Thiomalic Acid-Protected Water-Soluble Alloy Nanomaterials and Their Application in the Conversion of NAD to NADH

Abstract

The present invention discloses a thiosuccinic acid-protected water-soluble alloy nanomaterial and its application in the conversion of NAD to NADH. The thiosuccinic acid-protected water-soluble alloy nanomaterial is a gold-silver nanocluster with the molecular formula [Au3Ag5(MSA)3@5TOA-5H]Na. Here, MSA represents the ligand thiosuccinic acid; TOA represents the phase transfer agent tetraoctylammonium bromide. The present invention utilizes the formation of hydrogen bonds between the carboxyl group on the ligand thiosuccinic acid and the amino group of NAD to achieve the mutual binding of NAD and the cluster. The alloy nanocluster with electronic flexibility transfers electrons to NAD to achieve the generation of NADH. The purification of the finally generated product NADH is simple, and the product can be obtained only by simple ultrafiltration.

Description

Technical Field

[0001] The present invention relates to a thiosuccinic acid-protected water-soluble alloy nanomaterial and its application in the conversion of NAD to NADH, belonging to the field of materials. Background Art

[0002] Electron transfer is a fundamental chemical behavior in homogeneous systems and is ubiquitous in processes such as redox reactions, nucleophilic substitution reactions of free radicals, photosynthesis, and respiration. The 2021 Nobel Prize in Chemistry achieved an asymmetric organic catalytic reaction through single electron transfer, providing a new idea for designing catalysts used in catalytic reactions. There are also many chemical reactions in living organisms that utilize electron transfer. For example, oxidized coenzyme I (NAD) and its reduced form, reduced coenzyme I (NADH), are an important pair of coenzymes in living organisms, assisting in the occurrence of approximately one-quarter of redox reactions (NADH Regeneration: A Case danJoseph.W.H, Burnett.Russell.F, Howe.Panagiotis.N, Kechagiopoulos, Xiaodong.Wang, ACS.Catal.2021.11.283 - 289). The conversion of NAD to NADH can be achieved by NAD gaining electrons. Currently, complexes and metal particles are widely used in the conversion of NAD to NADH (Efficient NADHRegeneration by a Redox Polymer-Immobilized Enzymatic System, Mengwei.Yuan, Matthew.J.Kummer, Ross.D.Milton, Timothy.Quah, Shelley.D.Minteer, ACS.Catal.2019.9.5486 - 5495), but the reaction process is complex and the reaction conditions are harsh. There has been no report on the conversion of metal nanoclusters in this direction.

[0003] Ligand-protected ultrasmall (<2 nm) metal nanoclusters (NCs) are a class of emerging functional materials with rich coordination chemistry. Due to their high atomic precision, clear molecular structure, and unique molecular-like properties, they have received increasing attention in basic and applied research. Metal nanoclusters have a certain degree of electronic flexibility due to their special structural characteristics. For example, they can achieve redox reactions through electron transfer and thus realize the conversion between different metal clusters (Conversion of Anionic [Au25(SCH2CH2Ph18)]-Cluster to Charge Neutral Cluster via Air Oxidation, Manzhou.Zhu, William.T.Eckenhoff, Tomislav.Pintauer, Rongchao.Jin, J.Phys.Chem.C. 2008.112.37.14221–14224). It is precisely because of this property of the cluster itself to donate and accept electrons that it has been reported in the literature that metal nanoclusters can biomimetically catalyze the synthesis of some compounds required in organisms (Gold-Platinum Bimetallic Nanoclusters for Oxidase-like Catalysis, Chunxiao.Zhang, Yancai.Gao, Hongwei.Li, Yuqing.Wu, ACS.Appl.NanoMater. 2020.3.9318-9328).

[0004] Water-soluble metal nanoclusters have good biocompatibility while having electronic flexibility. Using water-soluble metal nanoclusters for the conversion of NAD to NADH will provide a new idea for the synthesis of NADH. Summary of the Invention

[0005] Based on the problems existing in the above technical background, the present invention proposes a thiosuccinic acid-protected water-soluble alloy nanomaterial and its application in the conversion of NAD to NADH. This method is convenient to operate, the synthesis of the alloy nanomaterial is simple, and it can effectively achieve the conversion of NAD to NADH.

[0006] The thiosuccinic acid-protected water-soluble alloy nanomaterial of the present invention is a gold-silver nanocluster, and its molecular formula is [Au 3 Ag 5 (MSA) 3 @5TOA-5H]Na. Where MSA represents the ligand thiosuccinic acid; TOA represents the phase transfer agent tetraoctylammonium bromide.

[0007]

[0008] The preparation method of the thio-malate-protected water-soluble alloy nanomaterial of the present invention comprises the following steps:

[0009] Step 1: Add thio-malate (i.e., mercaptosuccinic acid) into ultrapure water, and then add sodium hydroxide solution, and stir and mix evenly.

[0010] Step 2: Add silver nitrate solution and chloroauric acid solution into the mixture in Step 1, add sodium hydroxide solution again, and stir and mix evenly, with the stirring speed being 300 r / min.

[0011] Step 3: Add sodium borohydride solution after reacting for 15 minutes in Step 2, and stir for a predetermined time to obtain a crude product.

[0012] Step 4: Filter the reaction solution obtained in Step 3 with a filter head, and obtain a concentrated solution of the alloy nanomaterial after ultrafiltration.

[0013] Step 5: Purify the concentrated solution obtained in Step 4 by polyacrylamide gel electrophoresis to obtain the thio-malate-protected water-soluble alloy nanomaterial.

[0014] Specifically, electrophorese the concentrated solution obtained in Step 4, collect the first band, soak it in ultrapure water for 2 h to obtain the purified aqueous phase product; add an equal volume of tetraoctylammonium bromide and toluene to the purified aqueous phase product, stir vigorously, let it stand for layer separation, collect the organic phase, wash it with ultrapure water 5 - 6 times, and collect the organic phase; spin-dry the collected organic phase, dissolve it with dichloromethane, and perform electrospray ionization mass spectrometry test to obtain the molecular formula.

[0015] Each raw material used in the present invention: the concentration of thio-malate is ≥98%, the purity of sodium hydroxide is ≥97%, the purity of silver nitrate (AgNO 3 ) is ≥99%, and the purity of chloroauric acid (HAuCl 4 ·3H 2 O) is ≥99%.

[0016] In Step 1, the molar ratio of thio-malate to sodium hydroxide is 3:10. Specifically in the preparation, the concentration of thio-malate in the system of Step 1 is 0.94 mg / mL, and the concentration of sodium hydroxide is 0.8 mg / mL.

[0017] In Step 2, the molar ratio of chloroauric acid to silver nitrate is 4:1; the molar ratio of chloroauric acid to sodium hydroxide is 0.09:1; the molar ratio of silver nitrate to sodium hydroxide is 0.023:1. Specifically in the preparation, the concentration of silver nitrate in the system is 0.1133 mg / mL, the concentration of chloroauric acid is 0.8989 mg / mL, and the concentration of sodium hydroxide is 1.24 mg / mL.

[0018] In the reaction system of Step 2, the molar ratio between silver nitrate and thiomalic acid is 1:9.2; the molar ratio between chloroauric acid and thiomalic acid is 1:2.3;

[0019] In Step 3, the molar ratio of sodium borohydride to the thiomalic acid in Step 1 is 1:22.35. The concentration of the sodium borohydride solution in the system is 0.0134 mg / mL, the reaction temperature is 25 °C, the reaction time is 4 h, the pH of the reaction system is 11, and the stirring speed is 300 r / min.

[0020] In Step 4, the size of the filter head is 22 μm, and the specification of the cut-off filter used for ultrafiltration is 3 kDa.

[0021] In Step 5, when purification is carried out by polyacrylamide gel electrophoresis, dispersion and stacking gels are prepared from 30% and 4% acrylamide monomers. The parameters of the electrospray ionization mass spectrometry are in the anion mode, the source voltage is 2.4 kV, the source temperature is 80 °C, and the cone voltage is 40.

[0022] The application of the water-soluble alloy nanomaterial protected by thiomalic acid in the present invention is to use it to regulate the conversion of NAD to NADH.

[0023] Furthermore, in the presence of the water-soluble alloy nanomaterial protected by thiomalic acid, the conversion of NAD to NADH can be achieved in an aqueous solution at 20-25 °C. The conversion process is simple and the operation is safe.

[0024] Furthermore, the molar ratio of the water-soluble alloy nanomaterial protected by thiomalic acid to NAD is 2:1.

[0025] Compared with the prior art, the beneficial technical effects of the present invention are reflected in:

[0026] 1. The present invention directly synthesizes water-soluble alloy nanoclusters protected by thiomalic acid by a one-pot method, obtains water-soluble alloy nanoclusters with a narrow size distribution, and can obtain cluster precursors with precise atomic numbers through electrophoresis separation.

[0027] 2. The present invention utilizes the formation of hydrogen bonds between the carboxyl group on the ligand thiomalic acid and the amino group of NAD to achieve the mutual binding of NAD and the clusters. The alloy nanoclusters with electron flexibility transfer electrons to NAD to generate NADH. The purification of the finally generated product NADH is simple, and the product can be obtained only by simple ultrafiltration.

[0028] 3. The aggregate cluster precursors of the present invention have uniform morphology and size, and their molecular formulas can be obtained by mass spectrometry. Moreover, the cluster precursors have no fluorescence and do not interfere with the fluorescence of the subsequent NADH.

[0029] 4. The method of the present invention is simple to operate, time-consuming less, without high pressure and high temperature, and dangerous operations such as passing hydrogen, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the regulation of the conversion from NAD to NADH by the thioctic acid-protected alloy nanomaterial of the present invention.

[0031] Figure 2 It is the ultraviolet-visible absorption spectrum of the water-soluble alloy nanoclusters of the present invention. Among them, (a) is the ultraviolet-visible absorption spectrum of the crude nanoclusters (the inset is a photo of the sample under sunlight); (b) is the ultraviolet-visible absorption spectrum of the first band after the nanoclusters are purified and separated by electrophoresis (the inset is the band distribution of the sample in the polyacrylamide gel after electrophoresis).

[0032] Figure 3 It is the X-ray photoelectron spectrum of the water-soluble alloy nanoclusters of the present invention. Among them, (a) is the Au4f orbital diagram; (b) is the Ag3d orbital diagram.

[0033] Figure 4 It is the high-resolution transmission electron microscope image of the water-soluble alloy nanoclusters of the present invention.

[0034] Figure 5 It is the electrospray ionization mass spectrum of the present invention.

[0035] Figure 6 It is the structural formula of NAD (a) and the structural formula of NADH (b) in the application of the present invention.

[0036] Figure 7 It is the ultraviolet absorption spectrum (a) and the fluorescence emission spectrum (b) of the NAD and NADH standard samples used in the application of the present invention.

[0037] Figure 8 It is the ultraviolet absorption spectrum and fluorescence emission spectrum of the water-soluble alloy nanoclusters of the present invention before and after reacting with NAD. Among them, (a) is the ultraviolet absorption spectrum; (b) is the fluorescence emission spectrum.

[0038] Figure 9 It is the electrospray ionization mass spectrum of the NADH product obtained by ultrafiltration after the water-soluble alloy nanoclusters of the present invention react with NAD.

[0039] Figure 10 It is the ultraviolet absorption spectrum of the NADH product obtained by ultrafiltration after the water-soluble alloy nanoclusters of the present invention react with NAD and detected by the enzymatic method. DETAILED DESCRIPTION OF THE INVENTION

[0040] Example 1: Preparation of Thioctic Acid-Protected Water-Soluble Alloy Nanomaterials

[0041] Transfer 10 mL of ultrapure water into a 25 mL flask. Weigh 0.0094 g of thiomalic acid and add it to the ultrapure water (0.94 mg / mL). Use a pipette to sequentially add 200 μL of sodium hydroxide solution (0.8 mg / mL), 56 μL of chloroauric acid solution (0.8989 mg / mL), 68 μL of silver nitrate solution (0.1133 mg / mL), and 100 μL of sodium hydroxide solution (1.24 mg / mL) to the ultrapure water, and stir for about 15 min (300 r / min) to mix the reactants evenly. After 15 min, use a pipette to take 280 μL of sodium borohydride solution (0.0134 mg / mL), react at 25 °C for 4 h to obtain a pale yellow solution. Remove the sample with a syringe, filter out large-sized particles with a filter head, add the filtrate to an ultrafiltration tube and ultrafilter (8000 r / min) for 18 min, and take out the concentrated solution for standby.

[0042] Example 2: Separation of water-soluble alloy nanomaterials protected by thiomalic acid

[0043] Add 50% glycerol to the obtained concentrated solution (the volume ratio of the concentrated solution to glycerol is 10:1), mix the concentrated solution and glycerol evenly, and then separate it by polyacrylamide gel electrophoresis. The working voltage of the polyacrylamide gel electrophoresis is 160 V, the working current is 260 mA, and the working time is 2 h. After the electrophoresis is completed, different bands can be seen on the polyacrylamide gel. Counting from top to bottom, cut the first band and put it into a centrifuge tube, crush it with a glass rod, and then add 3.5 mL of distilled water. After soaking in distilled water for 2 h, take out the solution with a syringe, filter out the gel with a filter head to obtain the purified water-soluble alloy nanoclusters.

[0044] Example 3: Take 2 mL of the sample purified by electrophoresis and first measure its ultraviolet absorption, and then measure its fluorescence emission.

[0045] Example 4: Preparation of NAD solution

[0046] Weigh 0.0026 g of NAD into a centrifuge tube, and use a pipette to transfer 2 mL of deionized water to the above centrifuge tube for standby.

[0047] Example 5: Use of water-soluble alloy nanomaterials protected by thiomalic acid for the conversion of NAD to NADH

[0048] Adjust its pH = 11 with sodium hydroxide and hydrochloric acid, and then use a pipette to take 2 mL of the sample solution and 1 mL of the prepared NAD solution into a centrifuge tube. Place it in a mixer for reaction, with a reaction temperature of 25 °C, a reaction time of 2 h, and a rotation speed of 600 r / min.

[0049] Example 6:

[0050] Place the reacted solution in an ultrafiltration tube and ultrafilter at 8000 r / min for 12 min. The generated NADH has a small molecular weight and will be ultrafiltered into the lower-layer solution. Take out the lower-layer solution after ultrafiltration, which is the generated NADH product.

[0051] Example 7:

[0052] Take the generated NADH product for testing, and test its ultraviolet absorption spectrum, fluorescence emission spectrum, electrospray ionization mass spectrometry and enzymatic detection of NADH generation.

[0053] Figure 1 This is a schematic diagram of the regulation of NAD to NADH conversion by thiosuccinic acid-protected alloy nanomaterials in the present invention. The alloy nanomaterials act as reducing agents in the conversion to provide electrons to achieve the conversion of NAD to NADH. The structural formula of the ligand thiosuccinic acid used in this experiment, and the two carboxyl groups in the ligand are the key to interacting with the amino group in NAD; the ligand also contains a mercapto group, which has reducibility and can etch gold nanoparticles and form gold-sulfur or silver-sulfur bonds with metals.

[0054] Figure 2 (a) is the ultraviolet-visible absorption spectrum of the crude product of thiosuccinic acid-protected water-soluble alloy nanoclusters synthesized. It can be seen from the figure that the crude product has two absorption shoulder peaks at 270 nm and 360 nm, and there are no surface plasmon resonance peaks of gold nanoparticles and silver nanoparticles at 520 nm and 470 nm, indicating that no large particles are formed. Figure 2 (b) is the ultraviolet absorption spectrum of the first band after purification by polyacrylamide gel electrophoresis. It can be seen from the figure that the first band has an absorption shoulder peak at 360 nm, which can match the ultraviolet absorption spectrum of the crude product.

[0055] Figure 3 is the X-ray photoelectron spectroscopy of the synthesized thiosuccinic acid-protected water-soluble alloy nanoclusters. It can be seen from Figure (a) that there are zero-valent gold and monovalent gold; it can be seen from Figure (b) that there are zero-valent silver and monovalent silver. It conforms to the properties of gold-silver alloy nanoclusters, confirming the synthesis of the nanoclusters.

[0056] Figure 4 This is the high-resolution transmission electron microscopy image of the water-soluble alloy nanoclusters of the present invention. It can be seen from the figure that the size distribution of the water-soluble alloy nanoclusters is narrow, and the morphology distribution is uniform, with an average particle size of 1.06 nm.

[0057] Figure 5 This is the electrospray ionization mass spectrometry of the water-soluble alloy nanoclusters of the present invention, and the molecular formula matched by the mass spectrometry is [Au 3 Ag 5 (MSA) 3 @5TOA-5H]- , where MSA represents the ligand thiomalic acid used; TOA represents the phase transfer agent tetraoctylammonium bromide.

[0058] Figure 6 (a) is the structural formula of NAD and (b) is the structural formula of NADH in the application of the present invention. Both molecules contain a nicotinamide ring, two riboses, and one adenine, and the difference between NAD and NADH lies in the nicotinamide ring. NADH has one more H than NAD.

[0059] Figure 7 (a) is the ultraviolet absorption spectrum of NAD and NADH standard samples. It can be seen from the figure that NAD has only one obvious absorption peak at 260 nm, and NADH has obvious absorption peaks at 260 nm and 340 nm respectively. (b) is the fluorescence emission spectrum of NAD and NADH standard samples. As shown in Figure (b), NAD has no fluorescence emission, and NADH has fluorescence emission at 460 nm.

[0060] Figure 8 are the ultraviolet absorption spectrum and fluorescence emission spectrum of the water-soluble alloy nanoclusters before and after the reaction with NAD in the present invention. (a) is the ultraviolet absorption spectrum. It can be seen from the figure that the precursor of the water-soluble alloy nanoclusters has no obvious ultraviolet absorption. After reacting with NAD for 2 h, the ultraviolet absorption spectrum has obvious absorption at 260 nm and 340 nm, indicating the formation of NADH after the water-soluble alloy nanoclusters react with NAD. (b) is the fluorescence emission spectrum. It can be seen from the figure that the precursor of the water-soluble alloy nanoclusters has no fluorescence emission. After reacting with NAD for 2 h, the fluorescence emission spectrum has a fluorescence emission at 460 nm, which conforms to the fluorescence emission characteristics of NADH, indicating the formation of NADH after the water-soluble alloy nanoclusters react with NAD.

[0061] Figure 9 is the electrospray ionization mass spectrum of the NADH product obtained by ultrafiltration after the reaction of the water-soluble alloy nanoclusters with NAD in the present invention. The black line is the experimental spectrum, and the red line is the simulated spectrum. The two match well, confirming the formation of NADH.

[0062] Figure 10 is the ultraviolet absorption spectrum of the NADH product obtained by ultrafiltration after the reaction of the water-soluble alloy nanoclusters with NAD in the present invention after enzymatic detection. It can be seen from the figure that the ultraviolet absorption spectrum of the generated NADH after enzymatic detection has an obvious ultraviolet absorption at 570 nm, which is consistent with the literature reports. It confirms the formation of NADH.

Claims

1. Preparation method of thioctic acid-protected water-soluble alloy nanomaterials, characterized in that it includes the following steps: Step 1: Add thioctic acid to ultrapure water, then add sodium hydroxide solution, and stir and mix evenly; Step 2: Add silver nitrate solution and chloroauric acid solution to the mixed solution in Step 1, add sodium hydroxide solution again, and stir and mix evenly; Step 3: After reacting for 15 minutes in Step 2, add sodium borohydride solution, and stir for a predetermined time to obtain a crude product reaction solution; Step 4: Filter the reaction solution obtained in Step 3 with a filter head, add the filtrate to an ultrafiltration tube for ultrafiltration, and obtain a concentrated solution of alloy nanomaterials after ultrafiltration; Step 5: Purify the concentrated solution obtained in Step 4 by polyacrylamide gel electrophoresis. Specifically, perform electrophoresis on the concentrated solution obtained in Step 4, collect the first band, soak it in ultrapure water for 2 h to obtain the purified aqueous phase product; add an equal volume of tetraoctylammonium bromide and toluene to the purified aqueous phase product, stir vigorously, let it stand for layering, collect the organic phase, wash it with ultrapure water 5-6 times, and collect the organic phase; spin-dry the collected organic phase, dissolve it in dichloromethane, and perform electrospray ionization mass spectrometry test to obtain the molecular formula, and obtain thioctic acid-protected water-soluble alloy nanomaterials; The thiomalic acid-protected water-soluble alloy nanomaterial is a gold-silver nanocluster, and its molecular formula is [Au 3 Ag 5 (MSA) 3 @5TOA-5H]Na, where MSA represents the ligand thiomalic acid; TOA represents the phase transfer agent tetraoctylammonium bromide.

2. The preparation method according to claim 1, characterized in that: In Step 1, the molar ratio of thioctic acid to sodium hydroxide is 3:

10.

3. The preparation method according to claim 1, characterized in that: In Step 2, the molar ratio of chloroauric acid to silver nitrate is 4:1; the molar ratio of chloroauric acid to sodium hydroxide is 0.135:1; the molar ratio of silver nitrate to sodium hydroxide is 0.034:

1.

4. The preparation method according to claim 1, 2 or 3, characterized in that: In the reaction system of Step 2, the molar ratio of silver nitrate to thioctic acid is 1:9.2; the molar ratio of chloroauric acid to thioctic acid is 1:2.

3.

5. The preparation method according to claim 1, characterized in that: In Step 3, the molar ratio of sodium borohydride to thioctic acid in Step 1 is 1:22.

35.

6. The preparation method according to claim 5, characterized in that: In Step 3, the reaction temperature is 25 °C, the reaction time is 4 h, and the pH of the reaction system is 11.

7. The preparation method according to claim 1, characterized in that: In Step 5, when purifying by polyacrylamide gel electrophoresis, prepare dispersion and stacking gels with 30% and 4% acrylamide monomers as raw materials; the parameters of electrospray ionization mass spectrometry are anion mode, source voltage 2.4 kV, source temperature 80 °C, and cone voltage 40.

8. Application of thioctic acid-protected water-soluble alloy nanomaterials prepared by the preparation method according to any one of claims 1-7, characterized in that: The thio-malate-protected water-soluble alloy nanomaterials are used to regulate the conversion of NAD to NADH. Specifically, in the presence of the thio-malate-protected water-soluble alloy nanomaterials, the conversion of NAD to NADH can be achieved in an aqueous solution at 20-25 °C; the molar ratio of the thio-malate-protected water-soluble alloy nanomaterials to NAD is 2:1.

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