing, endogenous or exogenous bioactive mercaptan detection, two-photon fluorescence imaging, fluorescent labeling and two-photon photodynamic therapy.
Description
Technical Field
The invention relates to a fluorescent protein photosensitive dye, in particular to a red fluorescent protein two-photon photosensitive dye RFP-Ac and a preparation method and application thereof.
Background
Under the excitation of light, the photosensitive dye generates singlet oxygen at the tumor site through light-controlled energy transfer. The singlet oxygen has high toxicity to tumor cells, can react with DNA, RNA, lipid and protein molecules in the tumor cells to cause apoptosis, and realizes photodynamic therapy on the tumor cells. Cysteine and glutathione are the most representative active thiols in cells, and their concentration in tumor cells is higher than that in normal cells. Over-expression of cysteine and glutathione consumes reactive oxygen species within the cell, reducing the effectiveness of photodynamic therapy.
At present, most of photosensitive dyes for photodynamic therapy are located in a visible light region, have poor biocompatibility and lack of targeting groups, are difficult to meet the requirements of photodynamic therapy of a biological system, and greatly limit the application range of the dyes. The near-infrared two-photon photosensitive dye can effectively reduce light damage, has strong penetrating power in biological tissues, and is safe and reliable; the thiol targeting group facilitates the consumption of active thiol cysteines and glutathione that is overexpressed in tumor cells, thereby enhancing the photodynamic therapy effect. Therefore, designing and preparing a near-infrared two-photon photosensitive dye with a targeting group with excellent performance is a recent research hotspot.
The fluorescent protein is a bioluminescent macromolecule found in jellyfish, and the structure of a luminescent mother nucleus of the fluorescent protein is methylene imidazolinone. Because of the fluorophore in the organism, the fluorescent protein has excellent biocompatibility, low toxicity, large Stokes shift, small interference from the organism's own background, safety and reliability, but the near-infrared two-photon photosensitive dye based on the red fluorescent protein structure is rarely reported in the prior art.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a red fluorescent protein two-photon photosensitive dye RFP-Ac which has excellent performance, large Stokes shift, strong background interference resistance and good biocompatibility.
The second purpose of the invention is to provide a preparation method of the red fluorescent protein two-photon photosensitive dye RFP-Ac.
The third purpose of the invention is to provide the application of the red fluorescent protein two-photon photosensitive dye RFP-Ac in a near-infrared band fluorescent probe, two-photon fluorescence imaging, fluorescence sensing or fluorescence labeling.
The fourth purpose of the invention is to provide the application of the red fluorescent protein two-photon photosensitive dye RFP-Ac in serving as a near-infrared two-photon photodynamic therapy photosensitizer.
The technical scheme is as follows: in order to solve the technical problem, the invention provides a red fluorescent protein two-photon photosensitive dye RFP-Ac, which comprises the following structural formula:
the red fluorescent protein two-photon photosensitive material RFP-Ac is prepared by sequentially reacting a phenothiazine fluorescent protein analog RFP-Me with p-hydroxybenzaldehyde and acryloyl chloride, wherein the phenothiazine fluorescent protein analog RFP-Me has the following structural formula:
the invention relates to a preparation method of a red fluorescent protein two-photon photosensitive dye RFP-Ac, which comprises the following steps: under the protection of inert gas and at the temperature of 110 ℃, reacting phenothiazine fluorescent protein analogue RFP-Me with p-hydroxybenzaldehyde in dry toluene, then reacting with acryloyl chloride at room temperature, and purifying by column chromatography to obtain the red fluorescent protein two-photon photosensitive material RFP-Ac.
The yield of the red fluorescent protein two-photon photosensitive dye RFP-Ac prepared by the preparation method is 47-52 percent.
Wherein the molar ratio of the phenothiazine fluorescent protein analog RFP-Me to the p-hydroxybenzaldehyde is 1: 1.2-1: 2.0.
the invention also comprises the application of the red fluorescent protein two-photon photosensitive dye RFP-Ac in a near-infrared band fluorescent probe, two-photon fluorescence imaging, fluorescence sensing or fluorescence labeling.
The invention also discloses the application of the red fluorescent protein two-photon photosensitive dye RFP-Ac in a photosensitizer for near-infrared two-photon photodynamic therapy.
Wherein, the application is photodynamic therapy of tumor cells.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the red fluorescent protein two-photon photosensitive dye RFP-Ac comprises a conjugated structure of phenothiazine, vinylbenzene and a fluorescent protein chromophore methylene imidazolone, and an aryl acrylate is connected to the end group of a fluorophore to be used as a bioactive thiol recognition site and a photosensitizer targeting group. The fluorescent molecule can be used as an active mercaptan fluorescent probe, selective recognition of cysteine and glutathione can be realized, the Stokes displacement of the fluorescent probe molecule is up to 260nm, and the sensitivity is high. Under the irradiation of near-infrared laser, the red fluorescent protein two-photon photosensitive dye RFP-Ac can efficiently release singlet oxygen, inhibit cancer cell migration and promote tumor cell apoptosis, thereby realizing photodynamic therapy on tumor cells. The photosensitive dye molecule RFP-Ac has the characteristics of two-photon absorption, large Stokes shift, strong background interference resistance, high sensitivity, small cytotoxicity, good biocompatibility and high singlet oxygen yield, can be used as a near-infrared two-photon photosensitive dye with excellent performance, and has wide application in the aspects of near-infrared band fluorescent probes, two-photon fluorescence imaging, fluorescence sensing, fluorescence labeling, two-photon photodynamic therapy and endogenous or exogenous active thiol detection.
Drawings
FIGS. 1a 1-a 3 are fluorescence images of co-incubation of red fluorescent protein two-photon photosensitizing dye RFP-Ac with A-549 cells in the presence of bioactive thiol Cys/GSH, wherein a1 red light channel, a2 bright field channel, and a3 mixed field channel; FIG. 1b 1-b 3 shows fluorescence imaging of co-incubation of red fluorescent protein two-photon photosensitizing dye RFP-Ac and A-549 cells in the absence of thiol, wherein b1 red light channel, b2 bright field channel, and b3 mixed field channel.
FIG. 2 is a two-photon fluorescence imaging of red fluorescent protein two-photon photosensitive dye RFP-Ac incubated with zebra fish, with excitation wavelength of 800 nm.
Detailed Description
Example 1
(1) Preparation of red fluorescent protein two-photon photosensitive dye RFP-Ac:
0.23g of phenothiazine fluorescent protein analog RFP-Me is weighed and dissolved in 30mL of dry toluene under the protection of nitrogen and at the temperature of 110 ℃, 0.12g of p-hydroxybenzaldehyde is added, 5d of glacial acetic acid is added in a dropwise manner, and the mixture is stirred and reacted for 6 hours. After the reaction is finished, separating and purifying by using column chromatography. 0.08g of the resultant was weighed out, dissolved in 20mL of dry dichloromethane, and 0.15g of acryloyl chloride was added dropwise in an ice-water bath at 0 ℃ and then warmed to room temperature for reaction for 8 hours. After the reaction is finished, the red fluorescent protein two-photon photosensitive dye RFP-Ac is obtained through extraction, washing, drying and column chromatography separation, and the yield is 52%.
The synthetic route of the red fluorescent protein two-photon photosensitive dye RFP-Ac is as follows:
among them, the phenothiazine-fluorescent protein analog RFP-Me was prepared according to literature methods (Wen Hui, Zhangi, Chun Xue, Qian eagle, synthesis of phenothiazine-fluorescent protein chromophore analogs, S atom-promoted photodynamic therapy and two-photon fluorescence imaging. organic chemistry, 2021, 41(9), 3578-.
(2) Structural characterization of the red fluorescent protein two-photon photosensitizing dye RFP-Ac:
hydrogen spectrum of nuclear magnetic resonance1H NMR(600MHz，CDCl3)：δ8.08(s，1H)，8.06-7.93(m，2H)，7.63(d，J＝7.4Hz，1H)，7.22(d，J＝7.4Hz，2H)，7.13(d，J＝6.5Hz，3H)，6.84(s，3H)，6.63(t，J＝14.0Hz，2H)，6.34(dd，J＝17.3，10.4Hz，1H)，6.05(d，J＝10.4Hz，1H)，4.43(s，2H)，4.01(s，2H)，1.81(s，2H)，1.46(s，9H)，1.30(dd，J＝39.9，16.2Hz，2H)，0.97(t，J＝6.7Hz，3H).
High resolution mass spectrometry HRMS: m/z calcd for C37H37N3NaO5S[M+Na]+658.2346，found 658.2367
Therefore, the structural formula of the obtained red fluorescent protein two-photon photosensitive dye RFP-Ac is confirmed as follows:
EXAMPLE 2 preparation of two-photon photosensitizing dye RFP-Ac of Red fluorescent protein
Under the protection of nitrogen and at 110 ℃, 0.23g of phenothiazine fluorescent protein analog RFP-Me is weighed and dissolved in 30mL of dry toluene, 0.09g of p-hydroxybenzaldehyde is added, 5d of glacial acetic acid is added dropwise, the mixture is stirred and reacted for 6 hours, and the obtained product is separated after the reaction is finished. 0.08g of the resultant was weighed out, dissolved in 20mL of dry dichloromethane, and 0.15g of acryloyl chloride was added dropwise in an ice-water bath at 0 ℃ and then warmed to room temperature for reaction for 8 hours. After the reaction is finished, extracting, washing, drying and separating by column chromatography to obtain the red fluorescent protein two-photon photosensitive dye RFP-Ac with the yield of 50 percent.
EXAMPLE 3 preparation of two-photon photosensitizing dye RFP-Ac of Red fluorescent protein class
Weighing 0.20g of phenothiazine fluorescent protein analog RFP-Me under the protection of nitrogen and at the temperature of 110 ℃, dissolving in 30mL of dry toluene, adding 0.06g of p-hydroxybenzaldehyde, then dripping 5d of glacial acetic acid, stirring for reacting for 6h, finishing the reaction and separating the obtained product. 0.08g of the resultant was weighed out, dissolved in 20mL of dry dichloromethane, and 0.15g of acryloyl chloride was added dropwise in an ice-water bath at 0 ℃ and then warmed to room temperature for reaction for 8 hours. After the reaction is finished, the red fluorescent protein two-photon photosensitive dye RFP-Ac is obtained through extraction, washing, drying and column chromatography separation, and the yield is 47%.
Example 4 application of the Red fluorescent protein two-photon dye RFP-Ac as a photosensitizer for photodynamic therapy
The maximum ultraviolet-visible absorption wavelength of the red fluorescent protein two-photon photosensitive dye RFP-Ac prepared in the embodiment 1 is 498nm, the maximum fluorescence emission wavelength is 758nm, the Stokes shift is up to 260nm, and the probe RFP-Ac has the excellent properties of background interference resistance, high sensitivity and the like due to the extremely large Stokes shift.
By 1, 3-twoPhenylisobenzofuran (DPBF) as singlet oxygen scavenger, ruthenium tris-bipyridine dichloride (Ru (bpy)3Cl2) For reference, the singlet oxygen quantum yield was determined. Under the induction of active thiol cysteine (Cys) and Glutathione (GSH), the red fluorescent protein two-photon dye RFP-Ac rapidly generates singlet oxygen under the irradiation of near-infrared laser, the yield of singlet oxygen quantum is 36%, and the half-inhibition concentration IC50It was 4.2. mu.M.
Incubating red fluorescent protein two-photon photosensitive dye RFP-Ac and A-549 cells together, and taking a picture by using a confocal fluorescent microscope. The red fluorescent protein two-photon photosensitive dye RFP-Ac can quickly enter cells, has weak fluorescence in the absence of thiol, and shows strong red fluorescence in the presence of active thiol Cys/GSH. Under laser irradiation, the red fluorescent protein photosensitive dye RFP-Ac rapidly generates singlet oxygen in A-549 cells, effectively kills tumor cells, inhibits the migration of the tumor cells and promotes the apoptosis of the tumor cells, and the photodynamic therapy of the tumor cells is realized. FIGS. 1a 1-a 3 are fluorescence images of co-incubation of red fluorescent protein two-photon photosensitizing dye RFP-Ac with A-549 cells in the presence of bioactive thiol Cys/GSH, wherein a1 red light channel, a2 bright field channel, and a3 mixed field channel; FIG. 1b 1-b 3 shows fluorescence imaging of co-incubation of red fluorescent protein two-photon photosensitizing dye RFP-Ac and A-549 cells in the absence of thiol, wherein b1 red light channel, b2 bright field channel, and b3 mixed field channel.
Example 5 application of Red fluorescent protein two-photon photosensitive dye RFP-Ac as Zebra fish two-photon fluorescence imaging
The zebra fish eggs are incubated for 3 days, and then the zebra fish are evenly dispersed in a copolymerization coke tray. The red fluorescent protein two-photon photosensitive dye RFP-Ac (with the concentration of 2 mu M) prepared in the example 1 is added into a copolymerization focal dish containing the zebra fish for dyeing for half an hour, and the zebra fish is subjected to two-photon fluorescence imaging under the excitation of femtosecond laser with the wavelength of 800-. The near-infrared two-photon photosensitive dye RFP-Ac can quickly enter the zebra fish body and perform two-photon fluorescence imaging in a red light channel. FIG. 2 is a two-photon fluorescence imaging of zebra fish under 800nm laser excitation.
In conclusion, the red fluorescent protein two-photon photosensitive dye RFP-Ac comprises a conjugated structure of phenothiazine, vinylbenzene and fluorescent protein methylene imidazolidinone, and meanwhile, aryl acrylate is introduced into a terminal group of a fluorescent chromophore as an active thiol recognition site and a photosensitizer targeting group. The fluorescent molecule can be used as a bioactive thiol fluorescent probe, and can realize selective recognition on cysteine and glutathione. Under the excitation of near-infrared laser, the red fluorescent protein two-photon photosensitive dye RFP-Ac can efficiently release singlet oxygen, inhibit cancer cell migration and promote tumor cell apoptosis, thereby realizing photodynamic therapy on tumor cells. The red fluorescent protein photosensitive dye molecule RFP-Ac has the characteristic of two-photon absorption, the Stokes shift exceeds 200nm, the anti-interference capability is strong, the sensitivity is high, the cytotoxicity is small, the biocompatibility is good, the yield of singlet oxygen is high, and the red fluorescent protein photosensitive dye molecule RFP-Ac can be used as a two-photon photosensitive dye and a fluorescent probe with excellent performance and can be widely applied to the aspects of near-infrared band fluorescence sensing, endogenous or exogenous bioactive mercaptan detection, two-photon fluorescence imaging, fluorescent labeling and two-photon photodynamic therapy.
Claims (7)
1. A red fluorescent protein two-photon photosensitive dye RFP-Ac is characterized by comprising the following structural formula:
2. the red fluorescent protein two-photon photosensitizing dye RFP-Ac according to claim 1, wherein the photosensitizing dye RFP-Ac is prepared by reacting a phenothiazine fluorescent protein analog RFP-Me with p-hydroxybenzaldehyde and acryloyl chloride in sequence, and the RFP-Me has the following structural formula:
3. the method of claim 1 or 2, wherein the method comprises the following steps: reacting phenothiazine fluorescent protein analog RFP-Me with p-hydroxybenzaldehyde in anhydrous toluene under the protection of inert gas at 110 ℃, then reacting with acryloyl chloride at room temperature, and purifying by column chromatography to obtain the red fluorescent protein two-photon photosensitive dye RFP-Ac.
4. The method for preparing the red fluorescent protein two-photon photosensitive dye RFP-Ac according to claim 3, wherein the molar ratio of the phenothiazine fluorescent protein analog RFP-Me to p-hydroxybenzaldehyde is 1: 1.2-1: 2.0.
5. The use of the red fluorescent protein two-photon photosensitizing dye according to claim 1 or 2 in a near-infrared band fluorescent probe, two-photon fluorescence imaging, fluorescence sensing or fluorescence labeling.
6. Use of a red fluorescent protein two-photon photosensitizing dye according to claim 1 or 2 in the preparation of a medicament as a photosensitizer for near-infrared two-photon photodynamic therapy.
7. Use according to claim 6, wherein the medicament is a medicament for photodynamic therapy of tumour cells.
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