CN107501104B - Formaldehyde fluorescent nano probe intermediate with double-signal turn-on output and preparation and application thereof - Google Patents
Formaldehyde fluorescent nano probe intermediate with double-signal turn-on output and preparation and application thereof Download PDFInfo
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- CN107501104B CN107501104B CN201710650693.2A CN201710650693A CN107501104B CN 107501104 B CN107501104 B CN 107501104B CN 201710650693 A CN201710650693 A CN 201710650693A CN 107501104 B CN107501104 B CN 107501104B
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- C07C217/54—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
- C07C217/56—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains not further substituted by singly-bound oxygen atoms
- C07C217/58—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains not further substituted by singly-bound oxygen atoms with amino groups and the six-membered aromatic ring, or the condensed ring system containing that ring, bound to the same carbon atom of the carbon chain
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- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention discloses a preparation method and application of a mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe intermediate (III). the method comprises the steps of taking p-hydroxybenzaldehyde as an initial raw material, activating at the temperature of 60-70 ℃ in the presence of an acid binding agent, and then taking 3-bromopropyne as a nucleophilic reagent to perform nucleophilic substitution reaction in an acetone solvent at the temperature of 60-70 ℃ to obtain a compound (II); ammoniating the compound (II) by using an ammonia methanol solution at 0 ℃, adding propenyl boronic acid ortho-di-tert-alcohol ester at 0 ℃, mixing, and reacting at the temperature of 25-35 ℃ to obtain the compound shown in the formula (III). The compound shown in the formula (III) can be used as a formaldehyde fluorescent nano probe intermediate for preparing double-signal turn-on. The nano probe has almost no fluorescence in water, and two free fluorescent substances are obtained after the nano probe reacts with formaldehyde, so that the effect of double turn-on is realized, and the accuracy of a detection result is improved.
Description
Technical Field
The invention relates to a formaldehyde fluorescent nano probe intermediate with double-signal turn-on output, and preparation and application thereof.
Background
1. Formaldehyde, as a carcinogen, is the smallest gas molecule with carbonyl compounds. On the one hand, formaldehyde has a strong stimulating effect on the respiratory system and eyes of a human body, and the lung function of the human body can be reduced after the human body is exposed to the formaldehyde environment for a long time. On the other hand, formaldehyde is pathogenic to the human body. Excessive formaldehyde in vivo can cause various cancers such as digestive system cancer, lung cancer, etc., neurodegenerative diseases, Alzheimer's disease, etc. At present, most of the formaldehyde detection technologies can only detect formaldehyde in air or water, and cannot achieve the effect of real-time monitoring in living bodies or cells. Therefore, it is required to develop a technique capable of detecting the concentration of formaldehyde in cells in real time.
2. Fluorescent probes are used as a detection technology with the advantages of high efficiency, sensitivity, strong specificity, real-time monitoring and the like, and are applied by extensive researchers. Among a plurality of fluorescent probes capable of detecting formaldehyde in cells, no report is made on the effect of achieving a double-signal turn-on effect by realizing a nano fluorescent probe through self-assembly. The invention aims to develop an intermediate compound of a formaldehyde fluorescent nano probe output by a double-signal turn-on, the compound has two characteristics, one end of the compound has alkyne, and the compound can carry out click chemistry with azide and conveniently react with other compounds; the other end has homoallylic alcohol amine, which can generate aldehyde group with formaldehyde.
Disclosure of Invention
The invention aims to provide a formaldehyde fluorescent nano probe intermediate with double-signal turn-on output, and a preparation method and application thereof.
The invention adopts the following technical scheme for realizing the purpose:
a compound of formula (III):
a method for preparing a compound represented by formula (III), the method comprising: (1) activating p-hydroxybenzaldehyde serving as an initial raw material at the temperature of 60-70 ℃ in the presence of an acid-binding agent, then performing nucleophilic substitution reaction in an acetone solvent at the temperature of 60-70 ℃ by using 3-bromopropyne as a nucleophilic reagent, and performing aftertreatment on A to obtain a compound (II); (2) ammoniating the compound (II) obtained in the step (1) at 0 ℃, adding propenyl boronic acid ortho-di-tertiary alcohol ester at 0 ℃, mixing, reacting at 25-35 ℃, and performing post-treatment B to obtain the compound shown in the formula (III);
further, the acid-binding agent in the preparation method is potassium carbonate. The dosage of the acid-binding agent is 1.5 times of the equivalent of the p-hydroxybenzaldehyde.
Further, in the preparation method, the mass ratio of the p-hydroxybenzaldehyde to the 3-bromopropyne in the step (1) is 1: 1.5-3, preferably 1: 2.
Further, the ammonia concentration in the ammonia methanol solution in the step (2) in the above production method is 7 mol/L.
Further, the amount ratio of the theoretical substance of the compound (II) to ammonia in the ammonia methanol solution in the above production method is 1: 6-20, preferably 1: 10.
further, the amount ratio of the theoretical substance of the compound (II) to the ortho-di-tert-alcohol ester of propenylboronic acid in the above preparation method is 1: 1.2-2, preferably 1: 1.5.
Further, the post-treatment A in the preparation method is as follows: removing the solvent from the reaction liquid by rotary evaporation under reduced pressure, adding water, extracting with ethyl acetate, combining organic phases, washing the organic phases with water and saturated saline solution for a plurality of times, drying with anhydrous sodium sulfate, filtering, drying the solvent by rotary evaporation, and carrying out column chromatography separation to obtain a target product, wherein an eluant is ethyl acetate and petroleum ether with the volume ratio of 1: 10;
the post-treatment B comprises the following steps: and (3) carrying out reduced pressure rotary evaporation on the reaction liquid to remove the solvent, carrying out chromatographic column separation on the crude product to obtain a target product, wherein the eluent is dichloromethane and methanol with the volume ratio of 40: 1.
In addition, the invention also provides application of the compound shown in the formula (III) as a formaldehyde fluorescent nano probe intermediate for preparing the double-signal turn-on.
Further, the preparation method of the formaldehyde fluorescent nanoprobe with the double signal turn-on prepared by the compound shown in the formula (III) is as follows:
under an acidic condition, generating a Schiff base intermediate product by using the compound (III) and the compound (IV), and reducing a carbon-nitrogen double bond of the Schiff base intermediate product by using a reducing agent sodium triacetoxyborohydride to generate a compound (V); the compound (VI) and 3-azidopropylamine are subjected to amide reaction to generate a compound (VII); finally, the compound (V) and the compound (VII) are catalyzed by monovalent copper to generate a compound (I); dissolving the compound (I) in DMSO (dimethyl sulfoxide) to serve as mother liquor, diluting the mother liquor with ultrapure water or PBS (phosphate buffer solution) or cell culture solution, performing ultrasonic treatment for several minutes, then violently shaking, and performing self-assembly on the compound (I) to obtain the formaldehyde fluorescent nano probe with the double-signal turn-on;
the reaction route of the formaldehyde fluorescent nano probe with the double-signal turn-on is as follows:
furthermore, the method for preparing the formaldehyde fluorescent nano probe with the double signal turn-on by using the compound shown in the formula (III) specifically comprises the following steps:
(1) mixing the compound (IV), the compound (III) and sodium triacetoxyborohydride according to the mass ratio of theoretical substances of 1:1.2:4 at 0 ℃ under an acidic condition, then transferring to room temperature for reaction, and preparing a compound (V) through post-treatment C; the reaction time is 12 hours, and the reaction solvent is tetrahydrofuran;
(2) reacting the compound (VI) with 3-azidopropylamine according to the mass ratio of 1:1.2, and carrying out post-treatment D to prepare a compound (VII); the reaction temperature is room temperature, the reaction time is 10 hours, and the reaction solvent is dichloromethane;
(3) reacting a compound (V) with a compound (VII) according to the quantitative ratio of theoretical substances of 1:1 under the catalysis of monovalent copper, evaporating the solvent to dryness, and purifying the crude product by using a high performance liquid chromatography to obtain a compound (I); the reaction temperature is room temperature, the reaction time is 6 hours, and the reaction solvent is tetrahydrofuran and water;
(4) dissolving a compound (I) in DMSO to prepare a probe mother solution with the concentration of 0.1-2 mM, diluting the probe mother solution to 99 times of the original mother solution volume by using ultrapure water or PBS buffer solution or DMEM culture medium, performing ultrasonic treatment for several minutes, then violently shaking, and performing self-assembly on the compound (I) to obtain the fluorescent nano probe.
The compound (IV) of the present invention is a compound disclosed in the following references [ H.park, S. -K.Chang, signalling of water content in organic solvents by solvents, a-based meritorium, DyesPigm.122(2015) 324-330 ].
Further, the acidic condition in the step (1) of the method is realized by using acetic acid, and the mass ratio of the acetic acid to the compound (IV) is 8-20: 1, preferably 10: 1.
Further, 1-hydroxybenzotriazole and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride can be added in the step (2) of the method to serve as an amide reaction condensing agent, triethylamine serves as an acid-binding agent, and the recommended ratio of the three substances is 3:3: 4.
The post-treatment C in the method comprises the following steps: carrying out reduced pressure rotary evaporation on the reaction liquid to remove the solvent, carrying out chromatographic column separation on the crude product to obtain a target product, wherein an eluent is dichloromethane and methanol with the volume ratio of 20: 1;
the post-treatment D is as follows: separating the reaction solution by thin layer chromatography, wherein the developing agent is dichloromethane and methanol with the volume ratio of 20:1 to obtain the target compound.
In addition, the formaldehyde fluorescent nano probe with the double-signal turn-on can be used for detecting the concentration of formaldehyde.
Furthermore, the formaldehyde exists in the form of an aqueous solution, the concentration of the formaldehyde is 0-5 mmol/L, and the meaning of the concentration of 0 is infinitely close to 0 but not equal to 0.
Furthermore, the formaldehyde is preferably intracellular formaldehyde, and the concentration of the intracellular formaldehyde is 0-1 mmol/L.
Still further, the cell of the invention is a human breast cancer cell MCF-7.
The formaldehyde fluorescent nano probe with the double-signal turn-on is the mitochondria-targeted formaldehyde fluorescent nano probe nano-MTDF with the double-signal turn-on, which is called nano probe nano-MTDF for short.
The formaldehyde fluorescent nano probe which is self-assembled by the compound (I) and can be used as a mitochondrion-targeted double-signal turn-on can be applied to the fluorescent quantitative detection of formaldehyde. The fluorescence detection principle of the quantitative formaldehyde concentration is as follows: the method comprises the steps of taking a nano probe nano-MTDF as a fluorescent probe, reacting with formaldehyde to generate an intermediate product, then carrying out 2-aza-coppu rearrangement and hydrolysis to generate a fluorescent substance compound VIII and a fluorescent substance compound IX, and measuring the fluorescence intensity change under the excitation of 440nm and 535nm, thereby obtaining the formaldehyde concentration.
The principle of detecting the concentration of formaldehyde in water by using the novel mitochondrion-targeted double-signal turn-on formaldehyde fluorescent probe is as follows:
the mitochondrion-targeted double-signal turn-on formaldehyde fluorescent nano probe has almost no fluorescence in water, namely the fluorescence of two fluorophores 1, 8-naphthalimide and rhodamine B in the probe is quenched, wherein the principle is that the fluorescence of the 1, 8-naphthalimide is quenched due to fluorescence resonance energy transfer, meanwhile, the hydrophobicity of the 1, 8-naphthalimide causes the compound (I) to be assembled into nano particles in water, and the fluorescence of the rhodamine B is quenched due to aggregation-induced quenching. When the nano-probe nano-MTDF reacts with formaldehyde, two separated fluorophores are released, the nano-probe is self-assembled and removed, and the fluorescence of the 1, 8-naphthalimide and the rhodamine B is recovered simultaneously, so that the effect of a double-signal turn-on is realized. Wherein N in the rhodamine B structure+It also has the effect of targeting mitochondria.
Compared with the prior art, the invention has the following beneficial effects: firstly, the invention takes p-hydroxybenzaldehyde as raw material to synthesize the target intermediate compound through two steps. The reaction condition is mild, no inflammable and explosive reagent is used, and the industrial production scale is easy to realize. Secondly, the invention provides a new formaldehyde fluorescent nano probe intermediate with double-signal turn-on output, one end of the compound has alkyne, and the compound is easy to perform addition reaction with other compounds with azide groups; the other end of the catalyst has homoallylic alcohol amino group which is firstly reacted with formaldehyde to generate Schiff base, and then subjected to cope rearrangement and hydrolysis to generate aldehyde group, and the reaction has strong specificity to formaldehyde. In conclusion, the compound provides an effective synthetic intermediate for the formaldehyde fluorescent nano probe with double signal turn-on.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of compound (I) prepared in example 1 of the present invention.
FIG. 2 shows a nuclear magnetic carbon spectrum of Compound (I) prepared in example 1 of the present invention.
FIG. 3 shows the particle size of the nanoprobe nano-MTDF (1 μ M) prepared in example 1 of the present invention measured by dynamic light diffraction under DMSO/water (v/v: 1/99) and the nanoparticle imaging by transmission electron microscopy (standard size 1 μ M).
FIG. 4 is a fluorescence emission spectrum of the nano-probe nano-MTDF (1 μ M) prepared in example 1 in different DMSO/water ratios. The graph a shows the fluorescence emission spectrum with an excitation wavelength of 440 mm. Panel b shows fluorescence emission spectra with excitation wavelength of 535 nm.
FIG. 5 shows fluorescence emission spectra of the nanoprobe nano-MTDF (1 μ M) prepared in example 1 of the present invention under DMSO/PBS buffer (pH 7.4, v/v 1/99) with different equivalents of formaldehyde added. The graph a shows the fluorescence emission spectrum with an excitation wavelength of 440 mm. Panel b shows fluorescence emission spectra with excitation wavelength of 535 mm.
FIG. 6 is a fluorescence diagram showing the change with time of the nanoprobe nano-MTDF (1. mu.M) prepared in example 1 of the present invention in the presence of formaldehyde (1mM) in DMSO/PBS buffer (pH 7.4, v/v 1/99). Graph a excitation wavelength 440nm and emission wavelength 540 nm. Panel b excitation wavelength 535nm and emission wavelength 585 nm.
FIG. 7 is a fluorescence diagram showing the selective results of the nanoprobe nano-MTDF (1 μ M) prepared in example 1 of the present invention under the condition of DMSO/PBS buffer (pH 7.4, v/v 1/99). 1-17 are PBS, formaldehyde, acetaldehyde, methylglyoxal, benzaldehyde, p-nitrobenzaldehyde, p-hydroxybenzaldehyde, acetone, formic acid, sodium pyruvate, glucose, glutathione, homocysteine, cysteine, sodium hydrogen sulfate, hydrogen peroxide and tert-butyl hydroperoxide respectively. Graph a excitation wavelength 440nm and emission wavelength 540 nm. Panel b excitation wavelength 535nm and emission wavelength 585 nm.
FIG. 8 is a fluorescence image of the nanoprobe nano-MTDF (1. mu.M) prepared in example 1 of the present invention before and after reacting with formaldehyde under the condition of DMSO/different pH buffer (v/v. 1/99). Graph a excitation wavelength 440nm and emission wavelength 540 nm. Panel b excitation wavelength 535nm and emission wavelength 585 nm.
Fig. 9 shows a high performance liquid chromatogram and a mass spectrum of the nanoprobe nano-MTDF prepared in example 1 of the present invention before and after adding formaldehyde under the condition of DMSO/PBS buffer (pH 7.4, v/v 1/99).
FIG. 10 is the fluorescence imaging of formaldehyde in cells by the nanoprobe nano-MTDF prepared in example 1 of the present invention.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1(1) preparation of Compound (II)
1.22g of p-hydroxybenzaldehyde (10mmol) was dissolved in 50mL of acetone solution, followed by addition of 2.07g (15mmol) of potassium carbonate, addition of 2.37g of 3-bromopropyne (20mmol) after half an hour at 60-70 ℃ and reaction at 60-70 ℃ for 2 hours, and then the solvent was distilled off under reduced pressure. To the mixture was added water, extracted with ethyl acetate, the organic phases were combined, washed several times with water and saturated brine, dried over anhydrous sodium sulfate, filtered, the solvent was dried by spinning, and separated by column chromatography (ethyl acetate: petroleum ether ═ 1:10 as eluent) to give compound (II) as a white solid (1.53g, 95% yield).1H NMR(500MHz,CDCl3)δ9.88(s,1H),7.95–7.75(m,2H),7.18–6.98(m,2H),4.77(d,J＝2.4Hz,2H),2.58(t,J＝2.4Hz,1H).13C NMR(126MHz,CDCl3)δ190.69,162.30,131.81,130.51,115.11,77.51,76.35,55.87.ESI calcd.forC10H8O2[M+H]+161.05,found 161.18。
(2) Preparation of Compound (III)
0.64g of compound (II) (4mmol) was dissolved in 40mL of methanol, ice-cooled to 0 ℃ and,6mL of an methanolic ammonia solution (7mol/L, 42mmol) was added, the reaction was carried out at 0 ℃ for half an hour, and then 1g of vicinal-di-tert-butyl propenyl borate (6mmol) was added, the reaction was shifted to 25-35 ℃ and the reaction was carried out overnight. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was subjected to column chromatography (dichloromethane: methanol 40:1 as eluent) to give compound (III) as a colorless oily liquid (0.613g, 76% yield).1H NMR(500MHz,CDCl3)δ7.32–7.25(m,2H),6.99–6.91(m,2H),5.79–5.71(m,1H),5.16–5.05(m,2H),4.69(d,J＝2.4Hz,2H),3.98–3.95(m,1H),2.53(t,J＝2.4Hz,1H),2.49–2.41(m,1H),2.40–2.29(m,1H).13C NMR(126MHz,CDCl3)δ156.51,138.87,135.46,127.35,117.56,114.75,78.65,75.42,55.82,54.71,44.17.ESI calcd.for C13H15NO[M+H]+202.12,found 202.29。
(3) Preparation of Compound (V)
0.15g of compound (IV) (0.5mmol) was added to 15mL of anhydrous tetrahydrofuran, cooled to 0 ℃ and then 0.18g of compound (III) (0.6mmol), 0.3g of acetic acid (5mmol) and 0.42g of sodium triacetoxyborohydride (2mmol) were sequentially added. The reaction was switched to 20-30 ℃ and reacted overnight. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was subjected to column chromatography (dichloromethane: methanol ═ 20:1) to give compound (V) as an orange solid (0.14g, 59% yield).1H NMR(500MHz,CDCl3)δ8.38(t,J＝7.2Hz,2H),7.93(s,1H),7.53(t,J＝7.8Hz,1H),7.30(d,J＝8.6Hz,2H),7.03(d,J＝8.6Hz,2H),5.81-5.73(m,1H),5.22–5.13(m,2H),4.71(d,J＝2.4Hz,2H),4.13–4.05(m,2H),4.02(d,J＝14.2Hz,1H),3.91(d,J＝14.2Hz,1H),3.81(t,J＝7.0Hz,1H),2.70–2.59(m,2H),2.56(t,J＝2.3Hz,1H),1.70–1.64(m,2H),1.49–1.37(m,2H),0.97(q,J＝7.8Hz,3H).13CNMR(126MHz,CDCl3)δ164.60,163.76,162.42,157.39,133.86,132.93,132.48,131.08,128.81,128.29,124.93,123.02,121.68,118.77,115.34,111.71,78.39,75.74,61.41,55.85,49.83,41.43,39.98,30.21,24.81,20.39,13.85.ESI calcd.For C30H30N2O4[M–H]-481.22,found 481.27。
(4) Preparation of Compound (VII)
0.06g of Compound (VI) (0.1mmol) was dissolved in 5mLTo dichloromethane, 1-hydroxybenzotriazole (0.02g, 0.15mmol), 0.03g 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.15mmol), 0.02g triethylamine (0.2mmol) and 0.01g 3-azido-propylamine (0.12mmol) were added in this order, reacted at room temperature overnight, and after the reaction was completed, the crude product was chromatographed with preparative thin layer chromatography (dichloromethane: methanol 20:1) gave compound (VII) (0.057g, 82% yield) as a dark red solid.1H NMR(400MHz,DMSO)δ7.89(s,1H),7.83–7.66(m,3H),7.54(s,1H),7.13(dt,J＝9.7,5.6Hz,4H),6.95(d,J＝1.7Hz,2H),3.66(dd,J＝13.6,6.5Hz,8H),3.56–3.16(m,10H),3.08(dd,J＝12.2,6.2Hz,2H),2.50(d,J＝1.5Hz,3H),2.29(s,2H),1.73–1.49(m,2H),1.21(t,J＝6.9Hz,12H).13C NMR(75MHz,DMSO)δ171.70,170.45,166.94,159.23,158.77,158.30,157.83,157.44,155.95,155.51,135.64,132.13,131.05,130.76,130.14,127.87,118.34,114.63,113.41,96.29,48.73,47.13,45.75,44.63,41.44,36.12,30.59,28.80,28.07,12.76.ESI calcd.for C39H49N8O4[M]+693.39,found693.48。
(5) Preparation of Compound (I)
0.027g of Compound (VII) (0.04mmol) and Compound (V) (0.04mmol) were dissolved in 3mL of tetrahydrofuran, and an aqueous solution (3mL) of 0.004g of vitamin C (0.02mmol) and 0.003g of copper sulfate pentahydrate (0.02mmol) were added to the above mixture, followed by reaction at room temperature for 6 hours. The solvent was evaporated to dryness and the crude product was purified by preparative high performance liquid chromatography to give dark red old compound (I) (0.03g, 65% yield). Where vitamin C acts to reduce the divalent copper to monovalent copper.
1H NMR(300MHz,DMSO)δ8.43(d,J＝7.9Hz,1H),8.26(t,J＝3.5Hz,2H),7.98(s,2H),7.72(ddd,J＝13.2,7.3,4.6Hz,3H),7.46(ddd,J＝28.1,12.0,6.5Hz,4H),7.18–7.01(m,6H),6.91(d,J＝11.8Hz,2H),5.56(td,J＝17.0,6.8Hz,1H),5.16(s,2H),5.04(t,J＝12.8Hz,2H),4.38(t,J＝6.8Hz,2H),4.24(dd,J＝9.2,5.5Hz,2H),3.96(dd,J＝20.2,13.1Hz,6H),3.63(d,J＝6.9Hz,18H),3.25(dd,J＝33.1,17.3Hz,10H),3.04(d,J＝5.9Hz,3H),2.94–2.58(m,3H),2.30(s,2H),1.93(dd,J＝11.2,4.5Hz,2H),1.63–1.43(m,2H),1.30(dd,J＝14.8,7.4Hz,2H),1.19(dd,J＝12.5,5.8Hz,12H),0.90(t,J＝7.3Hz,3H).13C NMR(75MHz,DMSO)δ175.28,171.87,170.47,166.91,164.51,163.03,158.72,157.38,155.91,155.46,142.82,135.74,135.61,133.43,132.07,131.86,131.05,130.88,130.77,130.67,130.18,130.08,130.03,128.28,127.86,126.90,125.00,122.23,121.37,119.09,115.27,114.60,114.28,113.37,100.35,96.26,61.61,60.13,48.00,47.57,47.05,45.75,41.46,38.96,37.95,36.03,30.59,30.35,30.29,29.33,28.79,28.10,20.20,14.13,12.78.HRMS(ESI)calcd.for C69H79N10O8[M]+1175.6082,found1175.6067。
(6) Preparation of nano probe nano-MTDF
Dissolving the compound (I) in DMSO as a mother solution, adding the mother solution into ultrapure water, PBS buffer solution or cell culture solution, performing ultrasonic treatment for several minutes, and then performing vigorous shaking to obtain the nano-probe nano-MTDF.
Example 2 nanoprobe nano-MTDF particles were tested for particle size by dynamic light diffraction in DMSO/water buffer (pH 7.4, v/v 1/99) and nanoparticles imaged by transmission electron microscopy.
Accurately weighing a certain amount of the compound (I) prepared in example 1, preparing a probe mother solution with the concentration of 0.1mM by DMSO, sucking 0.02mL by a pipette gun, adding into 1.98mL of water, carrying out ultrasonic treatment for several minutes, then violently shaking to obtain a nano probe nano-MTDF, then measuring the particle size of the nano-MTDF in the water by using a nano-zs90particle analyzer, simultaneously dripping the mixed solution on a copper net, drying at 37 ℃ and carrying out projection electron microscope imaging, wherein the result is shown in figure 3.
Referring to FIG. 3(a), it can be seen that the dynamic light diffraction test results in that the average particle diameter of the particles was 161.9nm and the polydispersity PDI index was 0.262. Referring to FIG. 3(b), the nanoparticle size obtained from the transmission electron microscope (standard bar: 1 μm) was substantially identical to the data obtained from dynamic light diffraction, thus confirming that Compound (I) formed a nanomaterial in water.
Example 3 fluorescence spectroscopy detection of nanoprobe nano-MTDF (1. mu.M) at different DMSO/water ratios.
An amount of compound (I) prepared in example 1 was weighed out accurately, prepared with DMSO at 0.1mM concentration as a probe stock solution, pipetted 0.02mL into 1.98mL of different DMSO/water ratios (DMSO 1%, 5%, 10%, 20%, 40%, 60%, 70%, 80%, 90%), sonicated for several minutes, then shaken vigorously, and then the fluorescence spectrum of compound (I) was determined.
The experimental result shows that the fluorescence of the compound (I) is enhanced along with the increase of the proportion of DMSO, thereby proving that the aggregation effect of the compound (I) is enhanced along with the decrease of the proportion of DMSO, the fluorescence at different excitation wavelengths is weakened, and the fluorescence of rhodamine B is quenched due to aggregation. Meanwhile, the probe has only one emission peak under the excitation wavelength of 440nm no matter the proportion of DMSO, which indicates that the fluorescence of naphthalimide is quenched by rhodamine B through fluorescence resonance energy transfer. The fluorescence spectrum is shown in FIG. 4.
Example 4 the nanoprobe nano-MTDF (1 μ M) of the present invention was subjected to fluorescence spectroscopy under DMSO/PBS buffer (pH 7.4, v/v 1/99) with different equivalents of formaldehyde added.
Accurately weighing a certain amount of the probe (I) prepared in example 1, preparing a mother solution with the concentration of 0.1mM by using dimethyl sulfoxide, sucking 0.02mL by using a pipette gun, adding the mother solution into 1.96mL of PBS buffer solution, carrying out ultrasonic treatment for several minutes, then carrying out vigorous shaking to obtain the nano-probe nano-MTDF, sucking 396 mu L of nano-probe nano-MTDF solution each time, adding 4 mu L of formaldehyde solutions with different equivalent weights (the final concentrations of formaldehyde in water are respectively 0, 0.0025, 0.0075, 0.01, 0.025, 0.04, 0.05, 0.06, 0.075, 0.1, 0.15, 0.25, 0.4, 0.5, 0.6, 0.75, 1, 2and 5mM), reacting at 37 ℃ for 3 hours, and then determining the fluorescence value. The excitation wavelength was 440nm or 535nm, respectively, and the fluorescence spectrum is shown in FIG. 5.
The experimental result shows that with the increase of formaldehyde equivalent, two free fluorophores generated by the nano-probe nano-MTDF are increased, and the fluorescence intensity of the two fluorophores is respectively increased.
Example 5 fluorescence profiles of nanoprobes nano-MTDF (1 μ M) of the present invention with time during the action with formaldehyde (1mM) in DMSO/PBS buffer (pH 7.4, v/v 1/99).
Accurately weighing a certain amount of probe (I), preparing a mother solution with the concentration of 0.1mM by using dimethyl sulfoxide, sucking 0.02mL by a pipette gun, adding the mother solution into 1.96mL of PBS buffer solution, carrying out ultrasonic treatment for several minutes, then violently shaking to obtain the nano-probe nano-MTDF, sucking 396 mu L of nano-probe nano-MTDF solution each time, adding 4 mu L of formaldehyde aqueous solution (finally, the concentration of formaldehyde in water is 1mM), reacting at 37 ℃, and measuring the fluorescence value of the nano-probe at different time points (0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 hours respectively). Fluorescence spectrum 6 (a): excitation wavelength was 440nm, emission wavelength was 540nm, fluorescence spectrum is shown in FIG. 6 (b): the excitation wavelength was 535nm and the emission wavelength was 585 nm.
Experiments prove that the fluorescence intensity of the two fluorophores can be enhanced along with the increase of time, and the effect of detecting formaldehyde by the probe is met.
Example 6 fluorescence spectroscopy detection of the selectivity results of nanoprobes nano-MTDF (1 μ M) in the present invention under DMSO/PBS buffer (pH 7.4, v/v 1/99).
Accurately weighing a certain amount of probe (I), preparing mother liquor with the concentration of 0.1mM by using dimethyl sulfoxide, sucking 0.02mL by a pipette, adding the mother liquor into 1.96mL of PBS buffer solution, carrying out ultrasonic treatment for several minutes, then violently oscillating to obtain the nano-probe nano-MTDF, sucking 396 mu L of nano-probe nano-MTDF solution each time, respectively adding 4 mu L of formaldehyde aqueous solution (the final concentration of formaldehyde in water is 1mM) and biologically-related active small molecule aqueous solution (acetaldehyde, methylglyoxal, acetone, formic acid, 4-hydroxybenzaldehyde, 4-nitrobenzaldehyde, benzaldehyde, hydrogen peroxide, tert-butyl hydroperoxide, sodium hydrosulfide, glutathione, cysteine, homocysteine, sodium pyruvate and glucose, the final concentration is 1mM), reacting for 3 hours at 37 ℃, and determining the fluorescence value. Fluorescence spectrum 7 (a): excitation wavelength is 440nm, emission wavelength is 540nm, fluorescence spectrum is shown in FIG. 7 (b): the excitation wavelength was 535nm and the emission wavelength was 585 nm.
Experimental results show that the fluorescence intensity of the nano-probe nano-MTDF is basically unchanged in the presence of other related bioactive molecules except formaldehyde, and the anti-interference capability of the nano-probe nano-MTDF is very good, namely the specificity of the nano-probe nano-MTDF to formaldehyde is good.
Example 7 fluorescence spectra of the nanoprobe nano-MTDF (1 μ M) of the present invention before and after reaction with formaldehyde in DMSO/different pH buffer (v/v-1/99) were detected.
Accurately weighing a certain amount of probe (I), preparing a mother solution with the concentration of 0.1mM by using dimethyl sulfoxide, sucking 0.02mL by a pipette gun, adding the 0.02mL into 1.96mL of buffer solutions with different pH values (the pH values are respectively 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 and 10.5), carrying out ultrasonic treatment for a plurality of minutes, then violently shaking to obtain the nano-probe nano-MTDF, sucking 396 mu L of nano-probe nano-MTDF solution each time, adding 4 mu L of formaldehyde aqueous solution (the final concentration of formaldehyde in water is 0 and 1mM), reacting at 37 ℃ for 3 hours, and determining the fluorescence value. Fluorescence spectrum 8 (a): excitation wavelength was 440nm, emission wavelength was 540nm, fluorescence spectrum is shown in FIG. 8 (b): the excitation wavelength was 535nm and the emission wavelength was 585 nm.
Experiments prove that the change of the pH has little influence on the nano-MTDF in the pH range from 5 to 7.5, namely the nano-MTDF is suitable for detecting the concentration of formaldehyde in organisms.
Example 8 the mechanism of the nanoprobe nano-MTDF (1 μ M) of the present invention after the reaction with formaldehyde under the condition of DMSO/PBS buffer (pH 7.4, v/v 1/99) was demonstrated.
Accurately weighing a certain amount of probe (I), preparing a mother solution with the concentration of 2mM by using dimethyl sulfoxide, sucking 0.02mL by a pipette gun, adding the solution into 1.96mL of PBS buffer solution, carrying out ultrasonic treatment for a plurality of minutes, then carrying out violent oscillation to obtain a nano probe nano-MTDF, sucking 0.99mL of nano probe nano-MTDF solution, respectively adding 10 mu L of formaldehyde aqueous solution, reacting overnight, and then analyzing by using high performance liquid chromatography. The HPLC chromatogram is shown in FIG. 9.
Experiments prove that the mechanism of the reaction of the nanoprobe nano-MTDF and formaldehyde is correct. The nano-MTDF and formaldehyde generated two free fluorescent substances, compound VIII and compound IX, to test the effect of the dual signal turn-on.
Example 9 Formaldehyde imaging analysis of the Nanoprobe nano-MTDF of the present invention in cancer cells
Accurately weighing a certain amount of probe (I), preparing mother liquor with the concentration of 0.5mM by using dimethyl sulfoxide, sucking 0.02mL by a liquid transfer gun, adding the mother liquor into 1.98mL of DMEM culture medium, carrying out ultrasonic treatment for several minutes, and then violently shaking to obtain a nano probe nano-MTDF. Adding 1mL of culture solution containing nanoprobe nano-MTDF into MCF-7 cells, incubating at 37 ℃ for 0.5h, washing twice with fresh DMEM medium, incubating for 3h with different formaldehyde concentrations (final formaldehyde concentrations are 0 and 1 respectively), washing twice with fresh DMEM medium, adding commercial DMEM medium, and incubating
Deep Red FM, in which, incubation was 20min at 37 ℃, PBS washed twice, and finally imaged with Perkinelmer UltraView Vox Spinning Disk confocal fluorescence. FIG. 10 is a diagram showing the effect of confocal fluorescence imaging of cells. (a1, a2, a3): formaldehyde (0mM), (a1, a2, a3) formaldehyde (1 mM). Na-channel (a1, b1): lambdaex＝440nm,λem＝455–515nm；Rho-channel(a2,b2):λex＝514nm,λem＝524.5–649.5nm；Deep red-channel(a3,b3):λex＝640nm, λ em660 and 750 nm. c1 coincidence of b1 and b 3; c2 is the superposition coefficient of Na-channel and Deep red-channel; c3 superposition of b2and b 3; c4 coincidence coefficient of Rho-channel and Deep red-channel, scale bar, 20 μm.
The experimental result shows that under the condition of increasing the concentration of formaldehyde, the fluorescence signal in the cells is also enhanced, which indicates that the substance can detect the formaldehyde in the cells. At the same time, by commercial and commercial
Image comparison of deep FM, the Pearson correlation coefficients are 0.81 (lambda)ex440nm) and 0.85(λ)ex514nm), which demonstrates that nano-MTDF is able to detect formaldehyde in mitochondria within cells.
Claims (10)
1. A compound of formula (III):
2. a process for the preparation of a compound according to claim 1, characterized in that it comprises: (1) activating p-hydroxybenzaldehyde serving as an initial raw material at the temperature of 60-70 ℃ in the presence of an acid-binding agent, then performing nucleophilic substitution reaction in an acetone solvent at the temperature of 60-70 ℃ by using 3-bromopropyne as a nucleophilic reagent, and performing aftertreatment on A to obtain a compound (II); (2) ammoniating the compound (II) obtained in the step (1) at 0 ℃, adding propenyl boronic acid ortho-di-tertiary alcohol ester at 0 ℃, mixing, reacting at 25-35 ℃, and performing post-treatment B to obtain the compound shown in the formula (III);
3. a process for the preparation of a compound according to claim 2, characterized in that: the acid-binding agent is potassium carbonate, and the amount of the acid-binding agent is 1.5 times of the equivalent of p-hydroxybenzaldehyde.
4. A process for the preparation of a compound according to claim 2, characterized in that: the mass ratio of the p-hydroxybenzaldehyde to the 3-bromopropyne in the step (1) is 1: 1.5-3.
5. A process for the preparation of a compound according to claim 2, characterized in that: and (3) the ammonia concentration in the ammonia methanol solution in the step (2) is 7 mol/L.
6. A process for the preparation of a compound according to claim 2, characterized in that: the amount ratio of the compound (II) to the ammonia substance in the ammonia methanol solution is 1: 6 to 20.
7. A process for the preparation of a compound according to claim 2, characterized in that: the mass ratio of the compound (II) to the propenyl boronic acid ortho-di-tertiary alcohol ester substance is 1:1.2 to 2.
8. A process for the preparation of a compound according to claim 2, characterized in that:
the post-treatment A comprises the following steps: removing the solvent from the reaction liquid by rotary evaporation under reduced pressure, adding water, extracting with ethyl acetate, combining organic phases, washing the organic phases with water and saturated saline solution for a plurality of times, drying with anhydrous sodium sulfate, filtering, drying the solvent by rotary evaporation, and carrying out column chromatography separation to obtain a target product, wherein an eluant is ethyl acetate and petroleum ether with the volume ratio of 1: 10;
the post-treatment B comprises the following steps: and (3) carrying out reduced pressure rotary evaporation on the reaction liquid to remove the solvent, carrying out chromatographic column separation on the crude product to obtain a target product, wherein the eluent is dichloromethane and methanol with the volume ratio of 40: 1.
9. The use of compound (III) according to claim 1 as an intermediate for the preparation of a dual-signal turn-on formaldehyde fluorescent nanoprobe of formula (I);
10. the use of claim 9, wherein: the formaldehyde fluorescent nano probe with the double signal turn-on is used for detecting the concentration of formaldehyde.
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