Photoactive Compound, Photoactive Protein-Immobilizing Gel And Use

Abstract

Provided are a photoactive compound having a chemical structure of formula (I), a photoactive protein-immobilizing gel and use. The photoactive protein-immobilizing gel, in which the photoactive compound is contained or immobilized, can crosslink with a protein under the action of UV light, thereby achieving separation and/or immobilization of the protein.

##STR00001##

Claims

1. A photoactive compound, wherein the photoactive compound has a structure of formula (I): ##STR00026## wherein: R.sub.1 is selected from nitro, cyano, halogen, haloalkyl, —OR.sub.a, —S(═O).sub.q—R.sub.a, —C(═Y)—R.sub.a, —NR.sub.aR.sub.b, substituted or unsubstituted heterocyclyl and substituted or unsubstituted aryl, wherein R.sub.a and R.sub.b of each occurrence in the R.sub.1 are same or different and independently selected from hydrogen, nitro, hydroxyl, cyano, halogen, substituted or unsubstituted alkyl and substituted or unsubstituted cycloalkyl; R.sub.2 is selected from —NH—, —O—, —S—, —C(═Y)—, —C(═Y)—CR.sub.aR.sub.b—, —CR.sub.aR.sub.b—C(═Y)—CR.sub.aR.sub.b—, —Y—CR.sub.aR.sub.b—, —CR.sub.aR.sub.b—Y—CR.sub.aR.sub.b—, —C(═Y)—NR.sub.a—, —NR.sub.a—C(═Y)—NR.sub.a—, —S(═O).sub.q—NR.sub.a—, —NR.sub.a—S(═O).sub.q—NR.sub.a—, —S(═O).sub.q—CR.sub.aR.sub.b—, —CR.sub.aR.sub.b—S(═O).sub.q—CR.sub.aR.sub.b—, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl and substituted or unsubstituted aryl, wherein R.sub.a and R.sub.b of each occurrence in R.sub.2 are same or different and independently selected from hydrogen, nitro, hydroxyl, cyano, halogen, substituted or unsubstituted C.sub.1-6 alkyl, substituted or unsubstituted C.sub.2-6 alkenyl, substituted or unsubstituted C.sub.2-6 alkynyl, substituted or unsubstituted C.sub.3-6 cycloalkyl and substituted or unsubstituted C.sub.3-6 cycloalkenyl; R.sub.3 is selected from hydrogen, —NR.sub.aR.sub.b, —C(═Y)—R.sub.a, —CR.sub.aR.sub.b—C(═Y)—R.sub.a, —CR.sub.aR.sub.b—Y—R.sub.a, —C(═Y)—NR.sub.aR.sub.b, —NR.sub.a—C(═Y)—NR.sub.aR.sub.b, —S(═O).sub.q—NR.sub.aR.sub.b, —NR.sub.a—S(═O).sub.q—NR.sub.aR.sub.b, —S(═O).sub.q—R.sub.a, —CR.sub.aR.sub.b—S(═O).sub.q—R.sub.a, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl and substituted or unsubstituted aryl, wherein R.sub.a and R.sub.b of each occurrence in R.sub.3 are same or different and independently selected from hydrogen, nitro, hydroxyl, cyano, halogen, substituted or unsubstituted alkyl and substituted or unsubstituted cycloalkyl; Y of each occurrence is independently selected from O, S and NR.sub.a; and q of each occurrence independently represents 0, 1 or 2.

2. The photoactive compound of claim 1, wherein R.sub.1 is selected from any of following groups: —NO.sub.2, CN, —Cl, —F, —Br, —CH.sub.2Cl, —CHCl.sub.2, —CCl.sub.3, —CH.sub.2F, —CHF.sub.2, —CF.sub.3, —OCH.sub.3, —OCH.sub.2CH.sub.3, —COOH, —C(═O)—CH.sub.3, —C(═O)—CH.sub.2CH.sub.3, —C(═O)—CH.sub.2CN, —C(═O)—CH.sub.2NO.sub.2, —SCH.sub.3, —S(═O).sub.2—OH, —S(═O).sub.2—CH.sub.3, —S(═O).sub.2—CH.sub.2CN, —S(═O).sub.2—CH.sub.2CH.sub.2NO.sub.2, —NCH.sub.3CH.sub.3, —NHCH.sub.3, —NHCH.sub.2CN, — ##STR00027##

3. The photoactive compound of claim 1, wherein R.sub.2 is selected from any of following groups: —C(═O)—, —C(═O)—CH.sub.2—, —CH.sub.2—C(═O)—CH.sub.2—, —CH.sub.2—, —CH.sub.2—CH.sub.2—, —CH(CH.sub.3)—, —CH.sub.2—CH.sub.2—CH.sub.2—, —CH.sub.2—CH(CH.sub.3)—CH.sub.2—, —CH═CH—, —CH═C(CH.sub.3)—, —C(CH.sub.3)═C(CH.sub.3)—, —CH.sub.2—CH═CH—CH.sub.2—, —CH.sub.2—NH—CH.sub.2—, —NH—CH.sub.2—, —S(═O).sub.2—CH.sub.2—, —CH.sub.2—S(═O).sub.2—CH.sub.2—, —CH(OH)—, —CH.sub.2CH(OH)CH.sub.2—, —CH(CN)—, —CH.sub.2CH(CN)CH.sub.2—, —CH(NO.sub.2)—, —CH.sub.2CH(NO.sub.2)CH.sub.2—, —CH.sub.2—O—CH.sub.2—, —CH.sub.2—S—CH.sub.2—.

4. The photoactive compound of claim 1, wherein R.sub.3 is selected from any of following groups: —NH.sub.2, —NHCH.sub.3, —CH.sub.3, —CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3, —CH(OH)CH.sub.3, —C(═O)—CH.sub.3, —CH.sub.2—C(═O)—CH.sub.3, —C(═O)—NH.sub.2, —CH.sub.2—O—CH.sub.3, —CH.sub.2—S—CH.sub.3, —NH—S(═O).sub.2—NH—CH.sub.3, —S(═O).sub.2—CH.sub.2CH.sub.3, —CH.sub.2—S(═O).sub.2—CH.sub.3, ##STR00028##

5. The photoactive compound of claim 1, wherein R.sub.1 is substituted or unsubstituted heterocyclyl or substituted or unsubstituted aryl.

6. The photoactive compound of claim 1, wherein R.sub.2 is substituted or unsubstituted alkyl.

7. The photoactive compound of claim 5, wherein R.sub.2 is substituted or unsubstituted alkyl.

8. The photoactive compound of claim 1, wherein R.sub.3 is substituted or unsubstituted alkyl.

9. The photoactive compound of claim 5, wherein R.sub.3 is substituted or unsubstituted alkyl.

10. The photoactive compound of claim 6, wherein R.sub.3 is substituted or unsubstituted alkyl.

11. The photoactive compound of claim 7, wherein R.sub.3 is substituted or unsubstituted alkyl.

12. The photoactive compound of claim 1, wherein R.sub.2 is substituted or unsubstituted C.sub.1-6 alkyl and R.sub.3 is substituted or unsubstituted C.sub.1-6 alkyl.

13. The photoactive compound of claim 5, wherein R.sub.2 is substituted or unsubstituted C.sub.1-6 alkyl and R.sub.3 is substituted or unsubstituted C.sub.1-6 alkyl.

14. The photoactive compound of claim 12, wherein R.sub.1 is selected from any of following groups: ##STR00029##

15. The photoactive compound of claim 14, wherein R.sub.2 is unsubstituted C.sub.1-6 alkyl.

16. The photoactive compound of claim 14, wherein R.sub.3 is unsubstituted C.sub.1-6 alkyl.

17. The photoactive compound of claim 1, which is selected from any of following compounds: ##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036##

18. A photoactive protein-immobilizing gel, in which the photoactive compound according to claim 1 is contained or immobilized.

19. The photoactive protein-immobilizing gel of claim 18, wherein the gel is a polyacrylamide hydrogel.

20. Use of the photoactive protein-immobilizing gel of claim 18 for separating, immobilizing and/or detecting a protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 shows a synthetic route of a photoactive compound of Example 1.

[0054] FIG. 2 shows a proton nuclear magnetic resonance spectrum of Compound 1 of Example 1.

[0055] FIG. 3 shows a mass spectrum of Compound 2 of Example 1.

[0056] FIG. 4 shows a proton nuclear magnetic resonance spectrum of Compound 3 of Example 1.

[0057] FIG. 5 shows a UV-visible absorption spectrum of the photoactive compound (Compound 3) of Example 1.

[0058] FIG. 6 shows a comparison of bovine serum albumin protein immobilization efficiency of photoactive protein-immobilizing gels with concentrations of 0%, 0.75%, 1.5% and 3%.

[0059] FIG. 7 shows a comparison of bovine serum albumin protein immobilizing efficiency of photoactive protein-immobilizing gels under UV irradiation for different periods of time.

[0060] FIG. 8 shows an interaction principle between photoactive protein-immobilizing gel and protein under UV light excitation.

[0061] FIG. 9 shows a single-cell band obtained from the reaction of a photoactive protein-immobilizing gel with a protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The present invention will now be described in detail below with reference to the accompanying drawings.

[0063] General Method for Preparing Photoactive Compounds of the Present Invention

[0064] Photoactive compounds according to the present invention can be prepared by the method below. Unless otherwise indicated, variables (e.g., R.sub.1, R.sub.2 and R.sub.3), when used in the following formulae, are to be understood to represent those groups described above with respect to formula (I).

[0065] A general synthetic route of the photoactive compounds is illustrated by the following.

##STR00014##

[0066] A specific synthesis scheme may involve the following:

[0067] (1) An R.sub.1-containing monomer, (diacetoxyiodo)benzene and trifluoroethanol are weighed at a certain molar ratio. The R.sub.1-containing monomer is first dissolved in trifluoroethanol, (diacetoxyiodo)benzene is second added thereto at −40° C., and then the mixture is stirred for a period of time under nitrogen protection. The stirred mixture is concentrated to an oil and dissolved in dichloromethane, and ethyl tetrazole-5-carboxylate, copper(II) triflate and triethylamine weighed at a certain molar ratio are added thereto, and then the mixture is stirred at room temperature under nitrogen protection for a period of time. The resulting substance is washed separately with a saturated ammonium chloride solution and brine, and then is dried with anhydrous magnesium sulfate. Finally, it is filtered and further purified by silica gel chromatography (using PE/EA as an eluent), which results in an intermediate product with a structure of formula (Ia) below.

##STR00015##

[0068] (2) The intermediate product and lithium hydroxide are weighed at a certain mass ratio. The intermediate product is then dissolved in a 1:1 (v/v) MeOH/H.sub.2O solution, and lithium hydroxide is added thereto at 0° C. The system is stirred at room temperature under nitrogen protection to allow the reaction to proceed for a period of time. Finally, the temperature is lowered to 0° C., and the pH is adjusted to a desired value by adding HCl, which results in a mixed solution. An organic phase is then extracted from the mixed solution with an ethyl acetate solution, washed with brine, dried with anhydrous sodium sulfate, filtered and condensed to a solid, which is an electron-withdrawing group containing tetrazolyl compound with a structure of formula (Ib) below.

##STR00016##

[0069] (3) An R.sub.2 and R.sub.3-containing compound of formula (Ic) below, 1-hydroxybenzotriazole and N-(3-aminopropyl)methacrylamide hydrochloride are weighed at a certain mass ratio. N-(3-aminopropyl)methacrylamide hydrochloride is first dissolved in tetrahydrofuran, followed by addition thereto of the compound of formula (Ic) and 1-hydroxybenzotriazole, and the reaction is run for a period of time. The electron-withdrawing group containing tetrazolyl compound (formula (Ib)) and triethylamine are weighed at a certain mass ratio of N-(3-aminopropyl)methacrylamide hydrochloride to the electron-withdrawing group containing tetrazolyl compound to triethylamine, added to the reaction mixture, and mixed therewith by stirring. The reaction is then run under reflux for a period of time. After the reaction completes, a product thereof is purified by preparative HPLC to produce, as a final product, a photoactive compound with a structure of formula (I) below.

##STR00017##

[0070] Further description is made below by reference to specific Examples.

Example 1

[0071] After methyl-1H-pyrrole, (diacetoxyiodo)benzene and trifluoroethanol were weighed at a molar ratio of 1:11:200, methyl-1H-pyrrole was first dissolved in trifluoroethanol, and (diacetoxyiodo)benzene was added thereto at −40° C., and then the mixture was stirred under nitrogen protection for 3 h. The stirred mixture was concentrated to a black oil and dissolved in dichloromethane. After that, ethyl tetrazole-5-carboxylate, copper(II) triflate and triethylamine weighed at a molar ratio of 3:1:13 were added thereto, and then the mixture was stirred at room temperature under nitrogen protection for 24 h. The resulting substance was washed separately with a saturated ammonium chloride solution and brine, and then was dried with anhydrous magnesium sulfate. Finally, it was filtered and further purified by silica gel chromatography (using PE/EA=8:1 as an eluent), which resulted in a brown oil-like intermediate product (Compound 1) with a structure of the formula below.

##STR00018##

[0072] The yield of the above preparation approach was 9.6%. As shown in FIG. 2, proton nuclear magnetic resonance data of Compound 1 were as follows: 1H NMR (400 MHz, DMSO): δ 7.13-7.00 (m, 1H), 6.65 (dd, J=3.9, 1.9 Hz, 1H), 6.24 (dd, J=3.9, 3.0 Hz, 1H), 4.47 (q, J=7.1 Hz, 2H), 3.65 (s, 3H), 1.37 (t, J=7.1 Hz, 3H). Its mass spectrometric analysis suggested [M+H]+ 222.0.

[0073] The intermediate product and lithium hydroxide were weighed at a mass ratio of 1:1.8. The intermediate product was then dissolved in a 20 ml 1:1 (v/v) MeOH/H.sub.2O solution, and lithium hydroxide was added thereto at 0° C. The system was set to room temperature and stirred under nitrogen protection to allow the reaction to proceed for 1 h. Finally, the temperature was lowered to 0° C., and the pH was adjusted to 7 by adding 2N HCl, which resulted in a mixed solution. An organic phase was then extracted from the mixed solution with an ethyl acetate solution, washed with brine, dried with anhydrous sodium sulfate, filtered and condensed to a brown solid, which was an electron-withdrawing group containing tetrazolyl compound with a structure of the following formula (Compound 2). A final product of 370 mg was obtained in a yield of 94%. Mass spectrometric analysis of Compound 2 suggested [M+H]+ 194.1.

##STR00019##

[0074] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and N-(3-aminopropyl)methacrylamide hydrochloride were weighed at a mass ratio of 2:1:1. N-(3-aminopropyl)methacrylamide hydrochloride was first dissolved in 50 mL tetrahydrofuran, followed by addition thereto of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole at 0° C., and the reaction was run for 1 h. The electron-withdrawing group containing tetrazolyl compound (Compound 2) and triethylamine were weighed at a mass ratio of N-(3-aminopropyl)methacrylamide hydrochloride to the electron-withdrawing group containing tetrazolyl compound (Compound 2) to triethylamine of 1:1:2.5, added to the reaction mixture, and mixed therewith by stirring. The reaction was then run for 12 h under reflux. After the reaction completed, a product thereof was purified by preparative HPLC to produce, as a final product, a photoactive compound (Compound 3) with a structure of the following formula in the form of a white power which weighed 41 mg (in a yield of 6.7%).

##STR00020##

[0075] As shown in FIGS. 3 to 4, proton nuclear magnetic resonance data of Compound 3 were as follows: 1H NMR (400 MHz, MeOD) δ 6.97-6.82 (m, 1H), 6.60 (dd, J=4.0, 1.9 Hz, 1H), 6.23 (dd, J=3.9, 3.0 Hz, 1H), 5.78-5.64 (m, 1H), 5.50-5.23 (m, 1H), 3.71 (s, 3H), 3.50 (t, J=6.8 Hz, 2H), 3.36 (t, J=6.7 Hz, 2H), 2.03-1.92 (m, 3H), 1.87 (p, J=6.7 Hz, 2H). Mass spectrometric analysis of this photoactive compound suggested [M+H]+ 318.1.

[0076] The synthetic route of the photoactive compound of this Example is shown in FIG. 1. FIG. 5 shows a UV-Visible absorption spectrum of the photoactive compound of this Example, which indicates the light absorption characterization of the compound by an Evolution 220 UV-Vis spectrophotometer with a spectral scan range of 300 nm to 800 nm from Agilent Technologies (China) Co., Ltd. Longer excitation wavelength in a narrower UV spectral band means lower energy, which avoids UV-induced deactivation of a protein's antigenic site and reduces UV-induced background autofluorescence from the gel itself.

Example 2

[0077] A photoactive compound with a structure of the following formula (Compound 4) was prepared.

##STR00021##

[0078] Pyrrole, (diacetoxyiodo)benzene and trifluoroethanol were weighed at a molar ratio of 1:10:170. Pyrrole was first dissolved in trifluoroethanol, and (diacetoxyiodo)benzene was added thereto at −40° C., and then the mixture was stirred under nitrogen protection for 3 h. The stirred mixture was concentrated to a black oil and dissolved in dichloromethane. Subsequently, ethyl tetrazole-5-carboxylate, copper(II) triflate and triethylamine weighed at a molar ratio of 4:1:15 were added thereto, and then the mixture was stirred at room temperature under nitrogen protection for 24 h. The resulting substance was washed separately with a saturated ammonium chloride solution and brine, and then was dried with anhydrous magnesium sulfate. Finally, it was filtered and further purified by silica gel chromatography (using PE:EA=7:1 as an eluent), which resulted in a brown oil-like intermediate product.

[0079] After the intermediate product and lithium hydroxide were weighed at a mass ratio of 1:1, the intermediate product was first dissolved in a 20 ml 1:1 (v/v) MeOH/H.sub.2O solution, and lithium hydroxide was added thereto at 0° C. The system was set to room temperature and stirred under nitrogen protection to allow the reaction to proceed for 2 h. Finally, the temperature was lowered to 0° C., and the pH was adjusted to 7.5 by adding 2N HCl, which resulted in a mixed solution. An organic phase was then extracted from the mixed solution with an ethyl acetate solution, washed with brine, dried with anhydrous sodium sulfate, filtered and condensed to a brown solid, which was an electron-withdrawing group containing tetrazolyl compound.

[0080] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and N-(3-aminopropyl)methacrylamide hydrochloride were weighed at a mass ratio of 3:1:1. N-(3-aminopropyl)methacrylamide hydrochloride was first dissolved in 50 mL tetrahydrofuran, followed by addition thereto of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole at 0° C., and the reaction was run for 30 min. The electron-withdrawing group containing tetrazolyl compound and triethylamine were weighed at a mass ratio of N-(3-aminopropyl)methacrylamide hydrochloride to the electron-withdrawing group containing tetrazolyl compound to triethylamine of 1:1:2, added to the reaction mixture, and mixed therewith by stirring. The reaction was then run for 10 h under reflux. After the reaction completed, a product thereof was purified by preparative HPLC to produce, as a final product, a photoactive compound (Compound 4) in the form of a white power.

Example 3

[0081] A photoactive compound with a structure of the following formula (Compound 5) was prepared.

##STR00022##

[0082] Thiophene, (diacetoxyiodo)benzene and trifluoroethanol were weighed at a molar ratio of 1:12:150. Thiophene was first dissolved in trifluoroethanol, and (diacetoxyiodo)benzene was added thereto at −40° C., and then the mixture was stirred under nitrogen protection for 4 h. The stirred mixture was concentrated to a black oil and dissolved in dichloromethane. Subsequently, ethyl tetrazole-5-carboxylate, copper(II) triflate and triethylamine weighed at a molar ratio of 5:1:16 were added thereto, and then the mixture was stirred at room temperature under nitrogen protection for 27 h. The resulting substance was washed with a saturated ammonium chloride solution, and then was dried with anhydrous magnesium sulfate. Finally, it was filtered and further purified by silica gel chromatography (using PE:EA=6:1 as an eluent), which resulted in a brown oil-like intermediate product.

[0083] After the intermediate product and lithium hydroxide were weighed at a mass ratio of 1:2, the intermediate product was first dissolved in a 20 ml 1:1 (v/v) MeOH/H.sub.2O solution, and lithium hydroxide was added thereto at 0° C. The system was set to room temperature and stirred under nitrogen protection to allow the reaction to proceed for 1.5 h. Finally, the temperature was lowered to 0° C., and the pH was adjusted to 8 by adding 2N HCl, which resulted in a mixed solution. An organic phase was then extracted from the mixed solution with an ethyl acetate solution, washed with brine, dried with anhydrous sodium sulfate, filtered and condensed to a brown solid, which was an electron-withdrawing group containing tetrazolyl compound. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and N-(3-aminopropyl)methacrylamide hydrochloride were weighed at a mass ratio of 5:2:1. N-(3-aminopropyl)methacrylamide hydrochloride was first dissolved in 50 mL tetrahydrofuran, followed by addition thereto of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole at 0° C., and the reaction was run for 2 h. The electron-withdrawing group containing tetrazolyl compound and triethylamine were weighed at a mass ratio of N-(3-aminopropyl)methacrylamide hydrochloride to the electron-withdrawing group containing tetrazolyl compound to triethylamine of 1:1:4, added to the reaction mixture, and mixed therewith by stirring. The reaction was then run for 15 h under reflux. After the reaction completed, a product thereof was purified by preparative HPLC to produce, as a final product, a photoactive compound (Compound 5) in the form of a white power.

Example 4

[0084] A photoactive compound with a structure of the following formula (Compound 6) was prepared.

##STR00023##

[0085] Furan, (diacetoxyiodo)benzene and trifluoroethanol were weighed at a molar ratio of 1:13:130. Furan was first dissolved in trifluoroethanol, and (diacetoxyiodo)benzene was added thereto at −40° C., and then the mixture was stirred under nitrogen protection for 4 h. The stirred mixture was concentrated to a black oil and dissolved in dichloromethane. Afterwards, ethyl tetrazole-5-carboxylate, copper(II) triflate and triethylamine weighed at a molar ratio of 7:1:18 were added thereto, and then the mixture was stirred at room temperature under nitrogen protection for 30 h. The resulting substance was washed separately with a saturated ammonium chloride solution and brine, and then was dried with anhydrous sodium sulfate. Finally, it was filtered and further purified by silica gel chromatography (using PE:EA=5:1 as an eluent), which resulted in a brown oil-like intermediate product.

[0086] After the intermediate product and lithium hydroxide were weighed at a mass ratio of 1:3, the intermediate product was first dissolved in a 20 ml 1:1 (v/v) MeOH/H.sub.2O solution, and lithium hydroxide was added thereto at 0° C. The system was set to room temperature and stirred under nitrogen protection to allow the reaction to proceed for 2.5 h. Finally, the temperature was lowered to 0° C., and the pH was adjusted to 8.5 by adding 2N HCl, which resulted in a mixed solution. An organic phase was then extracted from the mixed solution with an ethyl acetate solution, washed separately with a saturated ammonium chloride solution and brine, dried with anhydrous sodium sulfate, filtered and condensed to a brown solid, which was an electron-withdrawing group containing tetrazolyl compound.

[0087] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and N-(3-aminopropyl)methacrylamide hydrochloride were weighed at a mass ratio of 6:2:1. N-(3-aminopropyl)methacrylamide hydrochloride was first dissolved in 50 mL tetrahydrofuran, followed by addition thereto of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole at 0° C., and the reaction was run for 1.5 h. The electron-withdrawing group containing tetrazolyl compound and triethylamine were weighed at a mass ratio of N-(3-aminopropyl)methacrylamide hydrochloride to the electron-withdrawing group containing tetrazolyl compound to triethylamine of 1:1:6, added to the reaction mixture, and mixed therewith by stirring. The reaction was then run for 17 h under reflux. After the reaction completed, a product thereof was purified by preparative HPLC to produce, as a final product, a photoactive compound (Compound 6) in the form of a white power.

Example 5

[0088] A photoactive compound with a structure of the following formula (Compound 7) was prepared.

##STR00024##

[0089] Toluene, (diacetoxyiodo)benzene and trifluoroethanol were weighed at a molar ratio of 1:15:100. Toluene was first dissolved in trifluoroethanol, and (diacetoxyiodo)benzene was added thereto at −40° C., and then the mixture was stirred under nitrogen protection for 5 h. The stirred mixture was concentrated to a black oil and dissolved in dichloromethane. Thereafter, ethyl tetrazole-5-carboxylate, copper(II) triflate and triethylamine weighed at a molar ratio of 8:1:20 were added thereto, and then the mixture was stirred at room temperature under nitrogen protection for 30 h. The resulting substance was washed separately with a saturated ammonium chloride solution and brine, and then was dried with anhydrous magnesium sulfate. Finally, it was filtered and further purified by silica gel chromatography (using PE:EA=4:1 as an eluent), which resulted in a brown oil-like intermediate product.

[0090] After the intermediate product and lithium hydroxide were weighed at a mass ratio of 1:5, the intermediate product was first dissolved in a 20 ml 1:1 (v/v) MeOH/H.sub.2O solution, and lithium hydroxide was added thereto at 0° C. The system was set to room temperature and stirred under nitrogen protection to allow the reaction to proceed for 2 h. Finally, the temperature was lowered to 0° C., and the pH was adjusted to 9 by adding 2N HCl, which resulted in a mixed solution. An organic phase was then extracted from the mixed solution with an ethyl acetate solution, washed with brine, dried with anhydrous magnesium sulfate, filtered and condensed to a brown solid, which was an electron-withdrawing group containing tetrazolyl compound.

[0091] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and N-(3-aminopropyl)methacrylamide hydrochloride were weighed at a mass ratio of 7:3:1. N-(3-aminopropyl)methacrylamide hydrochloride was first dissolved in 50 mL tetrahydrofuran, followed by addition thereto of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole at 0° C., and the reaction was run for 1 h. The electron-withdrawing group containing tetrazolyl compound and triethylamine were weighed at a mass ratio of N-(3-aminopropyl)methacrylamide hydrochloride to the electron-withdrawing group containing tetrazolyl compound to triethylamine of 1:1:8, added to the reaction mixture, and mixed therewith by stirring. The reaction was then run for 20 h under reflux. After the reaction completed, a product thereof was purified by preparative HPLC to produce, as a final product, a photoactive compound (Compound 7) in the form of a white power.

Example 6

[0092] A photoactive compound with a structure of the following formula (Compound 8) was prepared.

##STR00025##

[0093] p-Xylene, (diacetoxyiodo)benzene and trifluoroethanol were weighed at a molar ratio of 1:15:100. p-Xylene was first dissolved in trifluoroethanol, and (diacetoxyiodo)benzene was added thereto at −40° C., and then the mixture was stirred under nitrogen protection for 5 h. The stirred mixture was concentrated to a black oil and dissolved in dichloromethane. Thereafter, ethyl tetrazole-5-carboxylate, copper(II) triflate and triethylamine weighed at a molar ratio of 8:1:20 were added thereto, and then the mixture was stirred at room temperature under nitrogen protection for 30 h. The resulting substance was washed separately with a saturated ammonium chloride solution and brine, and then was dried with anhydrous magnesium sulfate. Finally, it was filtered and further purified by silica gel chromatography (using PE:EA=4:1 as an eluent), which resulted in a brown oil-like intermediate product.

[0094] After the intermediate product and lithium hydroxide were weighed at a mass ratio of 1:5, the intermediate product was first dissolved in a 20 ml 1:1 (v/v) MeOH/H.sub.2O solution, and lithium hydroxide was added thereto at 0° C. The system was set to room temperature and stirred under nitrogen protection to allow the reaction to proceed for 2 h. Finally, the temperature was lowered to 0° C., and the pH was adjusted to 9 by adding 2N HCl, which resulted in a mixed solution. An organic phase was extracted from the mixed solution with an ethyl acetate solution, washed with brine, dried with anhydrous magnesium sulfate, filtered and condensed to a brown solid, which was an electron-withdrawing group containing tetrazolyl compound. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and N-(3-aminopropyl)methacrylamide hydrochloride were weighed at a mass ratio of 7:3:1. N-(3-aminopropyl)methacrylamide hydrochloride was first dissolved in 50 mL tetrahydrofuran, followed by addition thereto of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole at 0° C., and the reaction was run for 1 h. The electron-withdrawing group containing tetrazolyl compound and triethylamine were weighed at a mass ratio of N-(3-aminopropyl)methacrylamide hydrochloride to the electron-withdrawing group containing tetrazolyl compound to triethylamine of 1:1:2, added to the reaction mixture, and mixed therewith by stirring. The reaction was then run for 12 h under reflux. After the reaction completed, a product thereof was purified by preparative HPLC to produce, as a final product, a photoactive compound (Compound 8) in the form of a white power.

Example 7

[0095] A photoactive protein-immobilizing gel containing the photoactive compound of Example 1 as a photoactive ingredient was prepared.

[0096] The photoactive compound of Example 1 (Compound 3) was dissolved in dimethyl sulfoxide to produce a 100 mM storage solution. In each of four 1.5 mL Ep tubes, 25 μL of a 1.5 M Tris-HCl buffer (pH=8.8), 166.7 μL of a 30% acrylamide/bis-acrylamide (29:1) solution and 265.3 μL of ddH.sub.2O were added, and 15 μL, 7.5 μL, 3.75 μL and 0 μL of the storage solution were then respectively further added therein, which resulted in gel precursor solutions at respective concentrations of 3%, 1.5%, 0.75% and 0%. After that, each Ep tube was further added therein with 10 μL 5% SDS, 10 μL 5% Triton X-100, 4 μL ammonium persulphate (APS) and 4 μL tetramethylethylenediamine (TEMED) and gently shaken until homogenous mixing was achieved. The resulting solutions were then dropped onto porous microarray molds and slowly and gently covered with glass slides to avoid the occurrence of bubbles. After the setups were left at rest for 20 minutes, the polyacrylamide hydrogels cured and the molds were peeled off. In this way, at least four sets of porous microarray gels with different concentrations were obtained.

[0097] 1 L of an electrophoresis buffer was prepared by weighing and mixing 500 mg 0.5% SDS, 100 μL 0.1% v/v Triton X-100, 250 mg 0.25% sodium deoxycholate, 1.514 g Tris and 7.2 g glycine and adjusting the pH to 8.3, and then stored at 4° C. This electrophoresis buffer was heated in a water bath to 55° C., and a UV light source was activated in advance to stabilize the light source.

[0098] Each glass slide with a gel coated thereon was placed in a clean vessel, with the gel-coated surface facing upward. A 5.12 mg/mL bovine serum albumin protein (BSA) solution in an amount of 200 μL was dropped thereon, and the glass slide was gently shaken to allow the BSA solution to evenly spread over the gel surface and then left at rest for 3 min. The gel was placed in an electrophoresis tank, and 10 mL of the electrophoresis buffer that had been preheated was gently and quickly poured into the electrophoresis tank at a corner thereof. Immediately after that, the power supply was turned on to apply a voltage of 200 V (E=40 v/cm.sup.2). After the electrophoresis was run 30 s for protein separation, the power supply was immediately turned off, and the gel was exposed to UV light for 10 min. Following the exposure, the gel was taken out and stained in a Coomassie brilliant blue solution (a mixed solution of 0.1 g of a Coomassie brilliant blue powder in 20 mL methanol, 16 mL water and 4 mL acetic acid) for 5 min. At this point, photographs were taken to record protein immobilization conditions of the four sets of gels (the left column in FIG. 6). Each gel was then washed under being shaken in a TBST buffer (100 mM Tris titrated to pH 7.5 with HCl, 150 mM NaCl, 0.1% Tween 20, 9480, EMD Millipore). The buffer was replaced with a fresh volume every 15 min in the first 2 hours, and the gel was again washed under being shaken in a TBST buffer for 12 h. Thereafter, photographs were again taken to record protein immobilization conditions of the four sets of gels (the right column in FIG. 6), as shown in FIG. 6. As can be seen from this figure, in contrast to the case without using a photoactive protein-immobilizing gel, where the protein was almost totally washed away within 12 h, even the low-concentration photoactive protein-immobilizing gel, i.e., the 0.75% photoactive protein-immobilizing gel, was demonstrated to immobilize the protein therein with high efficiency when excited by UV light.

[0099] In this Example, photoactive protein-immobilizing gels of the same concentration, each containing bovine serum albumin proteins of same concentration, were excited by UV light for different periods of time of 0 s, 15 s, 30 s, 1 min, 2 min, 4 min, 6 min and 10 min and each underwent the same shaken wash procedure as defined above, and the results are shown in FIG. 7. In this figure, the protein immobilization conditions after electrophoresis are shown in the left column, and the protein immobilization conditions after UV irradiation of different periods of time are shown in the right column. Even 15 s of UV excitation could trigger a protein-immobilizing effect, and an even stronger immobilizing effect was obtained when the period of excitation was extended to 30 s or longer. This demonstrates that the photoactive protein-immobilizing gel of the present invention requires a shorter period of UV excitation, which avoids deactivation of a protein's antigenic site and reduces UV-induced background autofluorescence from the gel itself.

[0100] FIG. 8 shows an interaction principle between a photoactive protein-immobilizing gel and a protein under UV light excitation. As can be seen from the figure, when the photoactive protein-immobilizing gel is excited by UV light with a wavelength of 300-400 nm, the tetrazolyl ring in the photoactive compound is split up and undergoes a nucleophilic addition reaction with a carboxyl group of the protein. Under the action of the electron-withdrawing group, 1,4-acyl group migrates, which eventually results in a stable structure between the gel and the protein, and thus immobilizes the protein. Therefore, the photoactive protein-immobilizing gel of the present invention can be excited within a short period of time, which is commonly within 30 s, when exposed to UV light. That is, it can more quickly polymerize with functional groups in protein molecules. This can avoid protein diffusion which may lead to a reduction in sensitivity due to a slow polymerization rate, which significantly promotes protein immobilizing efficiency. Moreover, since the photoactive protein-immobilizing gel reacts with carboxyl groups in proteins, it is not selective for any particular protein and can be used to immobilize all proteins. The photoactive protein-immobilizing gel of the present invention not only does not affect the molecular sieving of SDS-PAGE used in electrophoresis, which allows the method to retain its own specificity and separation resolution, and also the non-selectively immobilization of all proteins. Therefore, it can better work with electrophoretic separation and facilitate subsequent protein detection. FIG. 9 shows single-cell bands. Single cells placed in wells lysed and underwent electrophoresis. After the electrophoresis was completed, the photoactive protein-immobilizing gel reacted with proteins under UV excitation, and in-situ immobilized the proteins within the gel. Incubation was carried out with a primary antibody that could recognize a particular protein, followed by the use of a fluorescent secondary antibody to recognize the primary antibody. Finally, fluorescent band was detected. The protein under assay in the figure was the internal reference protein Tubulin. Obvious fluorescent bands were visible in the left half of the figure, which indicated no impact of the immobilized proteins on the antigen-antibody binding. A notable signal peak was detected in data analysis of Matlab, and an area under the curve (peak) represented an amount of the detected antigen. As can be seen from the figure, protein immobilization by the photoactive protein-immobilizing gel of the present invention does not affect antigen-antibody binding, receptor-ligand binding, enzymatic activity, aptamer binding, or the like in subsequent detection, and thus does not affect subsequent establishment of a detection system.

[0101] In the present invention, washing agents include saturated ammonium chloride and brine; drying agents include anhydrous magnesium sulfate and anhydrous sodium sulfate; purification processes involve the use of silica gel chromatography (using PE/EA=4-8:1 as an eluent) and preparative HPLC; and organic solvents include one or more of trifluoroethanol, dichloromethane, tetrahydrofuran or N,N-dimethylformamide. The foregoing technical features of the various embodiments may be combined in any way. Although not all such combinations have been described above for the sake of brevity, any of them is considered to fall within the scope of this specification as long as there is no contradiction between the technical features.

[0102] The embodiments disclosed herein are merely intended to illustrate the present invention without limiting the scope of the appended claims. Other substantively equivalent alternatives conceivable by those skilled in the art are all within the scope of the invention sought to be protected hereby.