CN107383078B - Phenylboric acid ester compounds and benzoyl peroxide detection kit comprising the compound - Google Patents
Phenylboric acid ester compounds and benzoyl peroxide detection kit comprising the compound Download PDFInfo
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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
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- C09K2211/1096—Heterocyclic compounds characterised by ligands containing other heteroatoms
Abstract
The present invention relates to benzoyl peroxide detection technique fields, and in particular to a kind of phenylboric acid ester compounds and the benzoyl peroxide detection kit comprising the compound.Benzoyl peroxide detection kit includes reagent stock liquid a and reagent stock liquid b；The reagent stock liquid a is the phosphate buffer for the ethyl alcohol that pH value is 6~8, and the reagent stock liquid b is the organic solvent solution of phenylboric acid ester compounds.Reagent stock solution b fluorescence itself is weaker in benzoyl peroxide detection kit of the invention；And then fluorescence intensity can be remarkably reinforced after reacting with benzoyl peroxide, strong absorption is generated at 690nm~750nm, and fluorescence significantly increases at 706nm, which is near-infrared region, background interference is few, therefore accuracy with higher and sensitivity；And the fluorescence probe has specificity to benzoyl peroxide, not by the interference of other common inorganic ions and oxide species.
Description
Technical field
The invention belongs to benzoyl peroxide detection technique fields, and in particular to phenylboric acid ester compounds and include the change
Close the benzoyl peroxide detection kit of object.
Background technique
In recent years, as the improvement of people's living standards, food-safety problem also has been to be concerned by more and more people.It ensures
Food safety is the important content built Health China, promote the well-being of the people, is the specific body using the people as center development thought
It is existing.Food safety sampling observation situation is classified as the key content of food safety job rating by " 13 " state food safety program,
Food safety sampling observation covering all foods classification, kind.Food additives are the main reason for causing food problems, numerous
In food additives, benzoyl peroxide is important food and brightens antistaling agent, is widely used in food, chemical industry and medicine neck
Domain.The effects of it is often used as the bleaching agent of wheat flour in field of food, and furthermore it can also play antibacterial, accelerate the ripening.
However, benzoyl peroxide (benzoyl peroxide) solve our daily lifes, production take it is hiding
Huge security risk.The excessive addition of benzoyl peroxide as active oxygen species will lead to the nutritional ingredient in food
The degradation of such as VitAVitE and vitamin B1, benzoyl peroxide can also aggravate burden of liver, inspire canceration, right
Peripheral blood can produce heredity toxic side effect.In addition, the benzoyl peroxide in food can generate in food heating process
Benzene radical, and then will form the noxious materials such as benzene, phenol, biphenyl, there is huge damage to human body, free radical accelerates human body
Cell ageing leads to atherosclerosis, or even induces a variety of diseases.Based on this, many countries and regions are all kept under strict control peroxidating
The use of benzoyl.China is in formally addition of the disabling benzoyl peroxide in flour from May 1,2011.But it can be small
It is used in flour and fungicide.
Currently, the method for benzoyl peroxide detection has high performance liquid chromatography (HPLC), gas-chromatography (GC), mass spectrum
With chromatograph joint used, ion chromatography, chemoluminescence method, electrochemistry etc..The illegal addition of benzoyl peroxide occurs mainly in food
In, food is the product that must disappear of national product life, even sampling observation also has a large amount of sample, it is therefore desirable to more convenient and quick
Detection method.
China application CN201110084232.6 discloses a kind of violated additive benzoyl peroxide of flour and quickly detects
Kit is made of reaction reagent, color developing agent, positive control agent stock solution and standard color comparison card；Reaction reagent is concentration
The ferrous ion acidic aqueous solution of 100mg/L to 300mg/L；Color developing agent is the thiocyanic acid of concentration 0.5mol/L to 0.75mol/L
Root aqueous solution；Positive control agent stock solution is the benzoyl peroxide ethanol solution of concentration 500mg/L to 1500mg/L；Standard
Colorimetric card includes several small color lumps, and each color lump corresponds to benzoyl peroxide concentration.Its testing principle is to utilize benzoyl peroxide first
The strong oxidizing property of acyl, by the ferrous ions in reaction reagent at ferric ion, ferric ion is again in an acidic solution
Chromogenic reaction occurs with the thiocyanate radical in color developing agent, generates orange red ferric rhodanate complex, orange red complex color
The depth carries out half-quantitative detection in correlation with Determination of benzoyl peroxide in wheat, but this method has biggish limitation, shows
Colour response is in visible light wave range, and many sample compositions are complex, and the pigment of itself has very big do for chromogenic reaction
Immunity, especially for the detection of biological tissue or living tissue, this detection kit can not be applicable in completely.
China application CN201611163372.1 discloses a kind of benzoyl peroxide rapid detection method, testing principle
The substance that can develop the color of visible light wave range can be formed with detection reagent also with the benzoyl peroxide in sample, using with mark
Quasi- colorimetric card carries out color and compares progress semi-quantitative analysis.In above-mentioned common method, the cumbersome time-consuming of sample pre-treatments is needed to sample
Product extract, and can just carry out chromogenic reaction；And need to carry out colorimetric twice and judge, it is at high cost, it is not easy to benzoyl peroxide first
The quick detection of acyl, is not easy to promote the use of.
Therefore, the new method for establishing a kind of sensitive quick detection Determination of benzoyl peroxide in wheat of energy, mentions for base's quality testing department
For technical support, realize that the safe handling of perbenzoic acid is significantly.
Optical probe is that one kind can react with target substance and cause optics (including shining, fluorescence, extinction) property
Variation.It, can be qualitative or even quantitatively environmental stimuli strong by means of means such as such as Fluorescence Spectrometer, fluorescence imaging microscopes
It is weak, to realize the purpose of analysis detection.It is detection means that fluorescence probe, which mainly relies on fluorescence signal, usually there is the increasing of fluorescence
By force, it is quenched or the variation of emission wavelength.Fluorescence probe is detected point by the variation of the luminescence phenomenon and absorption spectrum of molecule
Interaction between son, advantage can be summarized as follows: (1) it is convenient and efficient, there is very high sensitivity；(2)
It can use optical fiber technology and realize single celled real-time detection；(3) if having bigger variation, Ke Yi on absorption spectrum
In the case where without the help of any instrument, detection is directly achieved the purpose that by the variation of color.
Fluorescent spectrometry high sensitivity, selectivity are good, and the information of acquisition is intuitive, accurate, can the complicated sample of Scientific Expression explanation
Therefore structure, distribution, content and physiological function of product etc. obtain the concern of analysis detection field scientific research personnel.But fluorescence
There is also some difficult points, many organisms and its group, which are woven under the excitation of visible light, itself can emit fluorescence for the foundation of spectroscopic methodology,
The fluorescence detection of severe jamming biological sample and radiography, as haemocyanin in blood plasma fluorescent wavelength ranges be 325nm~
The fluorescent wavelength ranges of 350nm, reduced nicotinamide adenine dinucleotide phosphatase (NADPH) and bilirubin be 430nm~
470nm, so that the sensitivity of fluorescence analysis and accuracy receive very big influence.Launch wavelength is in the glimmering of visible light wave range
Light probe sensitivity and accuracy when being applied to biological living or biological sample analysis is insufficient, need to develop it is possible to prevente effectively from
The novel probe compound of biological tissue fluorescence interference itself.
The maximum absorption wavelength and launch wavelength of near infrared fluorescent probe are 600nm~900nm, and light infiltrating tissues are up to number
Centimetre, excellent biological tissue's penetration capacity and resistance background interference ability, near-infrared fluorescent detects in biological sample analysis
Have shown that obvious superiority.In consideration of it, exploitation launch wavelength belongs to the fluorescence detection reagent kit of near-infrared region, for biology
The detection of benzoyl peroxide has important value in sample.
Summary of the invention
In order to solve Detrmination of Benzoyl Peroxide poor selectivity in the prior art, sample pre-treatments are cumbersome, and colour developing is special
Property belongs to visible light section, sensitivity and accuracy can be greatly reduced by the interference of the chromogenic reaction of biological sample itself, because
And be not suitable for biology sample detection defect, on the one hand, the present invention using tricarbocyanine IR-780 skeleton derivative as fluorogen, with
As recognition group, design had synthesized one kind to be detected near infrared emission wave-length coverage the phenyl boronate of ortho position substitution
The compound of Benzoyl Oxide, on the other hand, the present invention also provides a kind of benzoyl peroxides based on fluorescence probe method
Detection kit and its detection method.
The present invention provides phenylboric acid ester compounds shown in formula (I),
The present invention provides the preparation method of phenylboric acid ester compounds shown in formula (I), includes the following steps:
In the presence of a base, formula (IV) compound represented and formula (V) compound represented are substituted point step (A)
Rearrangement reaction is solved, compound shown in formula (II) is obtained；
In the presence of a base, parent occurs step (B) for formula (II) compound represented and formula (III) compound represented
Core substitution reaction obtains phenylboric acid ester compounds shown in formula (I)；
Further, in the preparation method of above-mentioned phenylboric acid ester compounds, in step (A) and step (B), the alkali is equal
Selected from organic base or inorganic base；Wherein, the organic base in triethylamine, diisopropyl ethyl amine or pyridine at least one
Kind；The inorganic base is selected from least one of potassium carbonate, sodium carbonate, sodium hydroxide or sodium bicarbonate.
Further, in the preparation method of above-mentioned phenylboric acid ester compounds, in step (A), chemical combination shown in formula (IV)
The molar ratio of object, formula (V) compound represented and alkali is 1:1~5:0.5~5；In some embodiments, it feeds intake mole
Than for 1:2.5:2.5, the reaction time of step (A) according to inventory is not all 1 hour~24 hours, and inventory increases, instead
Accordingly extend between seasonable.Reaction is preferably in inert gas, as reacted in nitrogen or argon gas.
In step (B), the molar ratio of formula (II) compound represented, formula (III) compound represented and alkali is 1:1
~1.5:1.5~2, in some embodiments, molar ratio 1:1.5:1.5；When step (B) needs strict control to react
Between, every when carry out thin-layer chromatography TLC and detect reaction process, in some embodiments, reaction time of the step (B) according to
Inventory is not all 2 hours~4 hours, and inventory increases, and the reaction time accordingly extends.Reaction is preferably in inert gas, such as
It is reacted in nitrogen or argon gas.
Further, in the preparation method of above-mentioned phenylboric acid ester compounds, in step (A), reaction temperature be 30 DEG C~
60 DEG C, reaction dissolvent is selected from least one of n,N-Dimethylformamide, methylene chloride, acetonitrile, in some embodiments,
The reaction temperature of reaction is 40 DEG C~50 DEG C；In step (B), reaction temperature is 20 DEG C~60 DEG C, and reaction dissolvent is selected from N, N- bis-
One of methylformamide, acetonitrile, in some embodiments, reaction temperature are 40 DEG C~50 DEG C.In step (A) and step
(B) in, the dosage of reaction dissolvent, which is subject to, is completely dissolved reactant, without being particularly limited to its dosage.
The preparation method of phenylboric acid ester compounds (I) of the present invention, simple and easy to do, post-processing is simple, is easy to scale metaplasia
It produces.
The present invention also provides a kind of benzoyl peroxide detection kits, include reagent stock liquid a and reagent stock liquid b；
The reagent stock liquid a is the phosphate buffer for the ethyl alcohol that pH value is 6~8, and the reagent stock liquid b is benzene shown in formula (I)
The organic solvent solution of ylboronic acid ester compounds, the organic solvent are selected from dimethyl sulfoxide, n,N-Dimethylformamide, acetonitrile
At least one of.
Further, the phosphoric acid in benzoyl peroxide detection kit of the present invention, in the reagent stock liquid a
Salt is selected from Na2HPO4、NaH2PO4And KH2PO4At least one of, the concentration expressed in percentage by volume of ethyl alcohol is in reagent stock liquid a
10%, the phosphatic molar concentration is 5mM~15mM；Phenyl boronate chemical combination shown in formula (I) in the reagent stock liquid b
The concentration of object is 0.01mM~1mM.In some embodiments, the reagent stock liquid a in the kit and reagent storage
The volume ratio of standby liquid b is 200/1.
Further, in benzoyl peroxide detection kit of the present invention, the pH value of the reagent stock liquid a is
7.4, wherein phosphatic molar concentration is 10mM；Phenylboric acid ester compounds shown in formula (I) is dense in the reagent stock liquid b
Degree is 0.01mM~1mM.
The testing principle of benzoyl peroxide detection kit of the present invention:
Phenylboric acid ester compounds (I) in reagent stock liquid b are used as probe, and the fluorescence intensity of itself is lower, peroxide
Change benzoyl can lead to the frequency of the phenyl boric acid in compound (I), and where ester aoxidizes, electronics is reset, and finally discharges again with strong glimmering
The compound (II) of light blob, the fluorescence of the compound significantly increase at 690nm~750nm, have at 706nm most strong glimmering
Light, therefore this property can be used for benzoyl peroxide detection；The concentration of the variation of fluorescence intensity and benzoyl peroxide be in than
Example, the variation by measuring relevant fluorescence signal can measure the content of benzoyl peroxide.Reaction process is as follows:
In yet a further aspect, the present invention provides the peroxidating in the benzoyl peroxide detection kit measurement sample
The detection method of benzoyl content, it includes following steps:
(1) standard curve is prepared
Using 635nm~690nm as excitation wavelength, a series of benzoyl peroxide standard solution of various concentrations is measured
Fluorescence intensity at launch wavelength 690nm~750nm is denoted as F, fluorescence intensity of the measurement reagent blank in transmitted wave strong point, note
For F0, fluorescence intensity difference DELTA F=F-F0, using the concentration C of benzoyl peroxide as abscissa, fluorescence intensity difference DELTA F is vertical sits
Mark draws standard curve, wherein the benzoyl peroxide standard solution of the various concentration is by the reagent storage in kit
What standby liquid a, reagent stock liquid b and benzoyl peroxide Standard Stock solutions were prepared；
(2) in test sample benzoyl peroxide concentration
Benzoyl peroxide standard solution prepared by step (1) is replaced with into sample to be tested, reagent storage is added thereto
Standby liquid b, the fluorescence intensity according to step (1) the method detection sample to be tested in transmitted wave strong point identical with step (1), note
For F ', the F ' is substituted into standard curve obtained by step (1), and then obtains the concentration of benzoyl peroxide in sample to be tested.
When in some embodiments, using the kit, wherein a series of benzoyl peroxide of various concentrations
Formyl standard solution is matched by reagent stock liquid a, reagent stock liquid b and the benzoyl peroxide Standard Stock solutions in kit
It makes and obtains, in the benzoyl peroxide Standard Stock solutions, reagent stock liquid b additive amount is equal, and solvent is mainly reagent
Stock solution a；A series of solvent of the benzoyl peroxide standard solution of various concentrations is reagent stock liquid a, and volume is equal
For 2mL, concentration is followed successively by 0 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0
μM, 5.5 μM and 6.0 μM.After fluorescence intensity, find benzoyl peroxide concentration between 0.5 μM~4 μM when, concentration with
In a linear relationship between fluorescence intensity, degree of correlation R reaches 0.99 or more, can carry out quantitative detection.
When using heretofore described kit, the optional 630nm~690nm wavelength period of excitation wavelength, wherein in some realities
It applies in scheme, selective exitation wavelength 635nm；In other embodiments, selective exitation wavelength 670nm；Launch wavelength is optional
690nm~750nm wavelength period, wherein in some embodiments, selecting launch wavelength 706nm；In other embodiments,
Select launch wavelength 700nm.
In yet a further aspect, it is measured the present invention also provides another using the benzoyl peroxide detection kit
The detection method of benzoyl peroxide in sample, it includes following steps:
(i) fluorescence intensity or fluorescence imaging of blank control sample are measured
Using 635nm~690nm as excitation wavelength, a certain concentration is added in the control sample of Xiang Buhan benzoyl peroxide
Reagent stock liquid b, cleaned with reagent stock liquid a after a certain period of time to remove extra reagent stock liquid b, then in transmitted wave
The fluorescence intensity of system is measured at a length of 690nm~750nm with luminoscope, or carries out fluorescence imaging with laser co-focusing；
(ii) testing sample fluorescence intensity or fluorescence imaging are measured
The reagent stock liquid b with step (i) same concentrations is added into sample to be tested, uses reagent stock liquid after a certain period of time
A cleaning measures system in the fluorescence intensity of transmitted wave strong point identical with step (i) or shoots fluorescence imaging, and and step
(i) fluorescence intensity or the fluorescence imaging comparison measured；
When the fluorescence intensity of sample to be tested is greater than the fluorescence intensity or sample to be tested fluorescence imaging brightness ratio of control sample
When control sample fluorescence imaging is strong, then illustrate to contain benzoyl peroxide in sample to be tested.
Compared with prior art, benzoyl peroxide detection kit of the invention, has the following characteristics that
1, reagent stock solution b fluorescence itself is weaker in benzoyl peroxide detection kit of the invention；And and peroxidating
Then fluorescence intensity can be remarkably reinforced after benzoyl reaction, strong absorption be generated at 690nm~750nm, and fluorescence is aobvious at 706nm
Enhancing is write, which is near-infrared region, and background interference is few, therefore accuracy with higher and sensitivity.
2, benzoyl peroxide detection kit of the invention, fluorescence generation reaction speed is fast, can develop the color in 20 minutes
Stablize.
3, benzoyl peroxide detection kit of the invention, detection sensitivity is high, high sensitivity, and benzoyl peroxide is dense
Degree has fluorescence enhancement signal when being greater than 0.5 μM, and fluorescence intensity enhances with the increase of benzoyl peroxide concentration, in peroxide
When changing benzoyl concentration between 0.5 μM~4 μM, fluorescence intensity and benzoyl peroxide concentration, between it is in a linear relationship, it is related
Degree R reaches 0.99 or more.
4, benzoyl peroxide detection kit of the invention, benzoyl peroxide have the intensified response of fluorescence special
Property, not by other common inorganic salts such as calcium chloride (CaCl2), magnesium chloride (MgCl2), copper chloride (CuCl2), frerrous chloride
(FeCl2), zinc sulfate (ZnSO4), iron chloride (FeCl3), hypochlorite (ClO–), hydroxyl (OH–) or vitamin B6, dimension
Raw element C, glucose, arginine, serine, singlet oxygen (1O2), nitric oxide (NO), nitrite anions (NO2 –), the tertiary fourth of peroxide
Alcohol (TBHP), potassium permanganate, potassium bichromate, potassium chlorate, sodium chlorate, sodium perchlorate, Potassiumiodate, sodium hypochlorite, hydrogen peroxide etc.
The interference of typical oxidation active specy.
5, benzoyl peroxide detection kit of the invention has near-infrared fluorescent launch wavelength, can use fluorescence spectrum
Method detection, background signal is lower, and background noise is few, is adapted to detect for the biological sample of complicated component, is also adapted to monitoring living body sample
The distribution of benzoyl peroxide in this.
Detailed description of the invention
Fig. 1 is the fluorescence emission spectrum that kit is reacted with the benzoyl peroxide of various concentration.
Fig. 2 is standard curve when kit is used to detect benzoyl peroxide concentration.
Fig. 3 is the fluorescence emission spectrum that kit is used for the reaction of various interfering substances.
Fig. 4 is the fluorescence intensity change that kit detects various concentration benzoyl peroxide in Hela cell.
Fig. 5 is that the zebra fish fluorescence intensity change after benzoyl peroxide is bred in kit detection.
Specific embodiment
Further detailed description is done to the present invention combined with specific embodiments below, but embodiments of the present invention are not limited to
This.Those skilled in the art will realize that: chemical reaction described in the invention can be used to suitably prepare many
Other compounds of invention are used to prepare chemical combination of the invention for example, reaction condition makes some conventional modifications according to the present invention
Other methods of object are considered as within the scope of the present invention.
The structure of compound be by nuclear magnetic resonance (1H-NMR、13C-NMR it) determines.1H-NMR、13C-NMR chemical potential
(δ) is moved to provide with the unit of hundred a ten thousandths (ppm).1H-NMR、13The measurement of C-NMR is with Bruker Ultrashield-
600 nuclear magnetic resonance spectrometer of 400 nuclear magnetic resonance spectrometers and Bruker Avance III HD, measurement solvent are deuterated methanol
(CD3OD).Use TMS (0ppm) or chloroform (7.25ppm) as reference standard.When there is multiplet, it will use following
Abbreviation: s (singlet, unimodal), d (doublet, bimodal), t (triplet, triplet), m (multiplet, multiplet),
Br (broadened, broad peak), dd (doublet of doublets, double doublet), and brs (broadened singlet, it is wide
It is unimodal).Coupling constant is indicated with hertz (Hz).
Column chromatography is generally carrier using 200 mesh of Qingdao Haiyang chemical industry~300 mesh silica gel.
Experimental method described in following embodiments is unless otherwise specified conventional method；The reagent such as IR-780 iodine
It for object and biomaterial, unless otherwise specified, commercially obtains, can either use or according to known in the art
Method synthesize.
Without specified otherwise in embodiment, reaction carries out under nitrogen atmosphere；
Nitrogen atmosphere refers to that reaction flask connects the nitrogen balloon or steel kettle of an about 1L volume；
Without specified otherwise in specification, room temperature is 20 DEG C~30 DEG C, and temperature error described in embodiment is ± 5 DEG C.
The system of used solvent is reacted in the monitoring of reaction process in embodiment using thin-layered chromatography (TLC)
Have: the volume ratio of petroleum ether and ethyl acetate system, solvent is different according to the polarity of compound and is adjusted.
Embodiment 1: the preparation of formula (I) phenylboric acid ester compounds
The preparation of (1-1) compound (III)
Compound (III) can directly be commercially available or prepare in accordance with the following methods:
30mL toluene is added into a there-necked flask equipped with Dean-Stark water segregator, 2- methylphenylboronic acid is then added
(0.21g, 1.55mmol), pinacol (pinacol-H2O, 0.22g, 1.86mmol), gained mixture is heated to reflux two small
When.After reaction, (30mL × 3) are washed with water in mixture, remove unreacted pinacol and phenyl boric acid, then have what is separated
Removing toluene solvant is mutually evaporated under reduced pressure in machine.Gained crude product is dissolved with methylene chloride (50mL), after washing (30mL) again, to
Anhydrous sodium sulfate is added in gained organic phase to be dried, the solution after drying is evaporated under reduced pressure to obtain crude product 0.32g.
Take gained crude product (0.32g, 1.47mmol), NBS (N-bromosuccinimide, 0.39g, 2.2mmol), AIBN
In the acetonitrile (10mL) that (azodiisobutyronitrile, 0.003g, 0.018mmol) is dissolved into.Gained reaction solution flows back at 90 DEG C
Two hours.After cooling down at room temperature, evaporating solvent under reduced pressure obtains crude product.Gained crude product silica gel column chromatography separating purification, with
Petroleum ether (60~90 DEG C)/ethyl acetate (v/v)=1:1 makees eluent, and obtaining product is white solid (0.53g, yield
80%).
The preparation of (1-2) compound (II)
Resorcinol (86.0mg, 0.78mmol) and potassium carbonate (102mg, 0.78mmol) are dissolved in acetonitrile (20mL)
In, room temperature and stirred under nitrogen atmosphere 10 minutes, compound (IV) (the i.e. IR-780 that then will be dissolved in acetonitrile (1mL)
Iodo object, 207mg, 0.31mmol) it is added in system.Reaction mixture is reacted 4 hours at 50 DEG C, end of reaction subtracts
Solvent is evaporated off in pressure, obtains crude product.With silica gel column chromatography purifying crude product, with petroleum ether (60~90 DEG C)/ethyl acetate (1:1, v/
V) make eluant, eluent, obtaining product is cyan powders (67.3mg, yield 52.6%).
The structural characterization data result of compound (II) is as follows:
Hydrogen spectrum:1H NMR(400MHz,CDCl3): δ (ppm) 8.03 (d, 1H, J=14.2Hz), 7.28-7.23 (m, 3H),
7.19 (d, 1H, J=9.2Hz), 7.04 (t, 1H, J=7.6Hz), 6.81 (d, 1H, J=7.6Hz), 6.74 (d, 1H, J=
8.8Hz), 6.51 (s, 1H), 5.61 (d, 1H, J=14.2Hz), 3.76 (t, 2H, J=7.2Hz), 2.67 (t, 2H, J=
6.0Hz), 2.61 (t, 2H, J=6.0Hz), 1.91-1.78 (m, 4H), 1.66 (s, 6H), 1.06 (t, 3H, J=7.6Hz)
Carbon spectrum:13C NMR(400MHz,CH3OD):δ(ppm)174.5,173.7,163.8,158.5,144.0,142.2,
141.6,140.3,131.1,129.9,126.1,123.5,123.2,121.9,116.2,115.9,112.0,103.6,
100.1,50.4,46.6,29.6,29.0,25.3,22.1,21.8,11.9.
The preparation of compound shown in formula (II) can also be carried out according to the experiment condition of following table 1.
Table 1:
According to the experiment condition of upper table 1, available compound (II).
The preparation of (1-3) compound (I)
Compound (II) (40mg, 1.0mmol) and potassium carbonate (20mg, 1.5mmol) are dissolved in acetonitrile (4mL),
Room temperature and stirred under nitrogen atmosphere 10 minutes, the compound (III) (20mg, 1.0mmol) that then will be dissolved in acetonitrile (1mL)
It is added in mixed solution, reaction mixture is reacted 2.5 hours at 50 DEG C.It is dilute that methylene chloride (20mL) is added in end of reaction
Mixture is released, is successively washed with water (5mL) and saturated aqueous sodium carbonate (5mL).Then organic phase is dried with anhydrous sodium sulfate,
Evaporating solvent under reduced pressure obtains crude product.Crude product is purified with silica gel column chromatography, methylene chloride/methanol=10:1, it is deep for obtaining product
Blue solid (25mg, 36%).
The structural characterization data result of compound (I) is as follows:
Hydrogen spectrum:1H NMR (600MHz, MeOD) δ (ppm) 8.72 (d, J=14.9Hz, 1H), 7.84 (dd, J=7.4,
0.9Hz, 1H), 7.64 (d, J=7.4Hz, 1H), 7.58-7.43 (m, 6H), 7.39-7.34 (m, 2H), 7.03 (d, J=
2.1Hz, 1H), 7.00 (dd, J=8.5,2.3Hz, 1H), 6.51 (d, J=14.9Hz, 1H), 5.42 (s, 2H), 4.33 (t, J=
7.4Hz, 2H), 2.81-2.75 (m, 2H), 2.72 (t, J=6.0Hz, 2H), 1.98-1.91 (m, 4H), 1.81 (s, 6H), 1.28
(s, 12H), 1.08 (t, J=7.4Hz, 3H)；
Carbon spectrum:13C NMR(151MHz,MeOD)δ(ppm)179.19,164.13,163.07,155.83,146.95,
143.42,143.36,143.08,137.18,135.21,132.32,130.29,130.15,129.58,128.78,128.56,
128.44,123.80,117.24,115.77,115.20,114.05,104.88,102.50,85.16,71.83,52.00,
47.66,30.11,28.53,25.32,25.16,22.31,21.67,11.68.
The preparation of phenylboric acid ester compounds shown in formula (I) can also be carried out according to the experiment condition of following table 2.
Table 2:
According to the experiment condition of upper table 2, available compound (II).
Reaction temperature is 40 DEG C~50 DEG C, and formula (II) compound represented, formula (III) compound represented and alkali feed intake
Molar ratio is 1:1.5:1.5, and combined reaction effect is best.
Embodiment 2: the spectral property that fluorescence probe shown in formula (I) is reacted with various concentration benzoyl peroxide
The phosphate buffer (PBS) for first preparing the 10mM PH=7.4 containing ethyl alcohol, according to this field conventional practices
Preparation phosphate concn is 10mM, and pH is 7.4 phosphate buffer containing ethyl alcohol, and wherein the concentration expressed in percentage by volume of ethyl alcohol is
10%, PBS, which are prepared, uses NaHPO4-KH2PO4System, therefore phosphate concn refers to NaHPO4And KH2PO4Total concentration, so
Afterwards, prepared solution is as reagent stock liquid a.The phenylboric acid ester compounds (I) that embodiment 1 is prepared are configured to dense
The solution that degree is 1mM is acetonitrile as reagent stock liquid b, selected solvent, obtains reagent stock liquid b.It is verified, for matching
The type of reagent stock liquid b organic solvent processed does not influence detection effect, uses dimethyl sulfoxide instead or n,N-Dimethylformamide is
Effect is the same when solvent.The volume ratio of reagent stock liquid a and reagent stock liquid b in kit are 200/1.
It takes reagent stock liquid b (1mM, 20 μ L) to be dissolved in reagent stock liquid a, is arrived with reagent stock liquid a or ethyl alcohol constant volume
2mL.At this point, phenylboric acid ester compounds (I) i.e. concentration and probe concentration is 10 μM.Then, pass through the benzoyl peroxide of addition different volumes
Formyl Standard Stock solutions (1mM), final prepare obtain a series of benzoyl peroxide standard solution of various concentrations, wherein
The concentration of benzoyl peroxide be followed successively by 0 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μ
M, 4.5 μM, 5.0 μM, 5.5 μM and 6.0 μM.A series of volume of the reaction solution of benzoyl peroxide standard items containing various concentration
It is 2mL.Resulting mixed solution reacts 20min at 37 DEG C, and luminoscope F-4600 measures its fluorescence excitation spectrum and fluorescence
The slit width of emission spectrum, excitation and transmitting is 10nm.Using 670nm as excitation wavelength when fluorescence emission spectrum measures, obtain
The emission spectrum of the benzoyl peroxide standard solution of various concentration is as shown in Figure 1.
Using 670nm as excitation wavelength when fluorescence emission spectrum measures, a series of benzoyl peroxide of various concentrations is measured
The solution of standard items is denoted as F in the fluorescence intensity that launch wavelength is at 706nm；It is 706nm that reagent blank, which is measured, in launch wavelength
The fluorescence intensity at place, is denoted as F0, fluorescence intensity difference DELTA F=F-F0, using the concentration C of benzoyl peroxide as abscissa, fluorescence is strong
Degree difference DELTA F is ordinate, draws standard curve, it is as shown in Figure 2 to obtain standard curve.
The corresponding equation of linear regression of standard curve are as follows: Δ F=676.3 × [BPO] (μM) -100.1, (R2=0.993),
By standard curve it is found that linearly being closed between concentration and fluorescence intensity when benzoyl peroxide concentration is between 0.5 μM~4 μM
System, degree of correlation R reach 0.99 or more.In the linear range, reagent stock liquid b is added into sample to be tested, detects sample to be tested
In the fluorescence intensity of transmitted wave strong point, which is substituted into standard curve, so that it may obtain benzoyl peroxide first in sample to be tested
The concentration of acyl.Wherein, the concentration unit of benzoyl peroxide [BPO] is μM.
According to this field conventional methods, show that the detection limit (S/N=3) of the probe is after 11 repetitions are tested
47nM。
Phosphate buffer solution in the present embodiment is prepared according to the PBS preparation method of this field routine, reagent stock liquid a
In phosphate be selected from Na2HPO4、NaH2PO4And KH2PO4At least one of, phosphatic molar concentration can for 5mM~
15mM；The concentration of fluorescence probe shown in formula (I) can be 0.01mM~1mM in reagent stock liquid b.Common reagent stock liquid a
For concentration 10mM, pH 7.4, the PBS buffer solution containing ethyl alcohol 10%, reagent stock liquid b is the solution of concentration 1mM.
It is above-mentioned the experimental results showed that, the present invention in fluorescence probe phenylboric acid ester compounds (I) have the following characteristics that
1, fluorescence signal is extremely low in the solution for fluorescence probe phenylboric acid ester compounds (I), and background signal is lower；But with
The addition of benzoyl peroxide, compound (I) reacted with benzoyl peroxide after then fluorescence intensity can be remarkably reinforced, in 690nm
Strong absorption is generated at~750nm, and fluorescence is most strong at 706nm, which is near-infrared region, background interference
It is few, this probe accuracy with higher and sensitivity.
2, fluorescence probe phenylboric acid ester compounds (I) fluorescence generation reaction speed is fast, can color stability in 20 minutes.
3, fluorescence probe phenylboric acid ester compounds (I) have fluorescence when being greater than 0.5 μM in benzoyl peroxide concentration
Enhance signal, fluorescence intensity enhances with the increase of benzoyl peroxide concentration, in benzoyl peroxide concentration in 0.5 μM~4 μ
When between M, fluorescence intensity and benzoyl peroxide concentration are in a linear relationship, can carry out the quantitative detection of benzoyl peroxide.
Embodiment 3: the response situation (selection Journal of Sex Research) of the kit containing fluorescence probe (I) and other ions
The reagent stock liquid b of 1mM concentration is dissolved in be configured in reagent stock liquid a (10mM, pH 7.4) it is glimmering at 10 μM
Light probe solution is separately added into isometric various common inorganic salts, Biomedia, activating oxide and typical oxygen thereto
Compound, calcium chloride, magnesium chloride, copper chloride, frerrous chloride, zinc sulfate, iron chloride such as 30 μM；The vitamin B6 of 1.5mM, dimension life
Plain C, glucose, arginine, serine；30 μM of hypochlorite, hydroxyl, singlet oxygen, nitric oxide, nitrite anions, height
Potassium manganate, potassium bichromate, potassium chlorate, sodium chlorate, sodium perchlorate, Potassiumiodate, sodium hypochlorite, hydrogen peroxide and 6 μM of peroxidating
Benzoyl (as a control group).Interfering substance using concentration for 10mM reagent stock liquid a as solvent, it is molten to obtain one group of mixing
Liquid measures its fluorescence emission spectrum using luminoscope F-6000 after reacting 20min at 37 DEG C respectively.Fluorescence emission spectrum
With 670nm deexcitation when measurement；The slit width of excitation and transmitting is 10nm.
Experimental result is as shown in figure 3, Fig. 3 is the fluorescence emission spectrum that kit is used for the reaction of various interfering substances.
The experimental results showed that only benzoyl peroxide can cause fluorescence probe to generate apparent optical signal significant changes sound
It answers, it was demonstrated that the fluorescence probe has the selectivity of height to benzoyl peroxide；Other common inorganic salts, oxidation activity species
It is unobvious with typical oxidation object interference effect.
Embodiment 4: various concentration in the benzoyl peroxide detection kit detection Hela cell containing fluorescence probe (I)
The fluorescence intensity change of benzoyl peroxide
Hela cell adherent growth in glass culture dish；Culture solution be DMEM culture solution, wherein containing 10% fetal calf serum,
100mg/mL penicillin and 100mg/mL streptomysin；Condition of culture is 37 DEG C, 5%CO2；Incubation time is 12~24 hours.?
Before the fluorescence imaging for carrying out intracellular peroxidation benzoyl, one group of cell is as blank control, remaining four groups of Hela cell difference
It is incubated at 37 DEG C with the DMEM of the fetal calf serum of the benzoyl peroxide (0 μM, 2 μM, 4 μM, 6 μM) containing various concentration
10min is cleaned three times with reagent stock liquid a (concentration 10mM, pH 7.4).Again respectively with 10 μM of probe solution (by 1mM
Reagent stock liquid b be diluted with reagent stock liquid a) respectively with four groups of experimental group cell effect 20min, with same
Reagent stock liquid a is cleaned three times.Fluorescence reaction intensity is big after reagent stock liquid b is reacted with benzoyl peroxide, in biological sample
When detection, apparent detection effect can reach using low concentration, therefore, be generally diluted to 10 μM or so of concentration and use,
Reagent dosage is added according to this field conventional amount used.
With laser confocal microscope TCS SP5 under the excitation wavelength of 635nm fluorescence imaging.Experimental result such as Fig. 4 institute
Show, Fig. 4 is the fluorescence intensity change that kit detects various concentration benzoyl peroxide in Hela cell.
4A is the fluorescence imaging of the processed blank control of unused reagent stock liquid b in Fig. 4, and 4B is without addition peroxidating
Benzoyl only uses reagent stock liquid b treated the fluorescence imaging of cell, and 4C, 4D and 4E are respectively to use 2 μM, 4 μM, 6 μM of concentration
The processed cell sample of benzoyl peroxide, be added reagent stock liquid b after fluorescence imaging.Fig. 4 F~4J is Fig. 4 A~4E
Corresponding light field cytological map.
From in Fig. 4 4A and 4B comparison: Hela cell itself unstressed configuration is individually added into Hela cell after fluorescence probe
Less fluorescence.By 4C, 4D and 4E in Fig. 4 it is found that Hela cell fluorescence intensity compared with 4B have it is different degrees of significantly increase, say
It is bright in cell sample, benzoyl peroxide has different degrees of humidification to the fluorescence of probe, and with benzoyl peroxide first
The increase of acyl concentration, fluorescence enhancement；Fluorescence intensity change is obvious, can judge the dense of benzoyl peroxide by fluorescence intensity
Spend difference.By Fig. 4 F~4J it can also be seen that with benzoyl peroxide increasing concentrations, cell fluorescence intensity is gradually increased.
Embodiment 5: various concentration benzoyl peroxide in the benzoyl peroxide detection zebra fish containing fluorescence probe (I)
Fluorescence intensity change
Zebra fish is incubated in E3 embryo medium (15mM sodium chloride, 0.5mM potassium chloride, 1mM magnesium sulfate, 1mM chlorination
Calcium, 0.15mM sodium dihydrogen phosphate, 0.05mM disodium hydrogen phosphate, 0.7mM sodium bicarbonate, 5~10% methylenum careuleum, pH 7.5), it uses
The zebra fish of growth 1 day carries out fluorescence imaging.Before carrying out benzoyl peroxide processing, one group of zebra fish as blank control,
Remaining four groups of zebra fish respectively with (0 μM, 2 μM, 4 μM, 6 μM) cultivation 5min of benzoyl peroxide solution containing various concentration,
Then it is cleaned three times with reagent stock liquid a (concentration 10mM, pH 7.4)；Later with 10 μM of reagent stock liquid b (by the examination of 1mM
Agent stock solution b is diluted with reagent stock liquid a), 20min is cultivated, then cleaned three times with same reagent stock liquid a,
To wash off excess probes.Zebra fish after the completion of processing carries out fluorescence imaging, excitation wavelength 635nm, hair with living imaging instrument
A length of 690nm~the 750nm of ejected wave.
Experimental result is as shown in figure 5, Fig. 5 is with the variation of benzoyl peroxide concentration, zebra fish tail portion (A1~E1) and ovum
The variation of yellow capsule portion (A2~E2) fluorescence intensity.Fig. 5 A1 and Fig. 5 A2 are the processed blank control of unused reagent stock liquid b
Fluorescence imaging, Fig. 5 B1 and Fig. 5 B2 are the work for reagent stock liquid b only being used treated zebra fish without adding benzoyl peroxide
Body fluorescence imaging, Fig. 5 C1/5C2,5D1/5D2,5E1/5E2 are respectively to use 2 μM of concentration, 4 μM, the processing of 6 μM of benzoyl peroxide
The fluorescence imaging after reagent stock liquid b is added in the zebra fish biopsy samples crossed.As a comparison, Fig. 5 F1~5J1 is Fig. 5 A1~5E1
Original photo under non-fluorescence state, Fig. 5 F2~5J2 are original photo of Fig. 5 A2~5E2 under non-fluorescence state.
By Fig. 5 A1 and 5A2 it is found that under the conditions of same test, the tail portion and yolk bag portion of zebra fish itself without fluorescence, by
After Fig. 5 B1 and 5B2 is it is found that be individually added fluorescence probe in zebra fish living body, the tail portion of zebra fish and yolk bag portion fluorescence are micro-
It is weak.The zebra fish tail portion of various concentration benzoyl peroxide pre-treatment is carried out known to Fig. 5 C1/5C2,5D1/5D2,5E1/5E2
Enhanced with the fluorescence intensity in yolk bag portion, and the increase of fluorescence intensity and benzoyl peroxide concentration are proportional.
Meanwhile the variation for intuitive observation fluorescence intensity carries out quantization detection to the fluorescence intensity of zebra fish living body tail portion,
And Numerical analysis is carried out to fluorescence intensity level.Zebra fish (5B1) fluorescence intensity for adding 0 μM of benzoyl peroxide to handle is denoted as
1.0；Compared to this, processing group (5C1~5E1) fluorescence intensity of 2,4,6 μM of benzoyl peroxides is added to increase separately 0.77,
1.11 and 1.36 multiple illustrates the benzoyl peroxide that the probe compound in this kit is suitable for inside detection living body
Distribution.
Above embodiments 4 and embodiment 5 demonstrate well, using containing phenyl boronate shown in formula (I) of the present invention
Whether the kit of compound can intuitively judge biological sample to be measured by way of comparison fluorescence intensity or fluorescence imaging
Containing benzoyl peroxide, it is applicable not only to common biological sample (cell), it is equally applicable for complex biological biopsy sample, and
And in terms of detecting benzoyl peroxide, kit provided by the present invention is swift in response, and sensitivity level is high, easy to operate, fits
In extensive use.
In conclusion benzoyl peroxide kit of the invention is a kind of function admirable, benzoyl peroxide easy to use
Formyl detection device has huge application prospect in the fields such as food, medicine, living body biological imaging.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Claims (8)
1. phenylboric acid ester compounds, which is characterized in that shown in the structural formula of the compound such as formula (I):
2. the preparation method of phenylboric acid ester compounds described in claim 1, which comprises the steps of:
In the presence of a base, formula (IV) compound represented and formula (V) compound represented are substituted decomposition weight to step (A)
Row's reaction, obtains compound shown in formula (II)；
In the presence of a base, formula (II) compound represented occurs nucleophilic with formula (III) compound represented and takes step (B)
Generation reaction, obtains phenylboric acid ester compounds shown in formula (I)；
3. preparation method according to claim 2, which is characterized in that in step (A) and step (B), the alkali is selected from
Organic base or inorganic base；
Wherein, the organic base is selected from least one of triethylamine, diisopropyl ethyl amine or pyridine；
The inorganic base is selected from least one of potassium carbonate, sodium carbonate, sodium hydroxide or sodium bicarbonate.
4. preparation method according to claim 2 or 3, which is characterized in that in step (A), formula (IV) compound represented,
The molar ratio of formula (V) compound represented and alkali is 1:1~5:0.5~5；
In step (B), the molar ratio of formula (II) compound represented, formula (III) compound represented and alkali be 1:1~
1.5:1.5~2.
5. preparation method according to claim 2 or 3, which is characterized in that in step (A), reaction temperature is 30 DEG C~60
℃；Reaction dissolvent is selected from least one of N,N-dimethylformamide, methylene chloride, acetonitrile；
In step (B), reaction temperature is 20 DEG C~60 DEG C；Reaction dissolvent is selected from one of N,N-dimethylformamide, acetonitrile.
6. a kind of benzoyl peroxide detection kit, which is characterized in that described comprising reagent stock liquid a and reagent stock liquid b
Reagent stock liquid a is the phosphate buffer for the ethyl alcohol that pH value is 6~8, and the reagent stock liquid b is phenyl boron shown in formula (I)
The organic solvent solution of ester compound, the organic solvent is in dimethyl sulfoxide, n,N-Dimethylformamide, acetonitrile
The structural formula of at least one, the formula (I) is
7. benzoyl peroxide detection kit according to claim 6, which is characterized in that in the reagent stock liquid a
Phosphate be selected from Na2HPO4、NaH2PO4And KH2PO4At least one of, the concentration expressed in percentage by volume of ethyl alcohol in reagent stock liquid a
It is 10%, the phosphatic molar concentration is 5mM~15mM；
The concentration of phenylboric acid ester compounds shown in formula (I) is 0.01mM~1mM in the reagent stock liquid b.
8. benzoyl peroxide detection kit according to claim 7, which is characterized in that the reagent stock liquid a's
PH value is 7.4, wherein phosphatic molar concentration is 10mM；
The concentration of phenylboric acid ester compounds shown in formula (I) is 0.01mM~1mM in the reagent stock liquid b.
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