A biomimetic enzyme photocatalytic system based on tetraphenyl ethene-yl cation molecular cage

Abstract

This invention provides a method for efficiently and rapidly oxidizing NADH, thereby destroying NADH and NAD. + New materials for interphase conversion equilibrium processes, through two steps S N In a reaction design, an octacational molecular cage based on tetraphenylethylene (TPE)-alkenylpyridine was synthesized for constructing a biomimetic catalyst. The TPE unit endows the molecular cage with excellent fluorescence properties and the ability to adaptively switch chirality. The cavity of the molecular cage allows it to bind FAD molecules through host-guest interactions, thus achieving a dual response of FAD fluorescence and circular dichroism (CD) spectroscopy. The octacational nature not only ensures good water solubility of the molecular cage, but also promotes the formation of a highly efficient catalyst for the oxidation of NADH to NAD. + The oxidation process exhibits highly efficient catalytic activity, with a time-to-factor (TOF) frequency reaching 29.4 min. ‑1 This effectively solves the technical problem of the low conversion frequency in the current NADH catalytic oxidation process, which prevents the achievement of efficient catalysis for NADH oxidation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection, and particularly relates to a biomimetic enzyme catalytic system for photo-oxidation reaction of nicotinamide adenine dinucleotide. BACKGROUND

[0002] Coenzymes are a general term for a variety of organic cofactors, and coenzyme molecules play a crucial role in a plurality of physiological processes (such as tricarboxylic acid cycle, amino acid metabolism, redox reactions in cells, etc.). Reduced nicotinamide adenine dinucleotide (NADH) plays an important role in the metabolic process of cells. Since the oxidation of NADH in cancer cells will disturb the redox balance in cells, leading to the death of cancer cells, therefore, it is very urgent to develop new materials with high and fast oxidation of NADH to destroy the balance process for the diagnosis and treatment of cancer cells.

[0003] Supramolecular materials, especially macrocyclic supramolecular materials, have an unprecedented advantage in constructing efficient biomimetic catalytic systems due to their encapsulatable cavities, multifunctional structures, and dynamic adjustability of host-guest complexation. The common transition metal materials for NADH catalytic oxidation process are greatly limited in catalytic efficiency due to their single catalytic mode, and the turnover frequency (TOF) value is low, which cannot realize the efficient catalysis of the oxidation process of NADH. Macrocyclic molecules with adjustable structures are a kind of extremely attractive molecular tools for biomimetic catalytic oxidation of NADH. Compared with single molecular cages or FAD, the molecular cage can combine the FAD molecule through its limited cavity, so as to achieve the goal of synergistically improving the catalytic efficiency, and realize a deeper understanding of complex physiological processes. SUMMARY

[0004] To achieve the above-mentioned purpose, the present application is designed by two-step S N 2 reaction to synthesize an octacationic molecular cage based on tetraphenylethene (TPE) alkenyl pyridine for constructing a biomimetic catalyst. Among them, the TPE unit endows the molecular cage with excellent fluorescence performance and self-adaptive chiral conversion ability, the molecular cage can combine the FAD molecule through host-guest interaction and realize the dual response of fluorescence and circular dichroism (CD) spectrum of FAD; the octacationic property not only ensures the good water solubility of the molecular cage, but also ensures that the molecular cage and FAD can form a water-soluble host-guest complex. Compared with the single molecular cage and FAD, the host-guest complex has a higher catalytic effect, and has a high catalytic activity for the oxidation process of NADH to NAD + , and the turnover frequency can reach 29.4min -1 .

[0005] The present application provides the following technical scheme:

[0006] A kind of biomimetic enzyme photocatalysis system, which is based on the complex of tetraphenyl ethylene-based pyridine octacation molecular cage, the specific molecular cage structure formula is:

[0007] The system described in the application is the host-guest complex of tetraphenyl ethylene-based pyridine octacation molecular cage and FAD, which can realize the dual response of fluorescence and circular dichroism (CD) spectrum of FAD.

[0008] The conversion frequency of the system described in the application reaches 29.4 min -1 .

[0009] The molecular cage is synthesized by tetra (pyridyl-4-vinyl) styrene through two-step S N 2 reaction. First, 3 reacts with 1,4-bis (bromomethyl) -benzene (4) under reflux conditions for 3 days, and after ion exchange, 5·4PF6 – is obtained. Then, equimolar amounts of compounds 3 and 5·4PF6 – are reacted at 110℃ for 3 days in a pressure bottle under the catalysis of tetrabutylammonium iodide, and pure 1·8PF6 – is obtained by column chromatography. Then, water-soluble 1·8Cl - is obtained from 1·8PF6 – by ion exchange.

[0010] The application constructs an enzyme-like catalyst for catalyzing the oxidation of NADH to NAD + by the host-guest interaction between the molecular cage and FAD, which has a higher catalytic effect on the catalytic oxidation process compared with the molecular cage and FAD alone. The catalytic oxidation process is monitored by ultraviolet spectrophotometry and nuclear magnetic resonance hydrogen spectrum. The catalytic effects of different catalysts are evaluated by calculating their conversion frequency (TOF) values.

[0011] Based on the above technical solutions, the application has the following beneficial effects:

[0012] (1) The application designs and synthesizes a new type of octacation molecular cage, which can realize the dual response detection of fluorescence and CD of coenzyme molecule FAD, and has the advantages of simple operation, high sensitivity and low cost.

[0013] (2) The molecular cage used in the application can construct an efficient enzyme-like catalyst by combining with FAD through host-guest binding, which is used to catalyze the process of converting NADH to NAD + which is very important in physiological activities. Compared with existing catalysts containing transition metals, the enzyme-like catalytic system of the molecular cage and FAD complex has better catalytic oxidation effect, and its TOF value is as high as 29.4 min -1 .

[0014] (3) Compared with the prior art, the application can construct an enzyme-mimic catalytic system for catalyzing oxidation of NADH by host-guest interaction of a molecular cage and FAD, and realize high-efficiency oxidation of NADH by using a host-guest complex platform of the molecular cage and FAD, thereby opening up a new way for constructing an enzyme-mimic system to explore complex life activities. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The molecular cage structure formula involved in embodiment 1 of the application.

[0016] Figure 2 The molecular cage synthesis route and the structure formulas of FAD, NADH and NAD + involved in embodiment 1 of the application.

[0017] Figure 3 The fluorescence response graph of the molecular cage (10 μM) to FAD described in embodiment 2 of the application.

[0018] Figure 4 The CD response graph of the molecular cage (10 μM) to FAD described in embodiment 3 of the application.

[0019] Figure 5 The ultraviolet change graph of the complex of the molecular cage (5 μM) and FAD (10 μM) catalyzing oxidation of NADH (100 μM) described in embodiment 4 of the application.

[0020] Figure 6 The nuclear magnetic change graph of the complex of the molecular cage (100 μM) and FAD (50 μM) catalyzing oxidation of NADH (2 mM) described in embodiment 5 of the application.

[0021] Figure 7 The TOF comparison graph of the complex of the molecular cage (5 μM) and FAD (10 μM) and the single molecular cage and the single FAD to NADH (100 μM) oxidation catalysis and the control test of NADH (100 μM) without catalyst described in embodiment 5 of the application. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. However, this should not be understood as a limitation on the scope of the above-mentioned subject matter of the application to the following embodiments. Based on the embodiments in the application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the application.

[0023] Embodiment 1

[0024] Design and synthesis of the molecular cage:

[0025] by two steps S N 2 Reaction synthesis of molecular cage 1, weighed 3 (531 mg, 2.01 mmol) into a two-necked flask containing dry MeCN (50 mL) and the suspension was heated at 90 °C until all the compound was dissolved. 4 (100 mg, 134 μmol) was added to the solution of 3 in portions and the reaction was refluxed at 90 °C for three days. Then the mixture was cooled to room temperature, the precipitate was collected and washed with excess of acetone (3 x 40 mL) by centrifuge to obtain the crude product as an orange solid. The crude product was dissolved in water (25 mL) and then excess of NH4PF6 was added and stirred for 12 h. The precipitate was collected and washed with excess of H2O (3 x 20 mL) to obtain 5 · 4PF6 – orange solid. 5 · 4PF6 – (100 mg, 48.5 μmol) and tetrabutylammonium iodide (8.96 mg, 24.3 μmol) into a pressure bottle containing dry MeCN (100 mL) and the suspension was heated at 90 °C. 3 (40 mg, 53.4 μmol) was added to the suspension of 5 in portions and heated to 110 °C for three days. Then the mixture was cooled to room temperature, the precipitate was collected and washed with excess of MeCN (3 x 40 mL) by centrifuge to obtain the crude product as an orange solid. The crude product was dissolved in water (25 mL) and then excess of NH4PF6 was added and stirred for 12 h. The precipitate was collected and washed with excess of H2O (3 x 20 mL) to obtain the crude product as an orange solid (27 mg, 18.2 %). The crude product was purified by silica gel chromatography with CH2Cl2:MeCN (saturated NH4PF6) = 3:1 (v:v) as mobile phase to obtain pure 1 · 8PF6 – (10 mg, 6.7 %). 1 · 8Cl – .

[0026] Example 2

[0027] Fluorescence response of molecular cage to FAD:

[0028] Weighed 5.25 mg of molecular cage solid sample into a sample bottle and dissolved in 0.5 mL of ultrapure water, ultrasonic dissolution, prepared into a 4.8 mM concentration solution for standby. At the same time, weighed 1.99 mg of FAD into a sample bottle, added 0.5 mL of ultrapure water, ultrasonic dissolution, prepared into a 4.8 mM concentration solution of FAD. Used a pipette to take 6.25 μL of the concentrated solution of the molecular cage, added 2993.75 μL of ultrapure water to dilute the concentration to 10 μM, then took 2.4 mL of 10 μM dilute solution of the molecular cage into a quartz cuvette, and successively added 1.0 μL (0.2 equivalent) of the concentrated solution of FAD, and tested the fluorescence response behavior of the molecular cage to FAD by a fluorescence spectrometer.

[0029] Example 3

[0030] CD response detection of FAD by molecular cage:

[0031] The same operation process as in Example 2 was used to take 6.25 μL of a concentrated solution of the molecular cage, dilute it to 10 μM by adding 2983.75 μL of ultrapure water, then take 2.4 mL of a 10 μM dilute solution of the molecular cage in a quartz cuvette, and sequentially add 1.0 μL (0.2 equivalent) of a concentrated solution of FAD, and test the CD response behavior of the molecular cage to FAD by a circular dichroism spectrometer.

[0032] Example 4

[0033] UV detection of catalytic oxidation of NADH by the molecular cage:

[0034] A 1.7 mg solid sample of NADH was weighed and dissolved in 0.5 mL of ultrapure water, and ultrasonically dissolved to prepare a 4.8 mM concentrated solution for use. In 2928 μL of ultrapure water, 3.125 μL of a concentrated solution of the molecular cage was added, followed by 6.25 μL of a concentrated solution of FAD, mixed well, and then 62.5 μL of a concentrated solution of NADH was added to a quartz cuvette to dilute it to a 5 μM aqueous solution of the molecular cage, 10 μM of FAD, and 100 μM of NADH. A 200 W LED lamp was used to irradiate the cuvette every 5 s and detect the UV absorption spectrum, and each group of experiments was repeated 3 times.

[0035] TOF value calculation method:

[0036]

[0037] A = εbc

[0038] c = n / v

[0039] Where A is the absorbance, ε is the molar absorption coefficient, b is the thickness of the cuvette, c is the concentration, and n is the amount of substance. The ε of NADH at 339 nm 339nm = 6200 M -1 .

[0040] Example 5

[0041] NMR detection of catalytic oxidation of NADH by the molecular cage:

[0042] A 13.6 mg sample of NADH solid was weighed and dissolved in 0.5 mL of deuterium water, and ultrasonically dissolved, and prepared as a 38.4 mM concentrated solution for standby. A 2.19 mg sample of molecular cage solid was weighed and dissolved in 0.5 mL of deuterium water, and ultrasonically dissolved, and prepared as a 2 mM concentrated solution for standby. A 15.9 mg sample of FAD solid was weighed and dissolved in 0.5 mL of deuterium water, and ultrasonically dissolved, and prepared as a 38.4 mM concentrated solution for standby. In 442 μL of deuterium water, 12 μL of the concentrated solution of molecular cage in deuterium water was added, followed by 1.25 μL of the concentrated solution of FAD in deuterium water, mixed well, followed by 25 μL of the concentrated solution of NADH in deuterium water in an NMR tube, which was diluted to a 50 μM of molecular cage, 100 μM of FAD, and 2 mM of NADH in deuterium water solution, and the NMR tube was irradiated with a 200 W LED lamp every 5 mins and the NMR hydrogen spectrum was detected.

Claims

1. A biomimetic enzymatic photocatalytic system, characterized in that, The system is based on a host-guest complex of tetraphenylstyryl octapositively charged molecular cage and FAD, and the structural formula of the tetraphenylstyryl octapositively charged molecular cage is: 。 2. Use of a biomimetic enzyme photocatalytic system according to claim 1 for the preparation of catalytic oxidation of NADH to NAD + in an enzyme mimic catalyst.

3. The preparation method of the tetraphenylstyryl octapositively charged molecular cage according to claim 1, characterized in that: The tetraphenylstyryl octapositively charged molecular cage is synthesized by a two-step SN2 reaction of tetraphenylstyrene and then obtained by ion exchange, and the specific synthesis steps are as follows: In the first step, compound 3 is reacted with 1,4-bis(bromomethyl)-benzene under refluxing conditions for 3 days to give 5«4PF6after ion exchange – ; Second step, equimolar amounts of compound 3 and 5-4PF6 – were reacted in a pressure tube at 110 °C for 3 days under catalysis of tetrabutylammonium iodide and the pure 1-8PF6was isolated by column chromatography – and 1-8PF6was exchanged by ion exchange – to obtain water-soluble 1-8CI – The reaction scheme is as follows: 。

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