Preparation method and application of an amphiphilic tyrosinase copper competitive binding agent

Abstract

The application discloses a preparation method and application of an amphiphilic tyrosinase copper competitive binding agent. Two new compounds P4TA or P2TA are synthesized by coupling tyramine TA with 4,4',4'',4'''-(porphyrin-5,10,15,20-tetrayl) tetrakisbenzoic acid TCPP or 3,7,12,17-tetramethyl-8,13-diethyl-2,18-porphyrin dipropionic acid PPIX. An optimal synthesis process of P4TA and P2TA is provided, and an experience is provided for related synthesis. The obtained P4TA and P2TA can recognize tyrosinase and are good copper ion binding reagents, and the two compounds exhibit a significant inhibitory effect on TYR. The application can be used for tyrosinase targeted inhibition and copper ion metabolism intervention in biological medicines and food.

Description

TECHNICAL FIELD

[0001] The present application relates to an amphiphilic tyrosinase copper competitive binding agent, belonging to the field of tyrosinase recognition and inhibitors, and particularly relates to an amphiphilic multi-effect tyrosinase copper competitive binding agent and application thereof in tyrosinase inhibition, which can be used for tyrosinase targeted inhibition and copper ion metabolism intervention in biological medicine and food. BACKGROUND

[0002] The potential application of tyrosinase (TYR) recognized compounds in the treatment of cancer, neurodegenerative diseases, metabolic diseases and bacterial drug resistance has become a research hotspot. In recent years, it has been found that tyrosinase is involved in the degradation and metabolism of tyrosine, and the design and development of tyrosinase inhibitors can block the metabolic pathway of bacteria, thereby inhibiting their growth and reproduction [E.C.J. Syed, L.L. Grima, P.J. Magill, R. Bogacz, P. Brown, M.E. Walton, Action initiation shapes mesolimbic dopamine encoding of future rewards. Nat. Neurosci. 19 (2016) 34-36; S. Zolghadri, A. Bahrami, M.T. Hassan-Khan, J. Munoz-Munoz, F. Garcia-Molina, F. Garcia-Canovas, A. A. Saboury, A comprehensive review on tyrosinase inhibitors, J. Enzyme Inhib. Med. Chem. 34 (2019) 279-309.], Therefore, tyrosinase inhibitors have become a hot spot in the field of drug research and development.

[0003] TYR, as a kind of polyphenol oxidase, first catalyzes monophenol to diphenol, and then further oxidizes it to ortho-quinone; for example, under the catalysis of TYR, tyramine (TA) is sequentially converted into dopamine, dopamine ortho-quinone and other substances. This process has been applied to the development of responsive probes. In 2020, researchers developed a fluorescent probe with phenolic group as enzyme substrate [S. Yang, J. X. Jiang, A. X. Zhou, Y. B. Zhou, W. L. Ye, D. S. Cao, R. H. Yang, Substrate-photocaged enzymatic fluorogenic probe enabling sequential activation for light-controllable monitoring of intracellular tyrosinase activity. Anal. Chem. 92 (2020) 7194-7199.], the fluorescence of this probe can be "turned on" by ultraviolet light and TYR, and the results of fluorescence imaging confirmed that the probe can visualize the endogenous activity of cell TYR under light control.

[0004] Based on the recognition characteristics of TYR to TA, we choose TA as the recognition part, and adjust the coupling structure of TA and porphyrin to improve the amphiphilicity of porphyrin compounds and improve the multi-effect action point of the compounds with TYR. Although tyrosine inhibitors have been reported [T.N. Pham, E.A. Cazier, E. Gormally, P. Lawrence, Valorization of biomass polyphenols as potential tyrosinase inhibitors, Drug Discovery Today, 29 (2023) 103843], most of the synthetic heterocyclic tyrosinase inhibitors are toxic, and natural polyphenolic compounds have weak inhibition on tyrosinase and poor cell absorption [X.W. Zhang, G.L. Bian, P.Y. Kang, X.Y. Cheng, K. Yan, Y.L. Liu, Y.X. Gao, D.Q. Li, Recent advance in the discovery of tyrosinase inhibitors from natural sources via separation methods, J. Enzyme Inhibition Med. Chem. 36 (2021) 2104-2117], and no amphiphilic compound based on the competition binding of copper in the active center of tyrosinase has been reported. In order to develop copper binding agents targeting tyrosinase, new phenol-coupled porphyrin compounds and metal complexes are synthesized, and the research results will provide new ideas and methods for discovering more effective copper ion metabolism tyrosinase inhibitors and antibacterial agents. SUMMARY

[0005] In view of the problems in the prior art, the present application couples tyramine (labeled as TA) with 4,4',4'',4'''-(porphyrin-5,10,15,20-tetrayl) tetrakis(p-toluic acid) (labeled as TCPP) or 3,7,12,17-tetramethyl-8,13-diethyl-2,18-porphyrin dipropionic acid (labeled as PPIX) to synthesize two new compounds 4,4',4'',4'''-(porphyrin-5,10,15,20-tetrayl) tetrakis(p-hydroxyphenethyl) formamide (labeled as P4TA) and 3,3'-(3,7,13,17-tetramethyl-8,12-diethenylporphyrin-2,18-diyl) bis(N-(4-hydroxyphenethyl) propanamide) (labeled as P2TA). The optimal synthesis process of P4TA and P2TA is explored, providing an experience for related synthesis. Then, the combination of P4TA and P2TA with copper ions and tyrosinase (TYR) is studied, and it is found that P4TA and P2TA can recognize tyrosinase and are good copper ion binding reagents, and the two compounds exhibit significant inhibition effect on TYR. In addition, P4TA is soluble in dichloromethane and methanol and slightly soluble in water, and is a copper ion binding reagent with amphiphilic property and a good tyrosinase inhibitor. The structures of P2TA and P4TA are as follows:

[0006]

[0007]

[0008] The preparation method of P4TA or P2TA comprises the following steps:

[0009] 4,4',4'',4'''-(porphyrin-5,10,15,20-tetrayl) tetrakis(p-toluic acid) (labeled as TCPP) or 3,7,12,17-tetramethyl-8,13-diethyl-2,18-porphyrin dipropionic acid (labeled as PPIX) is mixed with pyridine in proportion, and then 1-hydroxybenzotriazole HOBt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI and N,N-diisopropylethylamine DIPEA are added dropwise; then the mixed solution is stirred at room temperature for 1-3 h in the dark, and then tyramine TA is added and stirred for 3-12 h; petroleum ether is added to the above system, and the precipitate is filtered, and then extracted with DCM and water, dried with anhydrous sodium sulfate, and then rotary evaporated or subjected to silica gel column chromatography to obtain P4TA purple red solid or P2TA purple red solid.

[0010] Further, when the raw material is TCPP,

[0011] The mass ratio of TCPP to pyridine is 1:10-1:60, preferably 1:40.

[0012] The molar ratio of TCPP:HOBt:EDCI:DIPEA is 1:1:1:1-1:8:8:8, preferably 1:3:3:3;

[0013] The molar ratio of TCPP:tyramine TA is 1:2-1:16, preferably 1:12;

[0014] Further, when the raw material is PPIX,

[0015] The mass ratio of PPIX to pyridine is 1:10-1:60, preferably 1:40.

[0016] The molar ratio of PPIX:HOBt:EDCI:DIPEA is 1:1:1:1-1:4:4:4, preferably 1:2:2:2;

[0017] The molar ratio of PPIX:tyramine TA is 1:2-1:8, preferably 1:6;

[0018] In the above step,

[0019] The mixing solution is stirred at room temperature for 1-3h, preferably 1h;

[0020] After adding tyramine, the stirring time is 3-12h, preferably 8h;

[0021] The volume ratio of petroleum ether to pyridine is 10:1-60:1, preferably 25:1,

[0022] When extracting, the volume ratio of DCM to water is 1:1.

[0023] The P4TA or P2TA prepared by the application is used for the binding of tyrosinase, wherein P4TA is a better tyrosinase binding reagent; the inhibition rate of 27μM P4TA to tyrosinase reaches 88.63%, indicating that P4TA is a tyrosinase targeting inhibitor; in addition, P4TA and P2TA have copper ion binding effect and can be used as a copper competitive binding reagent for tyrosinase, therefore, P4TA and P2TA have multiple effects of tyrosinase recognition, copper ion binding and tyrosinase activity inhibition, and the good liposolubility and water solubility make them have amphiphilic properties, accordingly, P4TA is a multi-effect tyrosinase inhibitor, and the tyrosinase inhibitor in the literature is to intervene in the catalytic activity of tyrosinase, and here we first report a multi-effect tyrosinase inhibitor with tyrosinase recognition and copper ion binding as a whole, which is first reported in the world.

[0024] The application has the following beneficial effects:

[0025] (1) The present application discloses a synthesis and purification process of P4TA or P2TA. It is found that when using pyridine as the basic solvent, the yield of the target product P4TA is significantly increased to 68.54%. Pyridine is selected as the best solvent for this reaction.

[0026] It is further found that there is a relationship between the TA / TCPP ratio and the yield. The results show that when the TA / TCPP ratio is in the range of 4 to 12, the yield increases with the increase of the ratio, and the maximum yield is reached when the ratio reaches 12.

[0027] (2) It is found that P4TA and P2TA have tyrosinase targeting binding properties. The binding constants of P4TA and P2TA to the tyrosine (Tyr) residues in tyrosinase are 1127 and 30.9, and the binding constants of P4TA and P2TA to the tryptophan in tyrosinase are 51.20 and 14.60, which indicates that P4TA is a better tyrosinase binding agent.

[0028] (3) P4TA and P2TA are inhibitors of tyrosinase catalyzed oxidation of L-tyrosine. Among them, the IC 50 of P4TA to tyrosinase is 9 μM (the inhibition rate of 9 μM P4TA to tyrosinase is 50.61), and the IC 50 of P2TA is 13 μM (the inhibition rate of 13 μM P2TA to tyrosinase is 49.43), and the inhibition rate of 27 μM P4TA to tyrosinase reaches 88.63, which indicates that P4TA is a better tyrosinase inhibitor.

[0029] (4) P4TA and P2TA can bind Cu 2+ , and the binding of copper ions is sensed by the change of fluorescence. Fluorescence titration of Cu 2+ binding of P4TA and P2TA is carried out, and the research results show that Cu 2+ significantly quenches the fluorescence emission of P4TA and P2TA, which indicates that P4TA and P2TA have copper ion binding sensing probes.

[0030] In summary, P4TA and P2TA have tyrosinase recognition, copper ion binding and tyrosinase activity inhibition multiple effects. Their good liposolubility and water solubility make them have amphiphilic properties. Therefore, we further propose that the competitive binding between P4TA and the copper ions in the active center of tyrosinase realizes the synergistic enhancement mechanism of TYR inhibition activity. The inhibition effect of phenolic compounds on tyrosinase is weak in the literature, and the tyrosinase inhibitor with copper ion binding effect is reported for the first time in the world. Copper ions and tyrosinase are related to diseases such as senile dementia, tumor and bacterial drug resistance. Therefore, P4TA, as a tyrosinase targeting copper ion and tyrosinase activity intervention reagent, is expected to be used for disease treatment. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The image shows the 1H NMR spectrum of P4TA in deuterated DMF.

[0032] Figure 2 The fluorescence emission spectra of P4TA (5 μM) with different copper ion concentrations are shown in the curves. The copper ion concentrations are 0, 1.50 μM, 3.0 μM, and 5.0 μM.

[0033] Figure 3 The image shows the 1H NMR spectrum of P2TA in deuterated DMSO.

[0034] Figure 4 The fluorescence emission spectra of P2TA (5 μM) with different copper ion concentrations are shown in the curves. The copper ion concentrations are 0, 1.50 μM, 3.0 μM, and 5.0 μM.

[0035] Figure 5 The excitation (ex) and emission (em) spectra of P2TA (5 μM) methanol solution are shown.

[0036] Figure 6 The effect of P4TA on the synchronous fluorescence emission of tyrosine (tyr) in tyrosinase (86.7 U / mL) is shown in the curves. The curves ah represent P4TA values ​​of 0 μM, 3.33 μM, 6.66 μM, 9.99 μM, 13.32 μM, 16.65 μM, 19.9 μM, and 26.64 μM, respectively, with Δλ = 15 nm.

[0037] Figure 7 The effect of P4TA on the synchronous fluorescence emission of tryptophan (try) in tyrosinase (86.7 U / mL) is shown in the curves. The curves ah represent P4TA concentrations of 0 μM, 3.33 μM, 6.66 μM, 9.99 μM, 13.32 μM, 16.65 μM, 19.9 μM, and 26.64 μM, respectively, with Δλ = 60 nm.

[0038] Figure 8 To investigate the inhibitory activity of different concentrations of P4TA on the oxidation of tyrosine (1 mM) catalyzed by tyrosinase (800 μL, 260 U / mL). Detailed Implementation

[0039] Reagents:

[0040] 3,7,12,17-tetramethyl-8,13-diethyl-2,18-porphyrindipropionic acid (common name: protoporphyrin, labeled as PPIX), 4,4',4",4"'-(porphyrin-5,10,15,20- tetrayl) tetrakisbenzoic acid (common name: porphyrin tetracarboxylic acid, labeled as TCPP), tyramine (TA), 1-hydroxybenzotriazole (HOBT), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N- diisopropylethylamine (DIPEA) were purchased from Shanghai Annjer Chemical Reagent Co., Ltd., tyrosinase (TYR) (1120 u / mg DW) was purchased from Yuan Ye Biotechnology Co., Ltd.

[0041] Example 1

[0042] Optimal synthesis scheme of 4,4',4",4"'-(porphyrin-5,10,15,20-tetrayl) tetrakis(p- hydroxyphenethyl) benzamide (labeled as P4TA):

[0043] TCPP (50 mg, 0.06 mmol) was dissolved in 2 mL of pyridine, 24 mg of HOBt (0.18 mmol), 35 mg of EDCI (0.18 mmol), and 0.12 g of DIPEA (0.18 mmol) were added respectively, and the mixture was stirred at room temperature in the dark for 1 h, then 137 mg of tyramine (0.72 mmol) was added. After stirring for 8 h, 50 mL of petroleum ether was added to the system, and after stirring for 5 min, the precipitate was filtered, and then extracted with 50 mL of DCM and 50 mL of water. After drying with anhydrous sodium sulfate, rotary evaporation was performed to obtain 54 mg of purple red solid, with a yield of 71.3%. The nuclear magnetic resonance of the compound is shown in the attached Figure 1 .

[0044] 1 H NMR (400 MHz, DMF-d7) δ 9.45 (s, 5H), 8.98 (d, J = 22.9 Hz, 12H), 8.50 - 8.22 (m, 16H), 7.21 (d, J = 8.3 Hz, 8H), 6.91 - 6.74 (m, 8H), 3.73 (t, J = 8.0 Hz, 8H), 2.74 (s, 8H). 13 CNMR (101 MHz, DMF-d7) δ 167.54, 166.54, 156.62, 144.59, 134.83, 131.74, 130.35, 129.97, 129.07, 126.13, 120.04, 115.53, 79.38, 67.79, 42.19, 38.91, 27.12, 23.83, 22.98, 22.67, 13.81, 10.77. HR-MS (ESI + , MeOH): Calcd for [M+H]+ [C 50 H 53 N6O4] + : 1266.5, found: 1267.51.

[0045] The mass spectrum data showed that the main ion of P4TA in methanol solution was [P4TA+H + ] + , m / z = 1267.51 (100), the molecular ion peak of P4TA combined with a proton; P4TA in DMSO solution at 410 nm appeared characteristic absorption peak of porphyrin ring S band, at 500-600 nm appeared four groups of porphyrin Q band absorption peaks, indicating the formation of porphyrin ring. Excited at 420 nm, P4TA appeared strong fluorescence emission at 650 nm and wide fluorescence emission peak at 720 nm Figure 2 , curve a) in the figure.

[0046] Example 2 (synthesis of P4TA)

[0047] TCPP (50 mg, 0.06 mmol) was dissolved in 3 mL of pyridine, 32 mg of HOBt (0.24 mmol), 46 mg of EDCI (0.24 mmol), 0.16 g of DIPEA (0.24 mmol) were added respectively, and the mixture was stirred at room temperature in the dark for 3 h, then 182 mg of tyramine (0.96 mmol) was added. After stirring for 12 h, 60 mL of petroleum ether was added to the system, and after stirring for 5 min, the precipitate was filtered, and then extracted with 50 mL of DCM and 50 mL of water respectively; dried with anhydrous sodium sulfate, and then rotary evaporated to obtain 12.1 mg of purple red solid, with a yield of 16.0%

[0048] Example 3 (synthesis of P4TA)

[0049] TCPP (50 mg, 0.06 mmol) was dissolved in 0.5 mL of pyridine, 21 mg of HOBt (0.06 mmol), 30 mg of EDCI (0.06 mmol), 0.11 g of DIPEA (0.06 mmol) were added respectively, and the mixture was stirred at room temperature in the dark for 2 h, then 23 mg of tyramine (0.12 mmol) was added. After stirring for 3 h, 30 mL of petroleum ether was added to the system, and after stirring for 5 min, the precipitate was filtered, and then extracted with 50 mL of DCM and 50 mL of water; dried with anhydrous sodium sulfate, and then rotary evaporated to obtain 10 mg of purple red solid, with a yield of 13.2%

[0050] Example 4 (synthesis of P4TA)

[0051] TCPP (50 mg, 0.06 mmol) was dissolved in 2 mL of pyridine, 64 mg of HOBt (0.48 mmol), 92 mg of EDCI (0.48 mmol), 0.32 g of DIPEA (0.48 mmol) were added respectively, and the mixture was stirred at room temperature in the dark for 1 h, then 137 mg of tyramine (0.72 mmol) was added. After stirring for 8 h, 20 mL of petroleum ether was added to the system, and after stirring for 5 min, the precipitate was filtered, and then extracted with 50 mL of DCM and 50 mL of water. After drying with anhydrous sodium sulfate, rotary evaporation was performed to obtain 39 mg of purple red solid, with a yield of 51.7%

[0052] Example 5

[0053] Optimal synthesis case of 3,3'-(3,7,13,17-tetramethyl-8,12-divinylporphyrin-2,18-diyl)bis(N-(4-hydroxyphenethyl)propanamide) (labeled as P2TA):

[0054] PPIX (34 mg, 0.06 mmol) was ultrasonically dissolved in 1.4 mL of pyridine; 16 mg of HOBt (0.12 mmol), 23.7 mg of EDCI (0.12 mmol), 0.08 mg of DIPEA (0.12 mmol) were added, and the mixture was stirred at room temperature in the dark for 1 h, then 91.3 mg of tyramine (0.48 mmol) was added. After stirring at room temperature under nitrogen protection for 8 h, 35 mL of petroleum ether was added to the system, and after stirring for 5 min, the precipitate was filtered, and then extracted with 50 mL of DCM and 50 mL of water. The precipitate was washed with 50 mL of deionized water for three times, dried with anhydrous sodium sulfate, and then separated by silica gel column chromatography. The product was eluted with dichloromethane: ethyl acetate = 4: 1 (v:v) to obtain 31.5 mg of purple red solid, with a yield of 65%. The nuclear magnetic resonance spectrum of P2TA is shown in the following figure: Figure 3 .

[0055] 1H NMR (400 MHz, DMSO-d6) δ 10.31 - 9.96 (m, 3H), 9.08 (s, 2H), 8.43 (dt, J = 17.8, 11.1 Hz, 2H), 8.09 (t, J = 5.6 Hz, 2H), 6.63 (d, J = 8.1 Hz, 4H), 6.50 - 6.41 (m, 4H), 6.40 (dd, J = 4.7, 1.6 Hz, 1H), 6.22 (ddd, J = 11.6, 5.1, 1.5 Hz, 2H), 4.30 (t, J = 7.7 Hz, 4H), 4.07 (d, J = 7.1 Hz, 1H), 3.66 (d, J = 6.7 Hz, 5H), 3.58 (d, J = 2.2 Hz, 5H), 3.14 (q, J = 6.9 Hz, 3H), 3.03 (t, J = 7.5 Hz, 3H), 2.23 (t, J = 7.6 Hz, 4H), 2.03 (s, 1H), 1.40 - 1.07 (m, 6H). 13 C NMR (101 MHz, DMF-d7) δ 172.27, 155.99, 140.30, 130.45, 129.62, 129.46, 129.35, 120.55, 114.95, 114.87, 97.77, 97.68, 97.45, 97.18, 41.03, 39.11, 26.96, 22.79, 22.50, 13.68, 12.27, 12.25, 10.88, 10.85. LR-MS (ESI + , MeOH): Calcd for [M+H] + [C 50 H 53 N6O4] + : 801.41, found: 801.79. HR-MS (ESI + , MeOH): Calcd for [M+MeOH+H] + [C 51 H 57 N6O5] + : 833.4385, found: 833.4377.

[0056] P2TA in methanol solution mainly ion [P2TA+H + ] + , m / z = 801.18 (100) and [P2TA+Na + ] +, m / z = 823.73 (173), The UV-Vis absorption spectrum of P2TA shows that it has a maximum absorption around 250-300 nm, an S-band absorption peak around 400 nm, and four absorption peaks in the Q-band between 500-680 nm. Upon excitation at 407 nm, P2TA has a strong fluorescence emission at 634 nm and a broad emission peak at 702 nm. Figure 4 , and curve a in Fig. 1 and Figure 5 ) in Fig. 1.

[0057] Example 6 (Synthesis of P2TA)

[0058] PPIX (34 mg, 0.06 mmol) was dissolved in 2.1 mL of pyridine under ultrasonic irradiation; 32 mg of HOBt (0.24 mmol), 46 mg of EDCI (0.24 mmol), and 0.16 g of DIPEA (0.24 mmol) were added, respectively. After stirring at room temperature for 3 h in the dark, 69 mg of tyramine (0.36 mmol) was added. After stirring for 3 h, 121 mL of petroleum ether was added to the system, and after stirring for 5 min, the precipitate was filtered. The precipitate was extracted with 50 mL of DCM and 50 mL of water, and then washed with 50 mL of deionized water three times. After drying with anhydrous sodium sulfate, the product was separated by silica gel column chromatography, eluted with dichloromethane: ethyl acetate = 4: 1 (v:v), and 15.8 mg of purple red solid was obtained, with a yield of 38%.

[0059] Example 7 (Synthesis of P2TA)

[0060] PPIX (34 mg, 0.06 mmol) was dissolved in 0.34 mL of pyridine under ultrasonic irradiation; 8 mg of HOBt (0.06 mmol), 11.5 mg of EDCI (0.06 mmol), and 0.04 g of DIPEA (0.06 mmol) were added, respectively. After stirring at room temperature for 3 h in the dark, 24 mg of tyramine (0.12 mmol) was added. After stirring for 8 h, 3.4 mL of petroleum ether was added to the system, and after stirring for 5 min, the precipitate was filtered. The precipitate was extracted with 50 mL of DCM and 50 mL of water, and then washed with 50 mL of deionized water three times. After drying with anhydrous sodium sulfate, the product was separated by silica gel column chromatography, eluted with dichloromethane: ethyl acetate = 4: 1 (v:v), and 5 mg of purple red solid was obtained, with a yield of 12%.

[0061] Performance test 1: effect of solvent and amount of TA on yield of P4TA

[0062] TCPP was covalently coupled with tyramine (TA) via amide bond to form compound P4TA. The effects of solvent and TA amount on the reaction yield were investigated (Table 1 and Table 2). First, we investigated the effects of different solvents on the reaction: when CH2Cl2and CH3CN were used as solvents, the amount of target product P4TA was extremely low; when DMF and methanol were used as solvents, the yield of P4TA was 19% and 36.75%, respectively; notably, when the basic solvent pyridine was used, the yield of target product P4TA was significantly increased to 68.54%. This phenomenon can be attributed to the fact that pyridine enhances the solubility of TCPP, in addition, the stability of the basic environment plays a key role in the formation and maintenance of intermediates, effectively reducing the generation of by-products, thereby significantly increasing the overall yield of the reaction. Therefore, pyridine was selected as the best solvent for this reaction. Further study on the relationship between TA / TCPP ratio and yield (Table 2) showed that when the ratio of TA / TCPP was in the range of 4 to 12, the yield increased with the increase of the ratio, and the maximum yield was obtained when the ratio reached 12.

[0063] Table 1 Effect of different solvents on the yield of P4TA [a]

[0064] Solvent Yield / % Pyridine 68.54 DMF (N,N-dimethylformamide) 36.75 MeOH (methanol) 19 CH3CN (acetonitrile) 8.62 CH2Cl2(dichloromethane) Trace

[0065] Note: [a] Reaction conditions: 0.06 mmol of TCPP, 0.54 mmol of HOBt, 0.54 mmol of EDCI, 0.54 mmol of DIPEA, 1.2 mmol of TA, N2 and solvent (4 mL) at room temperature.

[0066] Table 2 Effect of TA amount on the yield of P4TA

[0067] Ratio (TA / TCPP) Yield / % 4 39.23 8 53.36 12 71.26

[0068] Note: [a] Reaction conditions: 0.06 mmol of TCPP, 0.54 mmol of HOBt, 0.54 mmol of EDCI, 0.54 mmol of DIPEA, 1.2 mmol of TA, N2 and pyridine (4 mL) at room temperature.

[0069] Performance investigation 2: Cu 2+ Fluorescence titration experiments of P2TA and P4TA

[0070] The concentration of copper sulfate (CuSO4) aqueous solution was 1 mM; under the condition of constant stirring at 30°C, different concentrations (0 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM) of CuSO4 aqueous solution were added into P2TA or P4TA (7.5 μM, DMSO) solution every 17 min, and the fluorescence intensity of the solution was tested by fluorescence spectrophotometer (the excitation wavelength of P2TA was 406 nm, and the emission wavelength was 633 nm; the excitation wavelength of P4TA was 419 nm, and the emission wavelength was 587 nm).

[0071] The results of P2TA and copper ion interaction are shown in Figure 4 , which shows that Cu 2+ significantly quenches the fluorescence emission of P2TA; the results of P4TA and copper ion interaction are shown in Figure 2 , which shows that P4TA and P2TA are both copper ion binding responsive probes.

[0072] Performance test 3: evaluation of the interaction of P2TA and P4TA with tyrosinase

[0073] The concentration of tyrosinase (TYR) solution (PBS, pH = 6.8) was 86.7 U / mL, and different concentrations (0 μM, 3.33 μM, 6.66 μM, 9.99 μM, 13.32 μM, 16.65 μM, 19.98 μM, 23.31 μM, 26.64 μM, 29.97 μM, 33.3 μM, 36.63 μM, 39.96 μM, 43.29 μM, 46.62 μM) of P2TA and P4TA DMSO solution were added into the solution at room temperature, and the effect of P2TA and P4TA on tryptophan and tyrosine in TYR was explored by synchronous fluorescence method. The binding constant (Ka) and binding ratio (n) of biomolecules and compounds were calculated by using the fluorescence titration curve, and the equation was the improved Stern-Volmer equation:

[0074]

[0075] wherein F0 is the initial fluorescence intensity, F is the fluorescence intensity after adding the compound, and Q is the quencher concentration.

[0076] The data are shown in Table 3, and the results of P4TA and tyrosinase interaction are shown in Figure 6 and Figure 7 . The data in Table 3 show that P2TA and P4TA have good interaction with tyrosine groups (tyr) and tryptophan groups (try) in tyrosinase.

[0077] Table 3 Fitting parameters of luminescence spectra of P2TA and P4TA

[0078] Compound Ka (mL / μM) n [R 2 ]] [P4TA-Tyr] 1127 1.8743 0.98 [P2TA-Tyr] 30.9 0.9481 0.99 [P4TA-Try] 51.02 0.96 0.98 [P2TA-Try] 14.6 2.2723 0.99

[0079] Note: Tyr represents the tyrosine group in tyrosinase; Try represents the tryptophan group in tyrosinase; P4TA-Tyr and P2TA-Tyr represent the binding of the compound to the tyrosine group in the enzyme; P4TA-Try and P2TA-Try represent the binding of the compound to the tryptophan group in the enzyme.

[0080] Performance test 4: Effect of different concentrations of P2TA and P4TA on the activity of tyrosinase

[0081] DMSO-PBS (volume ratio 1:4) aqueous solutions of different concentrations of inhibitors P2TA or P4TA (0 μM, 3 μM, 6 μM, 9 μM, 13 μM, 16 μM, 31 μM, 47 μM, 63 μM, 78 μM, 94 μM) were prepared. Each sample was then added with tyrosinase (800 μL, 260 U / mL) to form a compound-enzyme complex system. After the complex system was incubated at 37°C in a water bath for 10 minutes, 2.4 mL of L-tyrosine (substrate of the enzyme) was added to the system, and after stirring for 17 min, the change in ultraviolet absorption spectrum at 475 nm of the system was measured to evaluate the effect of P2TA and P4TA on the activity of tyrosinase. The measurement was taken once every minute, and the inhibition rate I of the compound on the enzyme was calculated according to the following two formulas. The data of P2TA are shown in Table 4, and the data of P4TA are shown in Table 5. Figure 8 and Table 5.

[0082] Inhibition rate I of the enzyme = (A3-A4) / (A1-A2)

[0083] A1 is the absorbance without inhibitor and with substrate; A2 is the absorbance without inhibitor and without substrate; A3 is the absorbance with inhibitor and with substrate; A4 is the absorbance with inhibitor and without substrate.

[0084] Table 4 shows that the data show that the inhibition rate of 13 μM P2TA on tyrosinase is 49.43; Table 5 shows that the inhibition rate of 9 μM P4TA on tyrosinase is 50.61, and the inhibition rate of 27 μM P4TA on tyrosinase reaches 88.63, which indicates that P4TA is a better tyrosinase inhibitor.

[0085] Table 4 UV-Vis spectral values of A1-A4 of P2TA-enzyme-substrate system and inhibition rate I on the enzyme

[0086]

[0087] Table 5 UV-Vis spectral values of A1-A4 of P4TA-enzyme-substrate system and inhibition rate I on the enzyme

[0088]

[0089]

Claims

1. An amphiphilic tyrosinase copper competitive binding agent, characterized in that, The structural formula is as follows:

2. The method of claim 1, wherein the preparation of the amphiphilic tyrosinase copper competitive binding agent is characterized by, The method comprises the following steps: 4,4',4",4"'-(porphyrin-5,10,15,20-tetrayl) tetrakisbenzoic acid TCPP or 3,7,12,17-tetramethyl-8,13-divinyl-2,18-porphyrin dipropionic acid PPIX is mixed with pyridine in a certain proportion, and then 1-hydroxybenzotriazole HOBt, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride EDCI and N,N-diisopropylethylamine DIPEA are added drop by drop; then the mixed solution is stirred at room temperature in the dark, and then tyramine TA is added, and stirring is continued; petroleum ether is added to the above system, and the precipitate is filtered; then the precipitate is extracted with DCM and water, dried with anhydrous sodium sulfate, and then rotary evaporation or silica gel column chromatography is performed to obtain P4TA purple red solid or P2TA purple red solid.

3. The production method according to claim 2, wherein When the raw material is TCPP, The mass ratio of TCPP to pyridine is 1:10-1:60; The molar ratio of TCPP:HOBt:EDCI:DIPEA is 1:1:1:1-1:8:8:8; The molar ratio of TCPP:tyramine TA is 1:2-1:

16.

4. The production method according to claim 3, wherein When the raw material is TCPP, The mass ratio of TCPP to pyridine is 1:40; The molar ratio of TCPP:HOBt:EDCI:DIPEA is 1:3:3:3; The molar ratio of TCPP:tyramine TA is 1:

12.

5. The production method according to claim 2, wherein When the raw material is PPIX, The mass ratio of PPIX to pyridine is 1:10-1:60; The molar ratio of PPIX:HOBt:EDCI:DIPEA is 1:1:1:1-1:4:4:4; The molar ratio of PPIX:tyramine TA is 1:2-1:

8.

6. The production method according to claim 5, wherein When the raw material is PPIX, The mass ratio of PPIX to pyridine is 1:40; The molar ratio of PPIX:HOBt:EDCI:DIPEA is 1:2:2:2; The molar ratio of PPIX:tyramine TA is 1:

6.

7. The preparation method of claim 2, wherein the mixed solution is stirred at room temperature in the dark for 1-3 h; and after the addition of tyramine, the stirring time is 3-12 h. The volume ratio of petroleum ether to pyridine is 10:1-60:1; and the volume ratio of DCM to water is 1:1 during extraction.

8. The preparation method of claim 7, wherein the mixed solution is stirred at room temperature in the dark for 1 h; and after the addition of tyramine, the stirring time is 8 h. The volume ratio of petroleum ether to pyridine is 25:

1.

9. Use of the amphiphilic tyrosinase copper competitive binding agent of claim 1 in the preparation of a reagent for recognizing tyrosinase or inhibiting the activity of tyrosinase.

10. Use of the amphiphilic tyrosinase copper competitive binding agent of claim 9 in the preparation of a copper competitive binding reagent for tyrosinase. ​ ​ ​

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