CN108395372B - The metal complex and its synthetic method of double cup [4] arene derivatives and application - Google Patents

The metal complex and its synthetic method of double cup [4] arene derivatives and application Download PDF

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CN108395372B
CN108395372B CN201810215032.1A CN201810215032A CN108395372B CN 108395372 B CN108395372 B CN 108395372B CN 201810215032 A CN201810215032 A CN 201810215032A CN 108395372 B CN108395372 B CN 108395372B
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arene
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cho
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CN108395372A (en
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刘元忠
唐玉国
刘涛
董文飞
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Suzhou Guoke medical technology development (Group) Co., Ltd
Suzhou Institute of Biomedical Engineering and Technology of CAS
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Suzhou Institute of Biomedical Engineering and Technology of CAS
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Abstract

The invention discloses a kind of metal complexs of double cup [4] arene derivatives, have the chemical structure as shown in Formula II.Above-mentioned complex small toxicity, biocompatibility are high, suitable for the biomolecule covalent bond such as nucleic acid, peptide or protein, form the fluorescent probe molecule that fluorescent quantum rate is high, fluorescence lifetime is long, detection limit is low, suitable for the biomolecule detection in cell, tissue or living body, it is with a wide range of applications in biomedicine field.The invention discloses the synthetic methods of above-mentioned complex, provide multiple choices approach for the synthesis of complex, and synthesis path is simple and easy, and complex yield obtained is high, are adapted for engagement with the large-scale production preparation of object.

Description

The metal complex and its synthetic method of double cup [4] arene derivatives and application
Technical field
The invention belongs to technical field of biomedical detection, and in particular to a kind of double cup [4] arene derivatives and its metal Complex, synthetic method and application.
Background technique
Molecular recognition is main body (receptor) to object (substrate) selective binding and generates the process of specific function.It identified Journey may cause the variation of the magnetism, electricity, optical property and conformation of system, so as to cause chemical information storage, transmitting with And processing.Therefore, molecular recognition plays an important role in information processing transmitting, molecule and supermolecule device fabrication process.
It is main at present for causing magnetic or fluorescence property change to carry out molecular recognition using the combination of Subjective and Objective molecule Research direction, the extraction and separation of ion, bioanalysis, environmental project, material science and medicine etc. it is multi-field have extensively Application.Wherein, Fluorometric assay of fluorescence-labeled is a key area of molecular recognition, and fluorescence probe is established in molecular recognition and fluorescence It is a kind of molecule that molecular recognition process can be passed through to fluorescence signal effective expression, it is widely used in sun on the basis of technology The fluorescence identifying of the plurality of target substance such as ion, anion and neutral molecule detects.Fluorescent probe molecule is usually by recognition group It is formed with fluorophor two parts, recognition group decision realizes molecule by selective binding to the selectivity of different objects It identifies and obtains the information of molecule；Molecule combining information is converted to by the fluorescence signal transmission mechanism of phase fluorophor therewith It is easy to the fluorescence signal detected, to realize real-time detection in situ on a molecular scale.
In order to further increase the combination stability of subject and object molecule, selectivity in molecular recognition, new point is found The host molecule of son identification has become a hot topic of research.Calixarenes (calixarenes) as a kind of Novel macrocyclic compound, be by A kind of cyclic oligomer that phenol units are connected by methylene, because it exactly likes Greece sage by what aromatic ring was woven into Cup, therefore it is named as calixarenes.Calixarenes has synthesis material cheap and easy to get, and conformation is variable in a solvent, has unique space The advantages that structure, cavity size are adjustable, can form host-guest complex with cation, anion and neutral molecule；It can be with Guest molecule, therefore, calixarenes are identified by means of non-covalent bond effects such as hydrogen bond, electrostatic interaction, molecular force, pi accumulations " third generation host molecule " that has more potential for development being counted as after supramolecular chemistry relaying crown ether and cyclodextrin.
Talanova et al. reports the Hg based on cup [4] arene derivatives2+Chemical sensor.Wherein, cup [4] aromatic hydrocarbons spreads out Biology is to obtain the Derivatives Modified of naphthalene sulfonylamide on cup [4] aromatic hydrocarbons, and molecular structure is as follows:
The chloroformic solution of cup [4] arene derivatives has strong fluorescence at 520nm, and Hg is added2+Afterwards, cup [4] aromatic hydrocarbons spreads out Biological selectivity identifies Hg2+, and occur from sulfuryl amine group to Hg2+Electronics transfer, greatly reduce its fluorescence intensity, it is real Existing optical detection.But above-mentioned cup [4] arene derivatives selective binding Hg2+, it is imaged based on principle of fluorescent quenching, is examined in fluorescence There are background signals when survey it is big, sensitivity is lower the disadvantages of, be unsuitable for the biomolecule such as labeling nucleic acid or albumen for biological sample The detection of product limits its application in field of biomedicine.
Summary of the invention
Therefore, the technical problem to be solved by the present invention is to cup in the prior art [4] arene derivatives bind metal ions Have the defects that sensitivity is lower in fluorescence detection, be unsuitable for marking biomolecule, so that it is higher to provide a kind of sensitivity, fits In the metal complex of double cups [4] arene derivatives of biomolecular labeling.
For this purpose, having and being tied as shown in Formula II the present invention provides a kind of metal complex of double cup [4] arene derivatives Structure:
Wherein, M is rare earth ion；
R1~R8Separately it is selected from-H ,-NH2、-COOH、-CH2COOH、-CONH(CH2)XNH2、-CONH2、-SH、- CH2SH、-SO3H、 -SO3R、-NHCO(CH2)XCOOH、-NO2、-CH2PO(OH)2、With
R is alkali metal or benzene, R1~R8It is not simultaneously-H, the integer that X is 0~3；
A is selected from-(CH2-CH2)n-、-(CH2OCH2)n-、-CONH(CH2)nNHCO- andN be 1~ 3 integer.
Preferably, the metal complex of above-mentioned double cups [4] arene derivatives, the rare earth metal are terbium or europium.
It is further preferred that the metal complex of above-mentioned double cups [4] arene derivatives, double cup [4] aromatic hydrocarbons are derivative The metal complex of object is selected from any chemical structure of Formula II -1~Formula II -51.
The present invention provides a kind of synthetic methods of the metal complex of above-mentioned double cups [4] arene derivatives, including with Lower step:
(1) cup [4] arene compounds shown in cup [4] arene compounds shown in formula E and formula E ' are synthesized；Formula E ' institute Cup [4] arene compounds shown obtain intermediate shown in formula F ' through condensation reaction, introducing-O-A- group；
Wherein, R1'~R8' separately it is selected from-CHO ,-CH2CHO、-NHCO(CH2)XCHO、-SO3R、-H、-NH2、- CONH(CH2)XNH2、-CONH2、-SH、-CH2SH、-NO2、-CH2PO(OH)2、R is Alkali metal or benzene, the integer that X is 0~3, R1'~R8' it is not simultaneously-H；
TsO- is p-methyl benzenesulfonic acid base；
(2) intermediate shown in formula F ' is dissolved in the first organic solvent, p-methyl benzene sulfonic chloride is added, it is anti-at 0~4 DEG C It answers, after reaction pours into reaction solution in acid, precipitating, cup [4] arene compounds shown in as formula F are precipitated；
(3) [4] arene compounds of cup shown in formula E and alkali metal salt are dissolved in the second organic solvent, F is added after mixing Shown in cup [4] arene compounds, then second organic solvent is changed to third by the back flow reaction at 70~75 DEG C Organic solvent continues back flow reaction, filters after reaction, and the crystal in filtrate is precipitated；
(4) work as R1'~R8' it does not include-CHO ,-CH2CHO、-NHCO(CH2)XCHO and-SO3It is obtained in step (3) when R Crystal is double cup [4] arene derivatives shown in Formulas I；
Work as R1'~R8' in include at least one-CHO ,-CH2CHO、-NHCO(CH2)XCHO and/or-SO3When R, step (3) In the crystal obtained intermediate shown in ' shown in intermediate, by Formulas I ' that is Formulas I after peroxidating and/or hydrolysis, Formulas I is made Shown in double cup [4] arene derivatives；
(5) in an inert atmosphere, after rare earth metal salt being mixed with the 4th organic solvent that dehydration and deoxidation is handled It flows back at 75~80 DEG C, double cup [4] arene derivatives shown in Formulas I is then added, continues back flow reaction, cools down after reaction Reaction product filters, the metal complex of double cup [4] arene derivatives shown in isolated Formula II；
Synthetic route is as follows:
The present invention provides a kind of synthetic method of the metal complex of above-mentioned double cups [4] arene derivatives, features It is, comprising the following steps:
S1. cup [4] arene compounds shown in cup [4] arene compounds shown in formula E and formula E ' are synthesized；Wherein, R1'~R8' separately it is selected from-CHO ,-CH2CHO、-NHCO(CH2)XCHO、-SO3R、-H、-NH2、-CONH(CH2)XNH2、-CONH2、-SH、-CH2SH、-NO2、 -CH2PO(OH)2、R be alkali metal or Benzene, the integer that X is 0~3, R1'~R8' it is not simultaneously-H；
S2. by cup [4] arene compounds and alkali metal salt shown in cup shown in formula E [4] arene compounds, formula E ' It mixes, is dissolved in the 5th organic solvent, continuously adds compound TsO-A-OsT, under inert gas protection, at 105-120 DEG C Back flow reaction, cooled reaction solution, post separation obtain reaction product after reaction；Wherein, TsO- is p-methyl benzenesulfonic acid base；
S3. work as R1'~R8' it does not include-CHO ,-CH2CHO、-NHCO(CH2)XCHO and-SO3It is obtained in step S2 when R Reaction product is intermediate shown in Formulas I；
Work as R1'~R8' in include at least one-CHO ,-CH2CHO、-NHCO(CH2)XCHO and/or-SO3When R, step S2 In the reaction product obtained intermediate shown in ' shown in intermediate, by Formulas I ' that is Formulas I after peroxidating and/or hydrolysis, be made Double cup [4] arene derivatives shown in Formulas I；
S4. in an inert atmosphere, after rare earth metal salt being mixed with the 4th organic solvent that dehydration and deoxidation is handled It flows back at 75~80 DEG C, double cup [4] arene derivatives shown in Formulas I is then added, continues back flow reaction, cools down after reaction Reaction product filters, the metal complex of double cup [4] arene derivatives shown in isolated Formula II；
Synthetic route is as follows:
Preferably, above-mentioned synthetic method, first organic solvent are pyridine, and second organic solvent is acetonitrile, The third organic solvent is second alcohol and water with the mixed solution of the volume ratio of 1:1, and the 4th organic solvent is acetonitrile, described Alkali metal salt is potassium carbonate.
Preferably, above-mentioned synthetic method, the 5th organic solvent are toluene.
Preferably, above-mentioned synthetic method, cup shown in cup [4] arene compounds and the formula F shown in the formula E [4] molar ratio of arene compounds is 3:2~1:1, double cup [4] arene derivatives shown in the Formulas I and rare earth gold The molar ratio for belonging to salt is 1:1~2:3, the molar ratio of cup [4] arene compounds and the alkali metal salt shown in the formula E For 1:5.
Preferably, above-mentioned synthetic method, cup shown in cup [4] arene compounds, the formula E ' shown in the formula E [4] molar ratio of arene compounds, the compound TsO-A-OsT and the alkali metal salt is (2~3): (2~3): (2.5 ~3): (12.5~20).
Purposes the present invention provides the metal complex of above-mentioned double cups [4] arene derivatives as fluorescent dye.
The present invention provides a kind of fluorescent probe molecule, the fluorescent probe molecule is the biology point of fluorochrome label Son, the fluorescent dye be above-mentioned double cups [4] arene derivatives metal complex, the biomolecule selected from amino acid, At least one of polypeptide, albumen, nucleotide, oligonucleotides and nucleic acid.
Preferably, above-mentioned fluorescent probe molecule, the nucleotide, oligonucleotides and nucleic acid are amido modified nucleosides Acid, oligonucleotides and nucleic acid.
Preferably, above-mentioned fluorescent probe molecule, the metal complex of double cup [4] arene derivatives is in bridging agent In the presence of, it is connected with any group in the amino of the biomolecule, carboxyl and sulfydryl.
It is further preferred that above-mentioned fluorescent probe molecule, the bridging agent is n-hydroxysuccinimide, N- hydroxyl sulphur For succinimide salt, maleimide, glutaraldehyde or carbodiimide.
Above-mentioned technical proposal of the invention has the advantage that
1. the metal complex of double cups [4] arene derivatives provided by the invention has the structure as shown in Formula II.
Complex shown in Formula II is to be bonded based on double cups [4] arene derivatives I by object of rare earth ion Made of inclusion complex, wherein double cups [4] arene derivatives I is by two one cup [4] arene derivatives respectively by the hydroxyl of lower edge Base is connected double cups [4] aromatic hydrocarbons of four bridgings to be formed with bridging unit A, and A is separately selected from-(CH2-CH2)n-、- (CH2OCH2)n-、-(CH2PhCH2)nAnd-CONH (CH2)nThe integer that NHCO-, n are 1~3.Above-mentioned double cups [4] aromatic hydrocarbons is derivative Object conformational stability, double cup [4] arene derivatives form three-dimensional tube cavity at bridging position, in entrance tube cavity Metal ion forms certain space limit；On the one hand bridge linkage group A is formed to metal ion using non-covalent bond with cooperation With, on the other hand by adjust bridge linkage group A, the size of three-dimensional tube cavity can be adjusted, with formed to specific gold Belong to the position-limiting action of ion.Synergistic effect by space limit with non-covalent bond cooperation between the two, makes structure shown in Formulas I Double cups [4] arene derivatives Selective recognition rare earth ion, and have excellent mating capability.
The complex that double cup [4] arene derivatives selective complexation rare earth ions are formed has good water solubility, toxicity It is small, can be compatible with living cells etc. advantages.Meanwhile R1~R8Substituent group can be condensed by amidation or sulfuryl amine etc. The biomolecule covalent bond such as reaction and nucleic acid, albumen, polypeptide, obtains the conjugate of complex and biomolecule, with realization pair External, the detection of intracellular or biological living of specific protein, DNA molecular. R1~R8Amido bond in substituent group can increase Add double cup [4] arene derivatives to the selection Complex effect of rare earth ion, to improve complex as biomolecule detection The detection effect of probe.
Double cup [4] arene derivatives selective complexation rare earth ions make rare earth ion enter double cup [4] aromatic hydrocarbons In the three-dimensional tube cavity of derivative, the complex of conformational stability is formed.Energy in complex is transmitted by rare earth ion To double cup [4] arene derivatives, make double cup [4] arene derivatives that energy level transition occur, it finally will in the form of fluorescent photon Amount releases, and complex is made to generate characteristic fluorescence.Amide groups, carbonyl in double cup [4] aromatic hydrocarbons,Etc. structures be beneficial to further increase calixarenes to dilute The ability of earthmetal cations absorbs, and carries out effective transmitting of energy, makes finally with the fluorescence volume of the fluorescence probe of complex preparation Sub- yield height, fluorescence lifetime length, detection limit are low, luminosity is stablized, and can be realized long-time, real-time, dynamic METHOD FOR CONTINUOUS DETERMINATION.Separately Outside, due to fluorescence probe have it is preferable water-soluble, stable in physiological conditions, it is good to cell membrane penetration, and to cell toxicant Property it is small, be suitable for active somatic cell detect.
2. the present invention provides the synthetic method of the metal complex of two kinds of double cup [4] arene derivatives, mentioned for complex II Various selectable synthesis path is supplied, and synthesis process is simple and easy, it is cheap and easy to get to synthesize required raw material, with above two The yield that method prepares complex II is high, is suitable for large-scale industrial production and prepares.
The present invention provides the raw material proportioning for the metal complex for being suitable for double cup [4] arene derivatives of high yield and synthesis items Part can reduce cup [4] arene compounds and compound TsO-A- shown in cup shown in formula E [4] arene compounds, formula E ' Cup [4] arene compounds are in condensation reaction shown in cup [4] arene compounds and formula F shown in OsT or formula E Mispairing combine, improve the yield of double cup [4] arene derivatives I.
3. fluorescent probe molecule provided by the invention, wherein fluorescent probe molecule is the biomolecule of fluorochrome label, Fluorescent dye is the metal complex of above-mentioned double cups [4] arene derivatives, and biomolecule is selected from amino acid, polypeptide, albumen, core At least one of thuja acid, oligonucleotides and nucleic acid.Above-mentioned fluorescent probe molecule in conjunction with object to be measured molecular specificity (such as Object to be measured molecule be antigen, then biomolecule be the antibody specifically bound therewith), then pass through fluorescence detection with obtain to Survey target molecule information.It is produced since the metal complex that double cups [4] arene derivatives and rare earth ion are formed has both fluorescence The advantage that rate is high, fluorescence lifetime is long, fluorescence property is stablized and small toxicity, cell membrane penetration are good and bio-compatibility is high, can It is widely used in the detection of intraor extracellular substance, tissue and the imaging of living animal label, Pharmaceutical Analysis, pathological model research and disease Early diagnosis etc., it is with important application prospects in field of biomedical research.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the nuclear magnetic resonance figures of double cups [4] arene derivatives I-1 in the embodiment of the present invention 1；
Fig. 2 is the testing result of the fluorescence spectrum of complex II-1 in experimental example 1 of the present invention；
Fig. 3 is the testing result of the fluorescence spectrum of complex II-2 in experimental example 1 of the present invention；
Fig. 4 is the testing result of the fluorescence spectrum of complex II-3 in experimental example 1 of the present invention；
Fig. 5 is the testing result of the fluorescence spectrum of complex II-4 in experimental example 1 of the present invention；
Fig. 6 is the complex II-1 of various concentration in experimental example 2 of the present invention to prostate gland cancer cell DU-145 growth inhibition Testing result figure；
Fig. 7 is the complex II-2 of various concentration in experimental example 2 of the present invention to prostate gland cancer cell DU-145 growth inhibition Testing result figure；
Fig. 8 is the complex II-3 of various concentration in experimental example 2 of the present invention to prostate gland cancer cell DU-145 growth inhibition Testing result figure；
Fig. 9 is the complex II-4 of various concentration in experimental example 2 of the present invention to prostate gland cancer cell DU-145 growth inhibition Testing result figure；
Figure 10 is shown in experimental example 3 of the present invention marks EGFR antibody to detect cancer as fluorescent probe molecule using complex II-1 The fluorescence imaging result figure that EGFR is expressed in disease cell；
Figure 11 shows complex II-1~fluorescent stability of the complex II-4 as fluorescent dye in experimental example 5 of the present invention Testing result figure.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention. In addition, technical characteristic involved in invention described below different embodiments is as long as they do not conflict with each other It can be combined with each other.
The basic chemical industries raw material such as reagent used in the embodiment of the present invention chemical products can be commercially available at home, or Related intermediate preparation factory is customized.
Embodiment 1
The present embodiment provides a kind of metal complex and preparation method thereof of double cup [4] arene derivatives, double cup [4] aromatic hydrocarbons Derivative has the molecular structure as shown in Formulas I -1；Double cup [4] arene derivatives and rare earth ion Tb3+Matched Object is closed, molecular structure is as shown in Formula II -1:
With intermediate E -1 and intermediate F-1 synthetic intermediate I ' -1, double cup [4] aromatic hydrocarbons are obtained after the oxidation of intermediate compound I ' -1 Derivative I -1, double cup [4] arene derivatives I-1 complex rare-earth metal ion Tb3+, obtain double cup [4] arene derivatives I-1 with Tb3+Complex II-1, synthetic route are as follows:
(1) preparation of intermediate E -1
Step 1: using cup [4] aromatic hydrocarbons as reaction raw materials, taking cup [4] aromatic hydrocarbons (30.0g/70.7mmol), Anhydrous potassium carbonate (10.7g/77.4mmol) and p-methyl benzenesulfonic acid methyl esters (26.3g/141.4mmol), is dissolved in the CH of 500mL3It flows back in CN For 24 hours, it drains, the CH of 500mL is then added2Cl2And be added 1mol/L HCl (2 × 50mL), salt water 50mL, solvent is removed, is taken out It is dry, product I-1-1 is obtained after dry:
Step 2: taking compound I-1-1 (5.00g, 9.83mmol), NaH (1.60g, 39.30mmol, with 60% quality Score suspend in the oil), stir 30min after being dissolved in the DMF of 200mL, then be added 4-Nitrobenzenesulfonyl chloride (8.71g, 39.30mmol), it reacts at room temperature 7 days and obtains compound I-1-2:
Step 3: by compound I-1-2 (0.5g, 0.57mmol), hexamethylenetetramine (2.87g, 20.00mmol) is dissolved in In 30mL trifluoroacetic acid, 22h is reacted under -80 DEG C, nitrogen protection, obtains compound I-1-3, ethyl alcohol of the compound I-1-3 in KOH The 12h that flows back in solution obtains compound I-1-4, and compound I-1-4 is intermediate E -1.
The nuclear-magnetism characterize data of compound I-1-4 (intermediate E -1):1H-NMR(CDC13)8.23(s,2H,CHO),7.28 (s, 4H, Ar), 6.91 (s, 6H, Ar), 5.99 (s, 4H, OH), 3.81 (s, 8H, CH2)。
Mass spectrum mlecalcd 480.14, found (M-H+)481.20。
(2) preparation of intermediate F-1
Step 5: intermediate E -1 is dissolved in the ether of 250mL, is added NaH (33.5mmol), is stayed overnight in 0 DEG C of reaction, after Continue the BrCH that 26.8mmol is slowly added into reaction solution2CH2Precipitating is precipitated in OH, removes acetonitrile solvent, and chromatography is isolated Compound I-1-5, yield: 52%:
Step 6: at a temperature of 0 DEG C, compound I-1-5 (1.2mmol) being dissolved in the pyridine of 25ml, is then added to first Base benzene sulfonyl chloride (12.5mmol) saves 4 days in 4 DEG C of refrigerators, and solution pours into the HCl of 250mL 2mol/L frost, is precipitated Precipitating filters, and collects, dry intermediate F-1:
The nuclear-magnetism characterize data of intermediate F-1:1H-NMR(CDC13) 8.23 (s, 2H, CHO), 8.0-7.84 (d, 10H, Ar),7.55(s,4H,Ar),7.38 (s,4H,Ar),3.81(m,8H,CH2),1.28,1.11(s,12H,CH3)；
mass spectrum mlecalcd 1164.14,found(M-H+)1165.20。
(3) preparation of intermediate compound I ' -1
Intermediate E -1 (0.7mmol) and potassium carbonate (3.5mmol) are dissolved in acetonitrile, then intermediate is added in stirring 2h F-1 (0.7mmol) flows back 5 days at 70 DEG C, and after vacuum pump removes solvent, the ethyl alcohol-of 1:1 (v:v) is added into solid matter Then water mixed liquid dissolution, suspension heated overnight at reflux filter while hot, crude product is dissolved in 50mL chloroform, then uses filter paper Limpid solution is obtained by filtration, is then added the acetone of 40mL, intermediate compound I is precipitated to obtain in crystal ' -1, yield 74%:
The nuclear-magnetism characterize data of intermediate compound I ' -1:1H-NMR(CDC13) 9.92 (s, 4H, CHO), 7.89-7.17 (m, 20H, Ar),4.36(d,8H, PhOCH2CH2Ph),3.81(s,16H,PhCH2Ph),2.28(s,12H,CH3)；
mass spectrum mlecalcd 1069.38,found(M-H+)1070.45。
(4) double cup [4] arene derivatives I-1, double cups [4] arene derivatives I-1 and Tb3+Complex II-1 preparation
Intermediate compound I ' -1 is dissolved in acetonitrile, and it is derivative up to double cup [4] aromatic hydrocarbons that suitable trifluoroacetic acid (TFA) reaction 2h is added Under argon gas protective condition, TbCl is added into the 100mL two mouth flask of dehydration and deoxidation in object I-13With the processed second of dehydration and deoxidation The acetonitrile solution of double cup [4] arene derivatives I-1 is added dropwise into the white suspension after half an hour of flowing back at 75 DEG C for nitrile, TbCl3Molar ratio with compound I-1 is 1.2:1, reacts 4h.Cooled reaction solution after reaction, filtering, HPLC are separated To double cups [4] arene derivatives I-1 and Tb3+Complex II-1:
The nuclear-magnetism characterize data of double cup [4] arene derivatives I-1:1H-NMR(CDC13)7.84-7.26(m,20H,Ar), 4.36(d,8H, PhOCH2CH2Ph),3.89(s,12H,CH3), 3.81 (s, 16H, PhCH2Ph)；
mass spectrum mlecalcd 1132.33,found(M-H+)1133.26。
Embodiment 2
The present embodiment provides the synthetic methods of the metal complex II-1 of double cup [4] arene derivatives a kind of:
With intermediate E -1, intermediate E ' -1 (being equal to intermediate E -1) and TsO-CH2-CH2- OsT synthetic intermediate I '- 1, obtain double cup [4] arene derivatives I-1 after the oxidation of intermediate compound I ' -1, double cup [4] arene derivatives I-1 complex rare-earth metals from Sub- Tb3+, obtain double cup [4] arene derivatives I-1 and Tb3+Complex II-1, synthetic route are as follows:
(1) preparation of intermediate E -1
For synthesis process with the synthesis of the intermediate E -1 of embodiment 1, intermediate E ' -1 and intermediate E -1 are same compound.
(2) preparation of intermediate compound I ' -1
By intermediate E -1 (0.8mmol), intermediate E ' -1 (1mmol) and K2CO3(7mmol) is dissolved in toluene, then plus Enter TsO-CH2-CH2- OsT (1.1mmol), under nitrogen protection, flow back 3h at a temperature of 105 DEG C, is then added a certain amount of Polyethylene glycol, then flow back 2.5 days, cooling, the isolated intermediate compound I of silicagel column ' -1, yield 68%:
(3) double cup [4] arene derivatives I-1, double cups [4] arene derivatives I-1 and Tb3+Complex II-1 preparation
Intermediate compound I ' -1 is dissolved in acetonitrile, and it is derivative up to double cup [4] aromatic hydrocarbons that suitable trifluoroacetic acid (TFA) reaction 2h is added Under argon gas protective condition, TbCl is added into the 100mL two mouth flask of dehydration and deoxidation in object I-13With the processed second of dehydration and deoxidation The acetonitrile solution of double cup [4] arene derivatives I-1 is added dropwise into the white suspension after 80 DEG C of reflux half an hours for nitrile, TbCl3Molar ratio with compound I-1 is 1:1, reacts 4h.Cooled reaction solution after reaction, filtering, HPLC are isolated Double cup [4] arene derivatives I-1 and Tb3+Complex II-1:
Embodiment 3
The present embodiment provides a kind of metal complex and preparation method thereof of double cup [4] arene derivatives, double cup [4] aromatic hydrocarbons Derivative has the molecular structure as shown in Formulas I -2；Double cup [4] arene derivatives and rare earth ion Eu3+Matched Object is closed, molecular structure is as shown in Formula II -2:
It is synthesized with intermediate E -2 and intermediate F-2 (intermediate shown in formula F-2 is equal to intermediate shown in formula F-1) Intermediate compound I ' -2 obtain double cup [4] arene derivatives I-2, double cup [4] arene derivatives I-2 complexings after the oxidation of intermediate compound I ' -2 Rare earth ion Eu3+, obtain double cup [4] arene derivatives I-2 and Eu3+Complex II-2, synthetic route are as follows:
(1) preparation of intermediate E -2
Step 1: using cup [4] aromatic hydrocarbons as reaction raw materials, taking cup [4] aromatic hydrocarbons (30.0g/70.7mmol), Anhydrous potassium carbonate (10.7g/77.4mmol), p-methyl benzenesulfonic acid methyl esters (26.3g/141.4mmol), is dissolved in the CH of 500mL3It flows back in CN for 24 hours, It drains, the CH of 500mL is then added2Cl2And be added 1mol/L HCl (2 × 50mL), salt water 50mL, solvent is removed, is drained, is done Product I-1-1 is obtained after dry:
Step 2: taking compound I-1-1 (5.00g, 9.83mmol), NEH (1.60g, 39.30mmol, with 60% quality Score suspend in the oil), stir 30min after being dissolved in the DMF of 200mL, then be added 4-Nitrobenzenesulfonyl chloride (8.71g, 39.30mmol), it reacts at room temperature 7 days and obtains compound I-1-2:
The nuclear-magnetism characterize data of compound I-1-2:1H-NMR(CDC13) 8.34 (d, 4H, Er), 7.91 (m, 4H, Er), 7.7 (d, 6H, Er), 6.91 (m, 6H, Er), 3.81 (d, 14H, CH2, CH3), 1.2 (s, 2H, OH).
MEss spectrum mlecElcd 822.16, found (M-H+)823.17。
Step 3: compound I-1-2 (1.14mmol) is dissolved in 100mL methylene chloride and 12mL acetum, continues thereto The nitric acid of 4mL is added dropwise, reacts at room temperature 48h, is poured into water, then with the CH of 3 × 100mL2Cl2Extraction removes solvent and obtains compound I-2-1, yield 83%:
The nuclear-magnetism characterize data of compound I-2-1:1H-NMR(CDC13) 8.34 (d, 4H, Er), 8.02 (m, 4H, Er), 7.91 (d, 4H, Er), 6.91 (m, 6H, Er), 3.81 (d, 14H, CH2,CH3), 1.2 (s, 2H, OH).
MEss spectrum mlecElcd 822.16, found (M-H+)823.17。
Step 4: compound I-2-1 (1.0mmol), 1mol/L tetrabutylammonium fluoride tetrahydrofuran solution 0.0044mL with 4.4mmol acetic acid is mixed in jointly in the DMF of 7.2mL, reacts at room temperature 3h.Continue to be added 1.2mol/L's to reaction mixture HCl, solution layering, is extracted with chloroform, rotates dry compound I-2-2, yield 93%:
The nuclear-magnetism characterize data of compound I-2-2:1H-NMR(CDC13) 8.17 (d, 4H, Er), 7.24~6.77 (m, 6H, Er), 3.81 (d, 14H, CH2,CH3), 0.62 (s, 2H, OH).
MEss spectrum mlecElcd 542.16, found (M-H+)543.27。
Step 5: to catalyst nickel is added in compound I-2-2 (0.67mmol), being dissolved in the methanol containing 1.5mL hydrazine hydrate In (100mL) solution, reflux 7h is added, the hydrazine hydrate and catalyst nickel for adding 1.5mL continue the 15h that flows back, are cooled to room temperature, Filtering, chromatography separation, obtains compound I-2-3, compound I-2-3 is intermediate E -2:
The nuclear-magnetism characterize data of intermediate compound I -2-3 (intermediate E -2):1H-NMR(CDC13) 8.52 (s, 2H, OH), 7.16 (d, 4H, Er), 6.9 (m, 2H, Er), 6.68 (d, 4H, Er), 4.09 (s, 4H, NH2), 3.86 (s, 6H, CH3), 3.81 (s, 8H, CH2)；
MEss spectrum mlecElcd 482.22, found (M-H+)483.26。
(2) preparation of intermediate F-2
The intermediate F-1 prepared in intermediate F-2 and embodiment 1 is same substance.
(3) preparation of intermediate compound I ' -2
Intermediate E -2 (0.84mmol), potassium carbonate (4.2mmol) are dissolved in acetonitrile, then intermediate F-2 is added in stirring 2h (0.7mmol), 80 DEG C are flowed back 5 days, and vacuum pump removes solvent, and the solid matter ethanol-water mixture of 1:1 (V:V) is dissolved, Suspension heated overnight at reflux, reflux temperature are 75 DEG C, are then filtered while hot, and crude product is dissolved in 50mL chloroform, then with filter Limpid solution is obtained by filtration in paper, is then added the acetone of 40mL, intermediate compound I is precipitated to obtain in crystal ' -2, yield 72%:
The nuclear-magnetism characterize data of intermediate compound I ' -2:1H-NMR(CDC13) 8.23 (s, 2H, CHO), 7.84-6.50 (m, 22H, Ar),4.36(d,16H, PhOCH2CH2),4.14(s,2H,PhCH2),3.81(s,12H,CH2),3.55(s,2H,CH2),2.58 (m,2H,CH2),1.54(s,18H,CH3)。
mass spectrum mlecalcd 1042.15,found(M-H+)1043.25。
(4) double cup [4] arene derivatives I-2, double cups [4] arene derivatives I-2 and Eu3+Complex II-2 preparation
Intermediate compound I ' -2 are dissolved in acetonitrile, and it is derivative up to double cup [4] aromatic hydrocarbons that suitable trifluoroacetic acid (TFA) reaction 2h is added Under argon gas protective condition, EuCl is added into the 100mL two mouth flask of dehydration and deoxidation in object I-23With the processed second of dehydration and deoxidation The acetonitrile solution of double cup [4] arene derivatives I-2 is added dropwise into the white suspension after half an hour of flowing back at 80 DEG C for nitrile, EuCl3Molar ratio with compound I-2 is 1.5:1, reacts 5h.Cooled reaction solution after reaction, filtering, HPLC are separated To double cups [4] arene derivatives I-2 and Eu3+Complex II-2:
Embodiment 4
The present embodiment provides the synthetic methods of the metal complex II-2 of double cup [4] arene derivatives a kind of:
With intermediate E -2, intermediate E ' -2, (intermediate shown in intermediate shown in formula E ' -2 and formula E-1 is same object Matter) and TsO-CH2-CH2- OsT synthetic intermediate I ' -2 obtain double cup [4] arene derivatives I-2 after the oxidation of intermediate compound I ' -2, Double cup [4] arene derivatives I-2 complex rare-earth metal ion Eu3+, obtain double cup [4] arene derivatives I-2 and Eu3+Cooperation Object II-2, synthetic route are as follows:
(1) preparation of intermediate E -2
Synthesis of the synthesis process with the intermediate E -2 of embodiment 3.
(2) preparation of intermediate E ' -2
Preparation process of the synthesis process with the intermediate E -1 of embodiment 1.
(3) preparation of intermediate compound I ' -2
By intermediate E -2 (1.2mmol), intermediate E ' -2 (0.8mmol) and K2CO3(5mmol) is dissolved in toluene, then TsO-CH is added2-CH2- OsT (1mmol), under nitrogen protection, flow back 2h at a temperature of 120 DEG C, is then added a certain amount of Polyethylene glycol, then flow back 2 days, cooling, the isolated intermediate compound I of silicagel column ' -2, yield 73%:
(3) double cup [4] arene derivatives I-2, double cups [4] arene derivatives I-2 and Eu3+Complex II-2 preparation
Intermediate compound I ' -2 are dissolved in acetonitrile, and suitable trifluoroacetic acid (TFA) reaction 2.2h is added and spreads out up to double cup [4] aromatic hydrocarbons Under argon gas protective condition, EuCl is added into the 100mL two mouth flask of dehydration and deoxidation in biological I-23It is processed with dehydration and deoxidation Acetonitrile.It is flowed back after half an hour at 75 DEG C, the acetonitrile that double cup [4] arene derivatives I-2 are added dropwise into the white suspension is molten Liquid, EuCl3Molar ratio with compound I-2 is 1.2:1, reacts 5h.Cooled reaction solution after reaction, filtering, HPLC points From obtaining double cup [4] arene derivatives I-2 and Eu3+Complex II-2:
Embodiment 5
The present embodiment provides a kind of metal complex and preparation method thereof of double cup [4] arene derivatives, double cup [4] aromatic hydrocarbons Derivative has the molecular structure as shown in Formulas I -3；Double cup [4] arene derivatives and rare earth ion Eu3+Matched Object is closed, molecular structure is as shown in Formula II -3:
To obtain intermediate after intermediate E -3 and intermediate F-3 synthetic intermediate I ' -3-1, intermediate compound I ' -3-1 oxidation I ' -3-2, intermediate compound I ' -3-2 obtain double cup [4] arene derivatives I-3 through hydrolysis, and double cup [4] arene derivatives I-3 complexings are dilute Earthmetal cations Eu3+, obtain double cup [4] arene derivatives I-3 and Eu3+Complex II-3, synthetic route are as follows:
(1) preparation of intermediate E -3
Step 1: using cup [4] aromatic hydrocarbons as reaction raw materials, taking cup [4] aromatic hydrocarbons (30.0g/70.7mmol), Anhydrous potassium carbonate (10.7g/77.4mmol), p-methyl benzenesulfonic acid methyl esters (26.3g/141.4mmol), is dissolved in the CH of 500mL3It flows back in CN for 24 hours, It drains, the CH of 500mL is then added2Cl2And be added 1mol/L HCl (2 × 50mL), salt water 50mL, solvent is removed, is drained, is done Product I-1-1 is obtained after dry:
Step 2: compound I-1-1 (3.9mmol) is dissolved in the CH of 200mL2Cl2In the mixed liquor of the acetic acid of 2mL, then plus Enter the HNO of 0.5mL3, normal-temperature reaction 45min, the NaHCO being saturated with 150mL3Aqueous solution terminates reaction, continues to stir 10min, remove Solvent is removed, chromatography obtains compound I-3-1:
Step 3: compound I-3-1 (1.81mmol) is dissolved in the CHCl of 30mL3In, -13 DEG C are cooled to, is then added Cl2CHOCH3(5.43mmol) and SnCl43h is stirred at room temperature in (1.81mmol), then terminates reaction with the HCl of 1mol/L, after Continuous stirring 20min, then removes solvent, purification by chromatography obtains compound I-3-2:
Step 4: hydrazine hydrate (8.36mmol) and catalyst Pd/C are added to the second containing 0.418mmol compound I-3-3 In alcohol (25mL) solution, 3h is reacted at 60 DEG C, in N2It is filtered to remove Pd/C catalyst under protection, removes solvent, purifyingization Object E-3 is closed, yield 76%:
(2) preparation of intermediate F-3
Step 5: p-sulfonic acid sodium cup [4] aromatic hydrocarbons (13.6mol) derivative is dissolved in the ether of 215mL, and LiAlH is added4 (10mmol) is stayed overnight in 0 DEG C of reaction, the HCl of 2mol/L is slowly added into reaction solution, precipitating is precipitated, removes ether, chromatography Isolated compound I-3-4, yield 84%:
Step 6: at a temperature of 0 DEG C, compound I-3-4 (1.2mmol) is dissolved in the pyridine of 25ml, is then added to methyl Benzene sulfonyl chloride (14.5mmol) then saves 4 days in 4 DEG C of refrigerators, and solution pours into the HCl of 250mL2mol/L frost, and there have to be heavy Precipitation goes out, and filtering collects precipitating, is dried to obtain intermediate F-3:
(3) preparation of double cup [4] arene derivatives I-3
Intermediate E -3 (1.05mmol), potassium carbonate (5.25mmol) are dissolved in acetonitrile, then intermediate F- is added in stirring 2h 3 (0.7mmol) flow back 5 days at 72 DEG C, and vacuum pump removes solvent, by the solid matter ethanol-water mixture of 1:1 (V:V) Then dissolution, suspension heated overnight at reflux filter while hot, crude product is dissolved in 50mL chloroform, is then obtained by filtration with filter paper Then the acetone of 40mL is added in limpid solution, double cup [4] arene derivatives I ' -3-1 are precipitated to obtain in crystal, yield 73%:
Step 8: compound I ' -3-1 (1.72mmol) is dissolved in chloroform/acetone (60ml, 1/1, v/v), is cooled to 0 DEG C, so After H is added2NSO3H (545mg, 5.62mmol) and NaClO2(342mg, 3.78mmol) mixed liquor 15mL, is stirred at room temperature 18h, Solvent is removed, chromatography obtains compound I ' -3-2, yield 70%:
Step 9: compound I ' -3-2 (1.72mmol) being dissolved in acetonitrile, suitable trifluoroacetic acid is added, is stirred at room temperature 6h removes solvent, and dry, purifying obtains compound I-3:
The nuclear-magnetism characterize data of compound I-3:1H-NMR(CDC13)8.02-7.25(d,22H,Ar),6.81-6.44(s, 20H,Ar),6.02-4.70(s,22H, CH2),4.36(d,12H,CH2),3.95(d,8H,CH2),3.89(m,8H,CH2), 3.81(s,12H,CH3)；
Mass spectrum mlecalcd1331.23, found (M-H+)1332.25。
(4) double cup [4] arene derivatives I-3 and Eu3+Complex II-3 preparation
Under argon gas protective condition, a certain amount of EuCl is added into the 100mL two mouth flask of dehydration and deoxidation3And dehydration and deoxidation Double cup [4] arene derivatives I-3 are added dropwise into the white suspension after half an hour of flowing back at 79 DEG C for processed acetonitrile Acetonitrile solution, EuCl3Molar ratio with compound I-1 is 1.3:1.4h is reacted, cooling, filtering, the isolated double cups of HPLC [4] arene derivatives I-3 and Eu3+Complex II-3:
Embodiment 6
With intermediate E -3, intermediate E ' -3 (intermediate shown in formula E ' -3 is p-sulfonic acid sodium cup [4] aromatic hydrocarbons) and TsO- CH2-CH2- OsT synthesizes double cup [4] arene derivatives I ' -3-1, obtains double cups after double cup [4] arene derivatives I ' -3-1 oxidations [4] arene derivatives I ' -3-2, compound I ' -3-2 hydrolyze to obtain double cup [4] arene derivatives I-3, and compound I-3 complexing is dilute Earthmetal cations Eu3+, obtain double cup [4] arene derivatives I-3 and Eu3+Complex II-3, synthetic route are as follows:
(1) preparation of intermediate E -3
For synthesis process with the synthesis of the intermediate E -3 of embodiment 5, intermediate shown in formula E ' -3 is p-sulfonic acid sodium cup [4] arene derivatives.
(2) preparation of intermediate compound I ' -3-1
By intermediate E -3 (1mmol), intermediate E ' -3 (1.2mmol) and K2CO3(8mmol) is dissolved in toluene, then plus Enter TsO-CH2-CH2- OsT (1.2mmol), under nitrogen protection, flow back 2.5h at a temperature of 110 DEG C, is then added a certain amount of Polyethylene glycol, then flow back 2 days, cooling, isolated the intermediate compound I ' -3-1 of silicagel column, yield 78%:
(4) double cup [4] arene derivatives I-3, double cups [4] arene derivatives I-3 and Eu3+Complex II-3 preparation
Synthesis process is the same as embodiment 5.
Embodiment 7
The present embodiment provides a kind of metal complex and preparation method thereof of double cup [4] arene derivatives, double cup [4] aromatic hydrocarbons Derivative has the molecular structure as shown in Formulas I -4；Double cup [4] arene derivatives and rare earth ion Tb3+Matched Object is closed, molecular structure is as shown in Formula II -4:
With intermediate E -4 and intermediate F-4 synthetic intermediate I ' -4, double cup [4] aromatic hydrocarbons are obtained after the oxidation of intermediate compound I ' -4 Derivative I -4, double cup [4] arene derivatives I-4 complex rare-earth metal ion Tb3+, obtain double cup [4] arene derivatives I-4 with Tb3+Complex II-4, synthetic route are as follows:
(1) preparation of intermediate E -4
Step 1: following intermediate Es -2 is made with the synthetic method of the intermediate E -2 of embodiment 3:
Step 2: intermediate E -2 (0.6mmol) being dissolved in the acetonitrile of 50mL, appropriate trifluoroacetic acid is added, is then added 4- (2,5- dioxo -1- pyrrolidinyl) benzoic acid (1.2mmol), flow back 12h, removes solvent, and purifying obtains intermediate E -4:
(2) preparation of intermediate F-4
Intermediate F-4 is equal to intermediate shown in formula F-2, and synthetic method is shown in the preparation method in embodiment 3.
(3) preparation of intermediate compound I ' -4
Intermediate E -4 (1.04mmol) and potassium carbonate (5.2mmol) are dissolved in acetonitrile, then intermediate is added in stirring 2h F-4 (0.8mmol) flows back 5 days at 74 DEG C, and vacuum pump removes solvent, and the solid ethanol-water mixture of 1:1 dissolves, suspended Liquid heated overnight at reflux, is then filtered while hot, and crude product is dissolved in 50mL chloroform, is then obtained by filtration with filter paper limpid molten Liquid, is then added the acetone of 40mL, intermediate compound I is precipitated to obtain in crystal ' -4, yield 63%:
(4) double cup [4] arene derivatives I-4, double cups [4] arene derivatives I-4 and Tb3+Complex II-4 preparation
Intermediate compound I ' -4 are dissolved in acetonitrile, suitable trifluoroacetic acid (TFA) are added, reaction 2h is stirred at room temperature, filter, remove Solvent crosses isolated double cup [4] the arene derivatives I-4 of pillar.Double cup [4] arene derivatives I-4 are dissolved in acetonitrile, and argon gas is protected Under the conditions of shield, a certain amount of TbCl is added into the 100mL two mouth flask of dehydration and deoxidation3With the processed acetonitrile of dehydration and deoxidation, TbCl3Molar ratio with compound I-1 is 1.4:1, cooling after 77 DEG C of reflux half an hours, filtering, HPLC isolatedization Close object double cup [4] arene derivatives I-4's and Tb3+Complex II-4:
The nuclear-magnetism characterize data of compound I-4: 8.03 (s, 2H, NH), 7.73-7.40 (m, 28H, Ar), 6.89 (m, 8H, CH2), 4.36 (d, 16H, PhOCH2CH2), 3.81 (s, 8H, PhCH2)。
Embodiment 8
The present embodiment provides the synthetic methods of the metal complex II-4 of double cup [4] arene derivatives a kind of:
With intermediate E -4, intermediate E ' -4 (being equal to intermediate A -1) and TsO-CH2-CH2- OsT synthetic intermediate I '- 4, obtain double cup [4] arene derivatives I-4 after the oxidation of intermediate compound I ' -4, double cup [4] arene derivatives I-4 complex rare-earth metals from Sub- Tb3+, obtain double cup [4] arene derivatives I-4 and Tb3+Complex II-4, synthetic route are as follows:
(1) preparation of intermediate E -4
Synthesis of the synthesis process with the intermediate E -4 of the present embodiment 7.
(2) preparation of intermediate E ' -4
Intermediate E ' -4 and intermediate E -1 are same compound, and synthesis process is the same as embodiment 1.
(3) preparation of intermediate compound I ' -4
By intermediate E -1 (1.1mmol), intermediate E ' -1 (0.8mmol) and K2CO3(6.5mmol) is dissolved in toluene, so After TsO-CH is added2-CH2- OsT (0.8mmol), under nitrogen protection, flow back 2h at a temperature of 115 DEG C, is then added certain The polyethylene glycol of amount, then flow back 2 days, cooling, the isolated intermediate compound I of silicagel column ' -4, yield 64%:
(3) double cup [4] arene derivatives I-4, double cups [4] arene derivatives I-4 and Tb3+Complex II-2 preparation
Synthesis process is the same as embodiment 7.
Embodiment 9
The present embodiment provides a kind of fluorescent probe molecules, wherein fluorescent marker is the double cups [4] prepared in embodiment 1 Arene derivatives and Te3+The complex II-1 of formation, biomolecule are EGFR antibody (E1282 is purchased from Merck).
EGF-R ELISA EGFR (Epidermal Growth Factor Receptor, EGFR) is ErbB receptor One kind of family is a kind of glycoprotein, belongs to tyrosine kinase receptor, cell membrane perforation, molecular weight 170KDa.EGFR is located at Cell membrane surface, after activating EGF and TGF (transforming growth factor) activation with ligand binding, EGFR is by list Body activates it to be located at intracellular kinase pathway after being converted into dimer, guidance downstream (including MPAK, Akt and JNK access) Phosphorylation, induced cell proliferation.Research shows that high expression or unconventionality expression in many entity tumors there are EGFR, EGFR It is related with the inhibition of the proliferation of tumour cell, angiogenesis, tumor invasion, transfer and Apoptosis.The overexpression of EGFR is being disliked Property tumour development in play an important role, kidney, lung cancer, prostate cancer, cancer of pancreas, breast cancer and spongiocytoma etc. tissue in There is the overexpression of EGFR.The expression degree of EGFR and the generation, development and transfer of tumour have close correlation, It is of great significance in the clinical diagnosis and treatment of tumour.
Complex II-1 label EGFR antibody preparation method the following steps are included:
(1) suitable 1- ethyl -3- (3- dimethyl aminopropyl)-carbodiimides (EDC) and suitable N- hydroxyl are weighed Succinimide (NHS), is dissolved in respectively in the PBS buffer solution of 0.01M, and preparation obtains the NHS solution and 10mg/mL of 10mg/mL EDC solution.
(2) the complex II-1 of 5mg is weighed, the PBS buffer solution of 1mL is added thereto, complex II-1 is dissolved, is matched Close object II-1 solution.
(3) it is slowly added to EDC solution in complex II-1 solution obtained in step (2), shakes up reaction at room temperature 15min, the mass ratio that wherein complex II-1 and EDC are used are 1:2.
(4) NHS solution is added in the solution into step (3) after reaction, reacts 30min after mixing at room temperature, is lived The complex II-1 of change, the mass ratio that wherein complex II-1 and NHS are used are 1:4.
(5) into the complex II-1 solution after activation be added 30mg/mL EGFR antibody-solutions, complex II-1 with The mass ratio that EGFR antibody uses is 20:1, room temperature cross-linking reaction 16 hours on blending instrument.
(6) after reaction, reaction solution is dialysed at 4 DEG C 8h (changing liquid 4 times) with the PBS buffer solution of 0.01M, is collected, Obtain the EGFR antibody of complex II-1 label, -20 DEG C of preservations.
As alternative embodiment, the fluorescent marker for forming fluorescent probe molecule can also be II-2~II-51 The complex of double cups [4] arene derivatives and rare earth ion of structure shown in any.
Experimental example 1
1, experiment purpose: the fluorescence spectrum of the metal complex of double cup [4] arene derivatives and rare earth ion is detected.
2, experimental method:
(1) dimethyl sulfoxide stock solution (10 μ g/mL, 1mL), three hydroxyls of complex II-1 are added in 10.0mL volumetric flask Aminomethane-hydrochloric acid (Tris-HCl) buffer solution (1X10-3Mol/L, 1mL) and distilled water (3mL), with dimethyl Asia Sulfolane solution is diluted to scale, shakes up, and is placed at room temperature for l0min, moves into quartz colorimetric utensil (the Cary Eclipse fluorescence spectrophotometer of lcm Photometer, VARIAN company, the U.S.) carry out fluorescence spectrometry.
(2) in method shown in step (1), the fluorescence spectrum of complex II-2, II-3 and II-4 are measured respectively.
3, experimental result:
Fig. 2~Fig. 4 shows complex II-1~complex II-4 fluorescence spectrum testing result.In the light by specific wavelength After excitation, complex II-1~complex II-4 can generate apparent light absorption, then emit fluorescence, and wherein Fig. 2, which is shown, matches The fluorescence spectrum of object II-1 is closed, Fig. 3 shows that the fluorescence spectrum of complex II-2, Fig. 4 show the fluorescence spectrum of complex II-3, figure The fluorescence spectrum of 5 display complex II-4.The signal stabilization of complex II-1~complex II-4 fluorescent emission, can form spy Determine the fluorescence emission spectrum of peak shape, and the peak value of fluorescence emission peak is high, double cups [4] arene derivatives pair of structure shown in formula I Tb in rare earth ion3+And Eu3+With highly selective fluorescence signal energy transmission, to generate strong fluorescence signal Response is suitable for detection of the biomolecule such as luminescent dye molecule labeling nucleic acid, albumen for field of biomedicine.It is above-mentioned to match It closes object II-1~complex II-4 fluorescence exciting wavelength and launch wavelength statistics is as shown in table 1:
Table 1
Experimental example 2
1, experiment purpose: the metal complex for detecting double cup [4] arene derivatives (complex II-1, complex II-2, is matched Close object II-3 and complex II-4) cytotoxicity.
2, experimental method:
(1) metal complex of double cups [4] arene derivatives of various concentration is prepared
DU-145 cell strain (Human Prostate Cancer Cells) is put into 96 orifice plates and is cultivated.With DMSO (dimethyl sulfoxide) Decibel prepares complex II-1, complex II-2, complex II-3 and the complex II-4 of 40mg/mL, then by 40mg/mL Complex II-1~complex II-4 solution be diluted to 25 μ g/mL, 50 μ g/ with without the DMEM in high glucose culture solution of serum respectively The solution of mL, 100 μ g/mL, 200 μ g/mL, 400 μ g/mL and 800 μ g/mL, and be placed under conditions of temperature is 4 DEG C and save.To After cell in 96 orifice plates is adherent, take respectively the above-mentioned solution prepared of 10 μ L be put in containing 90 μ L serum-containing mediums 96 Culture in orifice plate (the calixarenes fluorescent chemicals concentration of the present invention in hole be respectively 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL and 80 μ g/mL).
(2) cell culture
DU-145 cell adherent growth can be passed on to 90~95%.In superclean bench, inhales and abandon in above-mentioned 96 orifice plate Culture solution, then rinse is carried out to the attached cell in 96 orifice plates with PBS (phosphate buffer) of the 2mL after high pressure degerming, Then be added the 1mL trypsase 0.25%Trypsin-EDTA of production (gibco company) to the attached cell in 96 orifice plates into Row digestion is added immediately 2mL culture solution and terminates digestion, then after being observed under the microscope to most cells diminution change after circle 15mL is drawn with centrifuge tube through postdigestive cell liquid and to put it into centrifuge, is centrifuged with the revolving speed of 1000r/min 5 minutes.After removing supernatant, 1mL culture solution is added, then the cell of bottom precipitation is broken up and is formed uniformly cell suspension.It inhales It takes 400 μ L cell suspensions to be put into another culture vessel to be cultivated, in addition draws 100 μ L cell suspensions again and be put in cell count Plate number number, being then diluted to 500 μ L cell suspensions of remainder makes its density reach 5.6*104/ mL, and by the cell after dilution Suspension, which is inoculated in 3 piece of 96 orifice plate (90 μ L cell suspensions are added in every hole), to be continued to cultivate.
(3) MTT is tested
The cell growth condition that next day is observed in 96 orifice plates is separately added into 10 μ L (25 μ g/mL), 10 μ after cell is adherent The sheet of L (50 μ g/mL), 10 μ L (100 μ g/mL), 10 μ L (200 μ g/mL), 10 μ L (400 μ g/mL) and 10 μ L (800 μ g/mL) Invention compound and DMEM in high glucose culture solution without serum are in hole.MTT (3- (4,5- dimethylthiazole -2) -2,5- hexichol Base tetrazole bromide) it is dissolved in formation MTT solution in PBS (phosphate buffer).After culture 1,2,3 day, add respectively in hole Enter 10 MTT solution of the μ L (5mg/mL) through filtration sterilization, after continuing culture 4 hours, inhales and abandon waste liquid in hole, 150 μ L are added in every hole DMSO (dimethyl sulfoxide) solution places shaking table (the TS-2 shaking table of its woods Bell's instrument manufacturing Co., Ltd production) shimmy Amplitude is rocked 20 minutes under conditions of being 70Rpm, finally with enzyme-linked immunosorbent assay instrument (the Epoch system of Bai Teng instrument company production Column) absorbance value is measured at 490nm wavelength, data are recorded, it is dense with the metal ion match of double cups [4] arene derivatives Degree is abscissa, and cell viability is that ordinate draws histogram.
3, experimental result:
Fig. 6 shows influence of the complex II-1 of various concentration to prostate gland cancer cell DU-145 growth activity, and Fig. 7 is shown Influence of the complex II-2 of various concentration to prostate gland cancer cell DU-145 growth activity, Fig. 8 show the cooperation of various concentration Influence of the object II-3 to prostate gland cancer cell DU-145 growth activity, Fig. 9 show the complex II-4 of various concentration to prostate The influence of cancer cell DU-145 growth activity.By result in figure it is found that matching in the control of complex concentration when within 10 μ g/mL It closes object II-1~complex II-4 and cell growth is inhibited without obvious；When complex concentration is 80 μ g/mL, the growth of cell is living Power is still in 50% or more, illustrates the cell of the metal ion match of double cups [4] arene derivatives provided by the invention Toxicity is low, the detection being suitably applied in active somatic cell.
Experimental example 3
1, experiment purpose: the glimmering of the metal complex II-1 coupling EGFR antibody formation of double cup [4] arene derivatives is detected Fluorescence imaging of the light probe in cell.
2, experimental method:
(1) in 6 well culture plates preset 25mm coverslip, the DU-145 cell of logarithmic growth phase, be inoculated in 6 holes training It supports in plate, using human embryonic kidney cells 293T as negative control, then in 5%CO2, 37 DEG C be incubated for, cultivate 48h；
(2) after having cultivated cell in culture plate, culture medium is absorbed, using PBS buffer solution fine laundering cell 3 times, every time 5min；
(3) PBS buffer solution is sucked, 4% paraformaldehyde (PBS preparation) room temperature is added and fixes 20 minutes；
(4) PBS containing 4% paraformaldehyde is sucked, is rinsed cell 3 times using PBS buffer solution, each 5min；
(5) PBS is sucked, 0.5%Triton X-100 (PBS preparation) the penetrating 20min of room temperature is used；
(6) Triton X-100 is removed, is rinsed cell 3 times using PBS, each 5min；
(6) PBS is sucked, the serum (PBS preparation) homologous with EGFR antibody of 10% volume fraction is added dropwise on the cover slip Room temperature closes half an hour；
(7) confining liquid is removed, does not wash, the complex II- of enough (1:200) volume dilutions is added dropwise into every coverslip The EGFR antibody-solutions (1mg/mL) of 1 label, are then placed in 4 DEG C of overnight incubations (or 37 DEG C, 60min) in wet box；
(8) the EGFR antibody of complex II-1 label is removed, PBST (1*PBS, contain 1 ‰ Tween20) embathes 3 times, every time 5min；
(9) it redyes core: 0.5 μ g/ml DAPI is added dropwise, be protected from light and be incubated for 5min, core dye is carried out to sample；
(10) PBST (1*PBS contains 1 ‰ Tween-20) embathes 3 times, each 5min；
(11) mounting liquid of the droplet containing anti-fluorescence quenching is dripped on glass slide, coverslip is taken out from hole, blotting paper Surplus liquid is blotted, faces on lower cover to mounting liquid, touches it gently on glass slide；Slide, which is placed in dark place 5min, makes it It dries, in fluorescence microscopy microscopic observation and acquires image.
3, experimental result:
Figure 10 is shown with the EGRF expression in the EGFR antibody test DU-145 cell and 293T of complex II-1 label Result figure.By Figure 10 result it is found that with the DU-145 after fluorescent probe molecule (complex II-1 modifies EGFR antibody) incubation After cell, apparent green fluorescence is able to detect that in DU-145 cell, fluorescence intensity is significantly stronger than 293T cell.Complex The penetrability of the EGFR antibody on cell film of II-1 label is good, and fluorescence intensity is high, can specifically bind and cross table in cancer cell The EGFR albumen reached.There is apparent fluorescence, complex II-1 after using complex II-1 as conjugated fluorescent dyes biomolecule Conjugated biological molecules have good fluorescence imaging, the high specificity of detection and the advantage of high sensitivity, are suitable for basic scientific research And the fields such as clinical medicine detection.
Experimental example 4
1, experiment purpose: the metal complex II-1 coupling EGFR antibody for measuring double cup [4] arene derivatives forms fluorescence The detection of probe molecule limits.
2, experimental method:
(1) fluorescent probe molecule is fixed on ELISA Plate surface
A. 96 hole elisa Plates of polystyrene or polypropylene material are selected, ELISA Plate is rinsed using preceding with PBS buffer solution, so The fluorescent probe molecule prepared in embodiment 5 is diluted to 3~10 μ g/mL afterwards, is added in ELISA Plate；
B. it covers diaphragm plate and is coated with 4 DEG C, place 6-24 hours；Then coating buffer is discarded, washs 3 times with PBST cleaning solution, gently Clapping ELISA Plate dries cleaning solution；
C. the BSA confining liquid sealing plate that 200 μ l mass fractions are 1% is added into every hole of ELISA Plate, in 37 DEG C of placement 1-2 Hour；
D. the solution inside orifice plate is got rid of, is patted dry after washing 2 times with PBST, 4 DEG C is placed in and saves backup.
(2) EGFR albumen is formulated as various concentration with PBS buffer solution, is added separately to be fixed with the enzyme mark of EGFR albumen In the different holes of plate, it is incubated for two hours at 37 DEG C, combines it sufficiently, then removes fluorescence probe solution, washed with PBST Wash 3 times, using microplate reader detect in different holes fluorescence intensity (instrument condition: excitation wavelength: 550nm, scanning range: 200~ 700nm, PMT Voltage:700V, EXSlit:5.0nm), using PBS buffer solution as blank sample solution.
(3) standard curve that different antibodies concentration corresponds to the linear fit of EGFR detection fluorescent intensity is drawn, with [LoD= The slope of (standard deviation of 3X blank value)/linear fit] come calculate detection limit (LoD).
3, experimental result:
EGFR protein concentration changes in 5.3x 10-1~9.2x 102In the range of μ g/mL, it is dense that EGFR albumen can be obtained Spend the linearity curve of corresponding fluorescent probe molecule fluorescence intensity, the sky that the standard deviation of blank value passes through 10 batches of measurement White sample solution is measured, and the detection of the EGFR antibody test EGFR albumen of complex II-1 label is obtained by linearity curve As shown in table 2, it is 3.3x 10 that fluorescent probe molecule is adapted to detect for minimum concentration to limit-2The protein molecular of μ g/mL, probe molecule Detection limit it is low, detection high sensitivity.
Table 2
The range of linearity (μ g/mL) Related coefficient Detection limit (μ g/mL)
Fluorescent probe molecule 5.3x 10-1~9.2x 102 0.997 3.3x 10-2
Experimental example 5
1, experiment purpose: the metal complex (complex II-1~II-4) of double cup [4] arene derivatives is detected in cell In fluorescent stability.
2, experimental method:
(1) by DU-145 cell in 5%CO2, temperature is culture in 37 DEG C of incubator to logarithmic phase；
(2) complex II-1, complex II-2, complex II-3 and complex II-4 are dissolved in dimethyl sulphoxide solution Making its concentration is respectively 1mg/mL；
(3) the DU-145 cell of logarithmic growth phase, is inoculated in 6 well culture plates, cell culture to the single culture of covering 60%~70% area in hole；
(4) culture medium in culture plate is removed, 1.0mL is added, the sterilizing PBS buffer solution of pH=7.4 is washed once.So Complex II-1~II-3 solution of 10 μ l and the cell culture medium of 1.9mL are separately added into single cell culture well afterwards. Continue to cultivate cell, has the culture plate of complex II-1~II-4 in culture 12h, for 24 hours with taking out to be incubated for respectively when 48h, will train The solution supported in hole is sucked out, and is carefully cleaned three times with PBS buffer solution.Then shoot fluorescence photo.
3, experimental result:
Figure 11, which is shown, carries out cell dyeing fluorescence intracellular after cultivating different time with complex II-1~II-4 Strength Changes result.Lateral first row successively show from left to right with complex II-1 culture 12h, for 24 hours with fluorescence when 48h at As situation, lateral second row successively shows from left to right with complex II-2 culture 12h, for 24 hours with fluorescence imaging feelings when 48h Condition, lateral third row is successively shown from left to right with complex II-3 culture 12h, for 24 hours with fluorescence imaging situation when 48h, horizontal To the 4th row successively show from left to right with complex II-4 culture 12h, for 24 hours with fluorescence imaging situation when 48h.By Figure 11 knot Fruit can detect apparent intracellular Fluorescence imaging, fluorescence it is found that in the cell dyed with complex II-1~II-4 Intensity is high, and invention does not occur because of the addition of complex and changes for cellular morphology, illustrates complex II-1~II-4 as glimmering The fluorescent quantum rate of photoinitiator dye molecule is high, cell membrane penetration is high, and good biocompatibility.With the increasing of cell culture time Add, significant change, the cooperation that double cup [4] arene derivatives and rare earth ion are formed do not occur for intracellular fluorescence intensity Object can provide stable fluorescence within 48 hours, and fluorescent stability is high, fluorescence lifetime is long.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or It changes still within the protection scope of the invention.

Claims (14)

1. a kind of metal complex of double cups [4] arene derivatives, which is characterized in that have the structure as shown in Formula II:
Wherein, M is rare earth ion；
R1~R8Separately it is selected from-H ,-NH2、-COOH、-CH2COOH、-CONH(CH2)XNH2、-CONH2、-SH、- CH2SH、-SO3H、-SO3R、-NHCO(CH2)XCOOH、-NO2、-CH2PO(OH)2、WithR For alkali metal or benzene, R1~R8It is not simultaneously-H, the integer that X is 0~3；
A is selected from-(CH2-CH2)n-、-(CH2OCH2)n-、-CONH(CH2)nNHCO- andN is 1~3 Integer.
2. the metal complex of double cups [4] arene derivatives according to claim 1, which is characterized in that the rare earth gold Belong to is terbium or europium.
3. the metal complex of double cups [4] arene derivatives according to claim 2, which is characterized in that double cups [4] The metal complex of arene derivatives is selected from following structures:
4. a kind of synthetic method of the metal complex of double cup [4] arene derivatives described in claim 1, which is characterized in that The following steps are included:
(1) cup [4] arene compounds shown in cup [4] arene compounds shown in formula E and formula E ' are synthesized；Shown in formula E ' For cup [4] arene compounds through condensation reaction, introducing-O-A- group obtains intermediate shown in formula F '；
Wherein, R1'~R8' separately it is selected from-CHO ,-CH2CHO、-NHCO(CH2)XCHO、-SO3R、-H、-NH2、-CONH (CH2)XNH2、-CONH2、-SH、-CH2SH、-NO2、-CH2PO(OH)2、R is alkali Metal or benzene, the integer that X is 0~3, R1'~R8' it is not simultaneously-H；
TsO- is p-methyl benzenesulfonic acid base；
(2) intermediate shown in formula F ' is dissolved in the first organic solvent, p-methyl benzene sulfonic chloride is added, is reacted at 0~4 DEG C, Reaction solution is poured into acid after reaction, precipitating, cup [4] arene compounds shown in as formula F are precipitated；
(3) [4] arene compounds of cup shown in formula E and alkali metal salt are dissolved in the second organic solvent, are added shown in F after mixing Cup [4] arene compounds, then it is organic to be changed to third second organic solvent by the back flow reaction at 70~75 DEG C Solvent continues back flow reaction, filters after reaction, and the crystal in filtrate is precipitated；
(4) work as R1'~R8' it does not include-CHO ,-CH2CHO、-NHCO(CH2)XCHO and-SO3When R, crystal obtained in step (3) As double cup [4] arene derivatives shown in Formulas I；
Work as R1'~R8' in include at least one-CHO ,-CH2CHO、-NHCO(CH2)XCHO and/or-SO3When R, system in step (3) Crystal be intermediate shown in Formulas I ' shown in intermediate, by Formulas I ' after peroxidating and/or hydrolysis, be made shown in Formulas I Double cups [4] arene derivatives；
(5) in an inert atmosphere, after rare earth metal salt being mixed with the 4th organic solvent that dehydration and deoxidation is handled 75~ It flows back at 80 DEG C, double cup [4] arene derivatives shown in Formulas I is then added, continue back flow reaction, after reaction cooling reaction Product filters, the metal complex of double cup [4] arene derivatives shown in isolated Formula II；
Synthetic route is as follows:
5. a kind of synthetic method of the metal complex of double cup [4] arene derivatives described in claim 1, which is characterized in that The following steps are included:
S1. cup [4] arene compounds shown in cup [4] arene compounds shown in formula E and formula E ' are synthesized；Wherein, R1'~ R8' separately it is selected from-CHO ,-CH2CHO、-NHCO(CH2)XCHO、-SO3R、-H、-NH2、-CONH(CH2)XNH2、- CONH2、-SH、-CH2SH、-NO2、-CH2PO(OH)2、R is alkali metal or benzene, X For 0~3 integer, R1'~R8' it is not simultaneously-H；
S2. cup [4] arene compounds shown in cup shown in formula E [4] arene compounds, formula E ' and alkali metal salt are mixed It is even, it is dissolved in the 5th organic solvent, continuously adds compound TsO-A-OsT, under inert gas protection, next time at 105-120 DEG C Stream reaction, cooled reaction solution, post separation obtain reaction product after reaction；Wherein, TsO- is p-methyl benzenesulfonic acid base；
S3. work as R1'~R8' it does not include-CHO ,-CH2CHO、-NHCO(CH2)XCHO and-SO3When R, reaction obtained in step S2 Product is intermediate shown in Formulas I；
Work as R1'~R8' in include at least one-CHO ,-CH2CHO、-NHCO(CH2)XCHO and/or-SO3When R, made in step S2 Reaction product be intermediate shown in Formulas I ' shown in intermediate, by Formulas I ' after peroxidating and/or hydrolysis, Formulas I is made Shown in double cup [4] arene derivatives；
S4. in an inert atmosphere, after rare earth metal salt being mixed with the 4th organic solvent that dehydration and deoxidation is handled 75~ It flows back at 80 DEG C, double cup [4] arene derivatives shown in Formulas I is then added, continue back flow reaction, after reaction cooling reaction Product filters, the metal complex of double cup [4] arene derivatives shown in isolated Formula II；
Synthetic route is as follows:
6. synthetic method according to claim 4, which is characterized in that first organic solvent be pyridine, described second Organic solvent is acetonitrile, and the third organic solvent is that second alcohol and water is organic with the mixed solution of the volume ratio of 1:1, the described 4th Solvent is acetonitrile, and the alkali metal salt is potassium carbonate.
7. synthetic method according to claim 5, which is characterized in that the 5th organic solvent is toluene.
8. the synthetic method according to claim 4 or 6, which is characterized in that cup [4] aromatic hydrocarbons chemical combination shown in the formula E The molar ratio of object and cup [4] arene compounds shown in the formula F is 3:2~1:1, double cup [4] aromatic hydrocarbons shown in the Formulas I The molar ratio of derivative and the rare earth metal salt is 1:1~2:3, cup [4] arene compounds shown in the formula E with it is described The molar ratio of alkali metal salt is 1:5.
9. the synthetic method according to claim 5 or 7, which is characterized in that cup [4] aromatic hydrocarbons chemical combination shown in the formula E The molar ratio of cup [4] arene compounds, the compound TsO-A-OsT and the alkali metal salt shown in object, the formula E ' For (2~3): (2~3): (2.5~3): (12.5~20).
10. use of the metal complex of described in any item double cup [4] arene derivatives of claim 1-3 as fluorescent dye On the way.
11. a kind of fluorescent probe molecule, the fluorescent probe molecule is the biomolecule of fluorochrome label, which is characterized in that The fluorescent dye is the metal complex of described in any item double cup [4] arene derivatives of claim 1-3, the biology point Son is selected from least one of amino acid, polypeptide, albumen, nucleotide, oligonucleotides and nucleic acid.
12. fluorescent probe molecule according to claim 11, which is characterized in that the nucleotide, oligonucleotides and nucleic acid For amido modified nucleotide, oligonucleotides and nucleic acid.
13. fluorescent probe molecule according to claim 11 or 12, which is characterized in that double cup [4] arene derivatives Metal complex in the presence of bridging agent, be connected with any group in the amino of the biomolecule, carboxyl and sulfydryl.
14. fluorescent probe molecule according to claim 13, which is characterized in that the bridging agent is that N- hydroxysuccinimidyl acyl is sub- Amine, N- hydroxy thiosuccinimide salt, maleimide, glutaraldehyde or carbodiimide.
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