CN111518262B

CN111518262B - Conjugated backbone polymer, application thereof and method for selectively inactivating basic protein

Info

Publication number: CN111518262B
Application number: CN201910108692.4A
Authority: CN
Inventors: 王树; 孙含; 刘礼兵; 吕凤婷
Original assignee: Institute of Chemistry CAS; University of Chinese Academy of Sciences
Current assignee: Institute of Chemistry CAS; University of Chinese Academy of Sciences
Priority date: 2019-02-03
Filing date: 2019-02-03
Publication date: 2021-03-16
Anticipated expiration: 2039-02-03
Also published as: CN111518262A

Abstract

The invention discloses a conjugated skeleton polymer, application thereof and a method for selectively inactivating basic protein. The polymer has a structure shown in a formula (I),

the polymer shown in the formula (I) can selectively inactivate basic proteins and effectively inhibit the toxicity of cardiotoxin.

Description

Conjugated backbone polymer, application thereof and method for selectively inactivating basic protein

Technical Field

The invention relates to a conjugated backbone polymer, application thereof and a method for selectively inactivating basic proteins.

Background

Biological macromolecules such as proteins, polysaccharides, nucleic acids, etc. play an important role in life activities as complex molecular machines. Many biological macromolecules such as proteins have recognizable "signals" in their own structures, and such recognizable signals are also commonly used in the art to construct compounds or polymers that selectively bind to specific biological macromolecules, which manipulate the activity of biological macromolecules by binding (or assembling) with natural biological macromolecules, and are of great importance in understanding life processes, diagnosis and treatment of diseases, and the like.

At present, there is increasing interest in constructing intelligent, biohybrid materials from responsive polymers and biomacromolecules. For example, a polymer containing phosphate group side chains is designed, and multivalent electrostatic interaction between the polymer and lysine or arginine on the surface of the protein is utilized to recognize basic protein or polypeptide, or supermolecular interaction between cucurbituril and N-terminal phenylalanine of insulin protein is utilized to construct an assembly body with the protein. In addition, synthetic polymeric nanoparticles have been designed to selectively recognize protein aggregates and help them refold into soluble proteins based on differences in the hydrophobicity of the protein surface.

However, the above compounds or polymers are bound or assembled with proteins by non-covalent bond action, such as electrostatic action, intermolecular force, hydrogen bond, etc., and the non-covalent bond action is easily affected by external environment, such as temperature, pH, etc., so that the formed binding or assembly is unstable. In addition, the compounds or polymers bound by non-covalent bonds in the prior art can be bound to various proteins without selectivity and specificity, and therefore, a compound or polymer stably bound to proteins is needed to ensure the stability and selectivity of binding, thereby realizing the regulation of the activity of proteins.

Disclosure of Invention

The invention aims to overcome the problems of unstable combination with protein, no selectivity of combination and the like in the prior art, and provides a conjugated skeleton polymer, application thereof and a method for selectively inactivating basic protein.

In order to achieve the above object, the present invention provides, in a first aspect, a conjugated skeleton polymer comprising a structural unit represented by the formula (I),

wherein R1 has a structure as shown in formula (II),

r2 has a structure as shown in formula (III),

wherein, the polymerization degree n is an integer of 10-200, k is an integer of 4-20, q is an integer of 1-5, m is an integer of 2-12, and represents the bonding position of R1 and R2 in the formula (I).

In a second aspect, the present invention provides a method of selectively inactivating an alkaline protein, the method comprising:

(1) adding a polymer according to the first aspect of the invention to a sample containing a basic protein to obtain a mixture,

(2) exposing the mixture obtained in step (1) to light.

In a third aspect, the invention provides the use of a polymer according to the first aspect of the invention to inhibit cardiotoxin toxicity.

The inventors of the present invention have found that the polymer represented by the formula (I) can selectively bind to and react with a basic protein. Meanwhile, the polymer shown in the formula (I) can sensitize oxygen molecules to generate active oxygen under the condition of illumination, and the active oxygen can destroy the structure of the protein and inhibit the activity of the protein. And because the generated active oxygen has short life, the active oxygen can only react with protein adjacent to the polymer, thereby realizing the selective light inactivation of the basic protein combined with the polymer shown in the formula (I).

While most biotoxin proteins in nature are basic, they generally cause apoptosis by disrupting cell membranes or cytoskeleton, affecting ion channel function. The polymer shown in the formula (I) provided by the invention can be combined with the toxin proteins and react, and further the toxin proteins can be selectively inactivated by illumination, so that the activity of the toxin proteins is inhibited.

More specifically, cardiotoxin is a toxin protein from the Chinese cobra, and surface electrostatic potential calculations show that a high density of positive charges is distributed on its surface. The polymer shown in the formula (I) can effectively inhibit hemolytic effect caused by cardiotoxin and greatly improve survival rate of mice injected with cardiotoxin.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a scheme for the synthesis of Polymer 1 (PPV-NHS);

FIG. 2 shows the zeta potential change and hydrated particle size change of PPV-NHS after interaction with different proteins;

FIG. 3 is a photograph of a gel electrophoresis of PPV-NHS after interaction with different proteins;

FIG. 4 is a graph showing the change of fluorescence intensity of DCFH with time after oxidation by PPV-NHS-generated active oxygen;

FIG. 5 shows the activity change of HRP and LPO under dark and white light irradiation before and after mixing with PPV-NHS respectively;

FIG. 6 is a plot of hemolysis of red blood cells as a function of time before and after CTX and CTX addition to PPV-NHS (in the dark and under white light illumination, respectively);

FIG. 7 shows the results of detoxification experiments of PPV-NHS in mice injected with CTX.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

Polymer and method for producing the same

The invention provides a polymer with a conjugated skeleton, which contains a structural unit shown as a formula (I),

wherein R1 has a structure as shown in formula (II),

r2 has a structure as shown in formula (III),

the polymerization degree n is an integer of 10-200, k is an integer of 4-20, q is an integer of 1-5, m is an integer of 2-12, and represents the bonding position of R1 and R2 in the formula (I).

In one embodiment, the degree of polymerization n is an integer from 10 to 50, k is an integer from 6 to 12, q is an integer from 1 to 5, and m is an integer from 2 to 8.

In one embodiment, preferably, the degree of polymerization n is an integer from 16 to 19, k is 8, q is 1, and m is 4, i.e. the polymer has the structure shown in formula (IV), referred to as polymer 1 (i.e. PPV-NHS):

according to the present invention, the polymer represented by the formula (I) can be produced according to the following scheme (V), wherein the compound 6 is subjected to substitution reaction with the compound 7 to obtain the compound 8, and the compound 8 is subjected to polycondensation reaction with the compound 10 to obtain the polymer 11. The reaction conditions for the reactions involved in the scheme of formula (V) can be selected according to the prior art, for example, in the step of polycondensation of compound 8 and compound 10, in the presence of palladium acetate catalyst, catalyst ligand P (o-Tol)₃And tri-N-butylamine in the presence of N, N-Dimethylformamide (DMF) as a solvent. Specifically, polymer 1 can be prepared according to the scheme shown in FIG. 1.

In the formula (V), Ts-in the compound 7 represents a 4-methylbenzenesulfonate group, NHS represents N-hydroxysuccinimide, and EDCI represents 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

As shown in formula (V), different polymers shown in formula (I) are obtained by selecting different raw material compounds 7 (different values of k), different raw material compounds 9 (different values of m), different raw material compounds 12 (different values of q), and limitations (different polymerization degrees n) of polycondensation reactions of the

compounds

8 and 10.

Polymer selective inactivation of basic proteins

Basic proteins, i.e. proteins which are positively charged under physiological conditions, such as alpha-chymotrypsin (alpha egg CT), papain (Pap), Lactoperoxidase (LPO), cytochrome c (cyt c), lysozyme (Lys); acidic proteins, which are proteins having a negative charge under physiological conditions and an isoelectric point (pI) of less than 7.4, include horseradish peroxidase (HRP), Bilirubin Oxidase (BOD), Glucose Oxidase (GOD), Bovine Serum Albumin (BSA), and glucose-6-phosphate dehydrogenase (G6 PD). The PPV-NHS surface is negatively charged by Zeta (Zeta) potential testing of the polymer of formula (I), e.g., Polymer 1(PPV-NHS), and the Zeta potential is-21.1 + -0.07 mV. As can be seen from FIG. 2, the zeta potential of PPV-NHS hardly changed when PPV-NHS was mixed with the acidic proteins BSA, GOD and HRP, respectively; after PPV-NHS is respectively mixed with basic proteins Pap, alpha-CT and Cyt c, the zeta potential of PPV-NHS is obviously moved in a positive direction, which shows that PPV-NHS is selectively combined with basic proteins Pap, alpha-CT and Cyt c. This is also demonstrated by the dynamic light scattering data results. Specifically, the hydrated particle size of PPV-NHS was 240nm as measured by dynamic light scattering. When acidic proteins BSA, GOD and HRP were added, respectively, the particle size of PPV-NHS was hardly changed as shown in FIG. 2; when the basic proteins Pap, α -CT and Cyt c were added, respectively, the particle size of PPV-NHS was significantly increased as shown in FIG. 2.

The results of denaturing gel electrophoresis tests, in turn, illustrate the reaction (i.e., covalent binding) of PPV-NHS with basic proteins. Acidic proteins (HRP, BOD, GOD, BSA, G6PD) and basic proteins (LPO, α -CT, Pap, Cyt c, Lys) (protein concentration 20 μmol/L) were reacted with different concentrations of PPV-NHS (20 μmol/L, 100 μmol/L,200 μmol/L, 400 μmol/L) at 37 ℃ for 30min in PBS solution at pH 7.4, followed by gel electrophoresis. And set the protein alone as a blank control. High molecular weight proteins HRP, BOD, GOD, BSA, G6PD, and LPO, electrophoresed using sodium dodecyl sulfate-polyacrylamide gel containing 12% separation gel and 5% concentration gel; the lower molecular weight proteins α -CT, Pap, Cyt c and Lys were electrophoresed using a trimethylglycine-sodium dodecylsulfate-polyacrylamide gel containing 16.5% separation gel, 10% sandwich gel and 4% concentrated gel. Protein bands were stained with coomassie brilliant blue. The resulting denatured gel electrophoresis pattern is shown in FIG. 3, and it can be seen from FIG. 3 that the protein band after the action of PPV-NHS with acidic protein is almost unchanged from that of the blank control, indicating that PPV-NHS does not react with acidic protein. And when PPV-NHS is mixed with the basic protein, the molar ratio of the basic protein to the polymer represented by the formula (I) is 1: (1-20), as the concentration of PPV-NHS increases, the original protein band gradually disappears and a new band appears in the high molecular weight region, indicating that the PPV-NHS and the basic protein undergo covalent reaction to increase the overall molecular weight.

In the present invention, the polymer represented by formula (I) may be selectively covalently bound (covalently reacted) to a basic protein, and may generate an active oxygen species under light irradiation. The inventors of the present invention verified the ability of the polymer of formula (I), such as PPV-NHS, to generate active oxygen using the active oxygen species probe 2, 7-dichlorofluorescein-3, 6-diethyl ester (DCFH-DA). DCFH-DA hydrolyzes to 2, 7-dichlorofluorescein-3, 6-diacetic acid (DCFH) under alkaline conditions, DCFH can be oxidized to highly fluorescent 2, 7-dichlorofluorescein in the presence of active oxygen, which has a strong emission at 525nm after 488nm excitation. FIG. 4 shows fluorescence intensity at 525nm (excitation wavelength 488nm) after adding PPV-NHS to freshly prepared DCFH solution (DCFH concentration of 40. mu. mol/L, PPV-NHS concentration of 10. mu. mol/L)Illumination time (white light density of 5mW cm)^-2) A variation diagram of (2). The fluorescence at 525nm was significantly higher than that of the control group (without PPV-NHS) with time, indicating that PPV-NHS has excellent active oxygen-generating ability.

Active oxygen can disrupt the structure of the protein, inactivating it, and active oxygen has a short lifetime, generally inactivating only adjacent proteins. The present inventors have found that the polymer of formula (I) can selectively inactivate basic proteins without affecting the activity of other proteins such as acidic proteins.

In the present invention, the "inactivation" is understood to mean a reduction in the activity of the protein to 0 to 30% of the activity of the original protein.

The inventors of the present invention investigated the effect of polymers of formula (I), such as PPV-NHS, on the activity of different proteins using two peroxidases with opposite surface charges. The polymer shown in formula (I) such as PPV-NHS is mixed with different proteins in dark, and treated in light and dark respectively (for example, lasting for 10min), then the activity of the polymer is detected respectively, and the single protein is set as a blank control group (the blank control group is used for explaining that the activity of the protein is not greatly influenced by pure light). As can be seen from FIG. 5, the activity of the acid protein HRP hardly changes after mixing with PPV-NHS, no matter in light condition or dark condition, which indicates that the polymer shown in formula (I) such as PPV-NHS does not affect the activity of HRP; while basic proteins such as LPO, after dark action, the activity decreased to 60% of the original protein activity, and after white light irradiation, the activity further decreased to 20%. Thus, polymers of formula (I), such as PPV-NHS, can selectively inactivate basic proteins.

In this regard, a second aspect of the invention provides a method of selectively inactivating a basic protein, the method comprising:

(1) mixing a sample containing basic protein with the polymer according to the first aspect of the invention to obtain a mixture;

(2) exposing the mixture obtained in step (1) to light for selective inactivation.

In the method for selectively inactivating a basic protein according to the present invention, the sample may be any sample containing a basic protein. In one embodiment, the sample may further contain an acidic protein selected from one or more of HRP, BOD, GOD, BSA, G6PD, and other acidic proteins.

In the method for selectively inactivating a basic protein according to the present invention, the basic protein may be a basic protein commonly used in the art, preferably the basic protein is selected from one or more of cardiotoxin, LPO, Pap, α -CT, Cyt c, Lys.

In the method for selectively inactivating a basic protein according to the present invention, in step (1), the basic protein and the polymer according to the first aspect of the present invention are used in a molar ratio of 1: (1-20), preferably 1: (3-12), more preferably 1: (5-10). In one embodiment, in step (1), the mixing is performed under physiological conditions and is maintained for 20-40 min. In another embodiment, in step (1), the mixing is performed in Phosphate Buffered Saline (PBS) at pH 7.4 and maintained for 20-40 min.

In the method for selectively inactivating a basic protein according to the present invention, in the step (1), the temperature of the mixing is 20 to 40 ℃.

In the method for selectively inactivating a basic protein according to the present invention, in the step (2), the wavelength of the light is 400-800nm, and the optical density is 50-150 mW-cm^-2。

In the method for selectively inactivating a basic protein according to the present invention, in the step (2), the exposure conditions include: the temperature is 25-40 deg.C, and the time is 5-30 min.

Use of polymers to inhibit cardiotoxin toxicity

Since most biotoxin proteins in nature are basic proteins, they generally cause apoptosis by disrupting the cell membrane or cytoskeleton to affect the function of ion channels. The polymers of formula (I) can inactivate basic proteins and can therefore also be used to inhibit the activity of these toxin proteins. The Cardiotoxin (CTX) is a toxin protein from Chinese cobra, and the calculation result of the surface electrostatic potential shows that the surface of the cardiotoxin is distributed with high-density positive charges, can be effectively combined with the polymer shown in the formula (I) and react, and is further inactivated by illumination, so that the damage of the cardiotoxin to erythrocytes is inhibited, and hemolysis is avoided.

In this regard, a third aspect of the invention provides the use of a polymer of formula (I) for inhibiting cardiotoxin toxicity.

The inventors studied the haemolysis of CTX alone and after addition of PPV-NHS using mouse and human erythrocytes. Specifically, fresh blood was collected in a sample tube containing heparin, washed twice with 10mmol/L PBS, centrifuged at 1500 rpm for 10 minutes, and then red blood cells were diluted to 2%. PPV-NHS (50. mu. mol/L) (and a control group without PPV-NHS) was added to a mixture of CTX (10. mu. mol/L) and erythrocytes at 80 mW. cm^-2Irradiating for 10min under white light, culturing at 37 deg.C for 8 hr, centrifuging each hour, and measuring hemoglobin absorption at 576nm of supernatant. A dark place was set for 10min (corresponding to 10min of light) as a control, only 10mmol/L PBS was added as a negative control, and only 1% triton-X100 was added as a positive control. As can be seen from fig. 6, with the extension of the incubation time, CTX showed significant hemolysis, reaching hemolysis rates of about 15% (mouse) and 33% (human) after 8 h. The addition of PPV-NHS reduced the hemolysis rate to below 5%, and after light exposure, the hemolysis effect induced by CTX was further inhibited and PPV-NHS alone was not destructive to erythrocytes.

The inventors have also demonstrated the detoxifying effect of polymers of formula (I), such as PPV-NHS, on CTX by mouse experiments simulating detoxification of animals bitten by venomous snakes. The survival of mice injected with CTX alone (25. mu. mol/L, 200. mu.L), PPV-NHS alone (125. mu. mol/L, 200. mu.L) and CTX alone (25mol/L, 200. mu.L) with continued injection of PPV-NHS (125. mu. mol/L, 200. mu.L) at 3min intervals is shown in FIG. 7. After injection of CTX, mice all died within 18 minutes, whereas two days after 3min interval between CTX injections, PPV-NHS injection, 57% of mice survived; although 43% of the mice died, their survival time was significantly extended to around 50 min. Therefore, PPV-NHS can obviously prolong the survival time of the mice, improve the survival rate of the mice and has good detoxification effect on CTX.

Therefore, the polymer shown in the formula (I) disclosed by the application, such as PPV-NHS, can effectively inhibit the toxicity of CTX and has good detoxification effect on CTX.

The present invention will be described in detail below by way of examples.

Example 1

Example 1 serves to illustrate the preparation of polymer 1, i.e., PPV-NHS.

PPV-NHS was prepared according to the process scheme in FIG. 1.

(1) Preparation of Compound 3

K was added to 10mL of acetone₂CO₃(0.23g), N was introduced₂To remove O in the solvent₂. In N₂The compound 2, 5-diiodo-1, 4-benzenediol (compound 1, 0.2g), 23-hydroxy-3, 6,9,12,15,18, 21-heptaoxatricosane 4-methylbenzenesulfonate (compound 2, 0.59g) was added under protection, the reaction temperature was raised to 60 ℃ under N₂And reacting for 30 hours under the atmosphere. After cooling to room temperature, the precipitate was filtered and washed. Obtaining anhydrous Na for filtrate₂SO₄Drying, filtering and removing the solvent. The crude product was isolated and purified by silica gel column chromatography to give a pale yellow liquid (0.13g, 22%). Performing nuclear magnetic hydrogen spectrum test, nuclear magnetic carbon spectrum test and mass spectrum test on the obtained light yellow liquid, wherein the obtained data are as follows:

¹H-NMR(400MHz,DMSO-d₆,δ):7.37(s,1H),4.55(t,J＝4.0Hz,1H),4.09(t,J＝6.0Hz,2H),3.74(t,J＝4.0Hz,2H),3.62(m,2H),3.50(m,24H),3.42(m,2H)；

¹³C-NMR(100MHz,CDCl₃,δ):153.01,123.36,87.35,72.79,70.61,70.32,70.23,70.17,69.41,60.66；

HR-MS(MALDI):[M+K]⁺a peak of 1105.2132 was found at 1105.2127.

From the above data, the resulting pale yellow liquid was compound 3.

(2) Preparation of Compound 5

13,13' - ((2, 5-diiodo-1, 4-phenylene) dioxy) bis (2,5,8, 11-tetraoxatridecane) (compound 4, 0.5g) was dissolved in 12mL of toluene and N was bubbled through₂To remove O in the solvent₂In N at₂To the solution were added tributylvinyltin (1.27g) and ethanol washed Pd (PPh) with protection₃)₄(16mg), and the temperature was raised to 100 ℃ to react for 16 hours. After the reaction mixture was cooled to room temperature, unreacted tributylvinyltin was removed with 40mL of a 2mol/L aqueous solution of KF. Collecting organic phase, and adding anhydrous Na₂SO₄Drying, filtering and removing the solvent. The crude product was isolated and purified by silica gel column chromatography to give a colorless oily liquid (0.21g, 59%). And (3) carrying out nuclear magnetic hydrogen spectrum test, nuclear magnetic carbon spectrum test and mass spectrum test on the obtained colorless oily liquid, wherein the obtained data are as follows:

¹H-NMR(300MHz,CDCl₃,δ):7.07-6.98(m,4H),5.74(dd,J＝16.5,1.2Hz,2H),5.26(dd,J＝9.9,1.2Hz,2H),4.13(t,J＝4.8Hz,4H),3.85(t,J＝5.4Hz,4H),3.75-3.63(m,20H),3.53(m,4H),3.37(s,6H)；

¹³C-NMR(100MHz,CDCl₃,δ):151.00,131.68,127.91,114.61,111.54,72.24,71.16,70.98,70.95,70.93,70.82,70.18,69.40,59.30；

HR-MS(MALDI):[M+K]⁺a peak of 581.2728 was found at 581.2723.

From the above data, the obtained colorless oily liquid was compound 5.

(3) Preparation of PPV-OH

In N₂Under protection, compound 3(0.21g), compound 5(0.11g), catalyst palladium acetate (10mg), catalyst ligand P (o-Tol)₃(20mg) and tri-N-butylamine (0.50mmol) were dissolved in 5mL of N, N-Dimethylformamide (DMF), and the mixture was dissolved in N₂To remove oxygen in the solvent, heating the reaction solution to 100 ℃, and reacting for 48 hours under the protection of nitrogen. After the reaction solution was cooled to room temperature, the reaction solution was dialyzed against methanol, the cut-off molecular weight of the dialysis bag was 7500, and finally methanol was removed by rotary evaporation to obtain a dark red oily liquid (0.18g, 56%). The obtained dark red oily liquid is subjected to nuclear magnetic hydrogen spectrum test and gel chromatography test, and the obtained data are as follows:

¹H-NMR(400MHz,DMSO-d₆,δ):7.52(br,2H),7.25(br,2H),4.53(br,1H),4.21-3.39(br,48H),3.19(s,3H)；

GPC:M_W29020, PDI 1.15, and degree of polymerization n is 16-19.

The nuclear magnetic hydrogen spectrum data shows that the obtained deep red oily liquid is PPV-OH.

(4) Preparation of PPV-COOH

Succinic anhydride (20mg) was added to a solution of 4mL of the polymer PPV-OH (15mg) in super dry dichloromethane under ice-bath conditions, and after stirring in an ice-bath for 1h, the reaction temperature was allowed to warm to room temperature and stirring was continued for 24 h. Diluted with dichloromethane and washed with saturated brine. Collecting the organic phase with anhydrous Na₂SO₄Drying, filtration and removal of the solvent gave an orange oily liquid (7.5mg, 44%). The orange oily liquid is subjected to nuclear magnetic hydrogen spectrum testing, and the obtained data are as follows:

¹H-NMR(300MHz,CDCl₃-d₆,δ):7.43(br,2H),7.17(br,2H),4.23-4.12(br,5H),3.93(br,4H),3.75-3.50(br,36H),3.34(s,3H),2.62(s,4H)。

the nuclear magnetic hydrogen spectrum data show that the obtained orange oily liquid is PPV-COOH.

(5) Preparation of PPV-NHS

PPV-COOH (20mg) was dissolved in 4mL of ultra-dry Dichloromethane (DCM), and N-hydroxysuccinimide (NHS) (4.5mg) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 6.32mg) were added at 0 ℃. The reaction was continued at 0 ℃ for 1h, then moved to room temperature and continued for 24 h. Dilute with 10mL of dichloromethane, wash with water, and dry Na₂SO₄After drying, concentration gave an orange-red oily liquid (18mg, 80%). The obtained orange-red oily liquid is subjected to nuclear magnetic hydrogen spectrum test, and the obtained data are as follows:

¹H-NMR(300MHz,CDCl₃-d₆,δ):7.42(br,2H),7.15(br,2H),4.25-4.12(br,5H),3.91(br,4H),3.73-3.49(br,36H),3.34(s,3H),2.93(t,2H),2.85(s,4H),2.75(t,2H)。

the nuclear magnetic hydrogen spectrum data shows that the obtained orange-red oily liquid is PPV-NHS.

Comparative examples 1 to 3 and examples 2 to 4

Comparative examples 1 to 3 and examples 2 to 4 are intended to illustrate that PPV-NHS can selectively inactivate basic proteins.

Comparative example 1

(1) Mixing acidic protein HRP and PPV-NHS at 37 deg.C in PBS solution with pH of 7.4 to obtain mixture, and maintaining for 30min, wherein the concentration of acidic protein HRP is 20 μmol/L, and the concentration of PPV-NHS is 200 μmol/L;

(2) the mixture obtained in the step (1) is subjected to white light (wavelength is 400-800nm, optical density is 80mW cm)^-2) Exposing at 37 deg.C for 10 min;

(3) the activity of the protein was measured, and the results are shown in FIG. 5.

Comparative example 2

Referring to the method of comparative example 1, except that, in step (2), the resulting mixture was placed in the dark instead of the light and the activity of the protein was measured, the results are shown in FIG. 5.

Example 2

(1) Mixing alkaline protein LPO and PPV-NHS at 37 deg.C in PBS solution with pH of 7.4 to obtain mixture, and maintaining for 30min, wherein the concentration of alkaline protein HRP is 20 μmol/L, and the concentration of PPV-NHS is 200 μmol/L;

As can be seen from FIG. 5, the activity of HRP was almost unchanged after the reaction with PPV-NHS, while the activity of LPO was reduced to 20% of the activity of the original protein. Thus PPV-NHS can selectively inactivate basic proteins while hardly affecting the activity of acidic proteins.

Comparative example 3

Referring to the method described in example 2, except that, in the step (2), the resulting mixture was placed in the dark instead of the light and the activity of the protein was measured, the results are shown in FIG. 5.

As can be seen from FIG. 5, the activity of the acid protein HRP hardly changes in both light and dark conditions; the activity of the alkaline protein LPO is reduced to 60% of the activity of the original protein after the action in the dark.

Example 3

With reference to the method described in example 2, except that, in the step (2) in which the concentration of PPV-NHS was 100. mu. mol/L, the activity of the protein was measured and the activity of the protein was decreased to 30% of the activity of the original protein, as in example 2.

Example 4

With reference to the method described in example 2, except that, in the step (2) in which the concentration of PPV-NHS was 140. mu. mol/L, the activity of the protein was measured and the activity of the protein was decreased to 25% of the activity of the original protein, as in example 2.

Example 5

Example 5 serves to illustrate the detoxification of the snake venom protein cardiotoxin by PPV-NHS.

(1) Finding 21 mice with the same mass and sign, injecting 7 single CTX (25 mu mol/L and 200 mu L), 7 single PPV-NHS (125 mu mol/L and 200 mu L), and continuing to inject PPV-NHS (125 mu mol/L and 200 mu L) after 3min interval of the 7 single CTX (25 mu mol/L and 200 mu L);

(2) survival of 21 mice was observed and the results are shown in fig. 7.

As can be seen from FIG. 7, the vital signs of 7 mice injected with PPV-NHS alone were good; 7 mice injected with CTX alone all died within 18 minutes of injection; and continuously injecting the PPV-NHS mice after the CTX injection interval is 3min, continuously observing for two days, wherein 4 mice survive, and the survival rate reaches 57%; although 43% of the mice died, their survival time was extended to around 50 min.

In conclusion, the polymer with the structure shown in formula (I), especially PPV-NHS, can selectively inactivate basic protein without affecting the activity of acidic protein, and can effectively inhibit the toxicity of cardiotoxin, thereby having good detoxification effect.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. A conjugated skeleton polymer contains a structural unit shown as a formula (I),

wherein R1 has a structure as shown in formula (II),

r2 has a structure as shown in formula (III),

2. The polymer according to claim 1, wherein the degree of polymerization n is an integer of 10 to 50, k is an integer of 6 to 12, q is an integer of 1 to 5, and m is an integer of 2 to 8.

3. The polymer according to claim 1 or 2, wherein the degree of polymerization n is an integer of 16 to 19, k-8, q-1, and m-4.

4. A method of selectively inactivating a basic protein, the method comprising:

(1) mixing a sample containing basic proteins with the polymer of any one of claims 1-3 to obtain a mixture;

(2) and (2) exposing the mixture obtained in the step (1) to light to inactivate the basic protein.

5. The method of claim 4, wherein said basic protein is selected from the group consisting of cardiotoxin, LPO, Pap, α -CT, Cyt c and Lys.

6. The method of claim 4 or 5, wherein the sample further comprises an acidic protein.

7. The method of claim 6, wherein the acidic protein is selected from one or more of HRP, BOD, GOD, BSA or G6 PD.

8. The method of claim 4 or 5, wherein the molar ratio of the basic protein to the polymer of claim 1 is 1: (1-20).

9. The method of claim 8, wherein the basic protein is present in a molar ratio to the polymer of claim 1 of 1: (3-12).

10. The method according to claim 4 or 5, wherein in the step (2), the wavelength of the light is 400-800 nm.

11. The method according to claim 4 or 5, wherein in step (2), the exposure conditions include: the temperature is 25-40 deg.C, and the time is 5-30 min.

12. Use of a polymer according to any one of claims 1 to 3 for inhibiting cardiotoxin toxicity.