CN117106189A - Metal organic framework artificial hydrolase and preparation method and application thereof - Google Patents

Metal organic framework artificial hydrolase and preparation method and application thereof Download PDF

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CN117106189A
CN117106189A CN202310881834.7A CN202310881834A CN117106189A CN 117106189 A CN117106189 A CN 117106189A CN 202310881834 A CN202310881834 A CN 202310881834A CN 117106189 A CN117106189 A CN 117106189A
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zinc
artificial
acid
tetrazole
hydrolase
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娄文勇
袁欣
吴晓玲
熊隽
刘姝利
宗敏华
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South China University of Technology SCUT
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/008—Supramolecular polymers
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
    • B01J31/1691—Coordination polymers, e.g. metal-organic frameworks [MOF]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
    • B01J31/22—Organic complexes
    • B01J31/2204—Organic complexes the ligands containing oxygen or sulfur as complexing atoms
    • B01J31/2208—Oxygen, e.g. acetylacetonates
    • B01J31/2217—At least one oxygen and one nitrogen atom present as complexing atoms in an at least bidentate or bridging ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
    • C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
    • C07C227/20—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters by hydrolysis of N-acylated amino-acids or derivatives thereof, e.g. hydrolysis of carbamates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
    • B01J2531/20—Complexes comprising metals of Group II (IIA or IIB) as the central metal
    • B01J2531/26—Zinc

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Abstract

The invention belongs to the field of nano biotechnology, and discloses a metal organic framework artificial hydrolase, a preparation method and application thereof. The artificial hydrolase is prepared from metal salt, amino acid and azole compound ligand, wherein the azole compound ligand does not participate in the formation of hydrogen bond between coordinated nitrogen atom or oxygen atom and the amino acid. The artificial hydrolase is a hydrogen bond mediated catalytic pathway which is formed by taking metal ions as active centers to mediate catalytic pathways and a secondary coordination environment to cooperatively catalyze the hydrolysis of amide bonds. Compared with natural enzymes, the metal organic framework artificial hydrolase provided by the invention has the characteristics of low-cost and easily available raw materials, high yield, high enzyme-like catalytic activity, good stability and the like, and can be used for completing the hydrolysis of amide compounds. Meanwhile, the artificial hydrolase can be repeatedly used for a plurality of times, can adapt to complex use environments, and provides a direction for industrial application.

Description

Metal organic framework artificial hydrolase and preparation method and application thereof
Technical Field
The invention belongs to the technical field of nano biology, and particularly relates to a metal organic framework artificial hydrolase with hydrolytic catalytic activity and activity which can be regulated and controlled through a second coordination sphere, and a preparation method and application thereof.
Background
Enzymes are highly efficient biomacromolecule catalysts that are widely distributed in living systems and have complex three-dimensional structures. The catalytic activity and specificity of enzymes are closely related to their complex active site, geometry and structure of functional amino acid residues. The hydrolase has important research and application values in both industrial fields and scientific research fields. Taking an amide bond as an example, the half-life period of the amide bond under the neutral condition of normal temperature and normal pressure is 350-600 years, so that the amide bond is difficult to break by itself. The natural hydrolase can realize the efficient cleavage of the amide bond in the room-temperature aqueous phase solution. For example, during food processing, ochratoxins are often readily produced with a strong toxicity and a broad distribution, creating a great threat to food safety and human health. Under the condition of aqueous solution, carboxypeptidase A can hydrolyze amide bonds in ochratoxin A with great toxicity, and the toxicity is obviously reduced. Therefore, the enzyme catalysis has wide application prospect in the fields of biological medicine, food industry, environmental management and the like.
However, most enzymes are expensive, and their high flexibility makes them susceptible to inactivation in practical application environments. Thus, increasing the stability of enzymes in an application environment is an important research direction of current academia and industry. Nanomaterial-based artificial enzyme design is one of the effective methods to overcome the limitations described above. Nano materials such as metal oxides, metal hybrids, carbon-based materials, graphene-based materials, metal Organic Frameworks (MOFs) and the like are found to have activities of mimic enzymes, and catalytic stability of the nano materials is remarkably improved compared with that of free natural enzyme molecules. Among them, MOFs have attracted a lot of attention due to their structural stability and high designability.
The versatility of metal-organic frameworks (MOFs) in metal nodes and organic ligands can mimic the active centers of natural enzymes, conferring MOFs with simulated enzymatic activity. In addition, MOFs have a rich and ordered pore structure that provides channels resembling the mass transfer of natural enzyme substrates. MOFs have outstanding characteristics of high specific surface area, excellent stability, rich porous characteristics and the like, so that MOFs become ideal carriers for preparing and researching artificial enzymes in recent years.
Although more than 100 of the chinese and english literature on artificial enzymes of MOFs is concerned, currently more than 90% of the MOFs artificial enzymes are concentrated on oxidoreductases (Huang X, zhang S, tang Y, zhang X, bai Y, pang h. Advances in metal-organic framework-based nanozymes and their applications, coordination Chemistry Reviews,2021, 449:214216.), and only a few MOFs-based artificial hydrolases have been developed (Wang S, ly HGT, wahiduzzaman M, et al a zirconium metal-organic framework with SOC topological net for catalytic peptide bond hydrolases, 2022,13 (1): 1284.; li S, zhou Z, tie Z, et al data-informed discovery of hydrolytic nanozymes. Nature communication, 2022,13 (1): 827.). However, the enzymatic activity of the artificial enzymes synthesized in the prior art is still far from satisfactory. This is mainly because of the high complexity of the structure of the native enzyme and its catalytic site, and how to construct artificial enzyme catalysts using simple chemical modules to mimic the chemical and electronic properties of the native enzyme activity pocket remains a major challenge. Currently, artificial hydrolases are mainly used for microenvironment control of metal single-node electronic states from the perspective of primary coordination spheres. However, the microenvironment regulation of the second coordination sphere, consisting of weak interactions (such as hydrogen bonds), is critical to the catalytic efficiency of the enzyme. Taking chymotrypsin as an example, the formation of tetrahedral intermediates formed by participation of hydrogen bonds in the second coordination sphere in the catalytic reaction process is key to efficient catalytic reaction of enzyme molecules. Therefore, the chemical environment of the second coordination sphere of the active site of the artificial hydrolase is accurately controlled, so that the efficient regulation of the activity of the artificial hydrolase is further improved and hopefully realized.
Disclosure of Invention
The invention provides a method for preparing metal organic framework artificial hydrolase based on bionic design. The method comprises the step of self-assembling metal ions and amino acid and azole compound ligands into MOF by adopting a one-step or two-step method in a solution. Wherein the metal center is used as a Lewis acid site catalytic site; the amino acid and azole ligand provide hydrogen bond donors and acceptors, forming a hydrogen bond mediated catalytic center.
The invention utilizes the structural advantages of MOFs to simulate and construct the active centers of two different types of hydrolases, improves the hydrolysis catalysis efficiency, and compared with artificial enzymes prepared by using traditional nano materials (such as metal oxides, carbon nano materials, noble metal nano particles and the like), the artificial enzymes obtained by the invention have definite chemical compositions and structures, and can more clearly research the catalysis mechanism. Can provide theoretical reference for the commercial development of different types of artificial hydrolytic enzymes.
The invention provides a metal organic frame artificial hydrolase, which is a metal organic frame material containing amino acid and azole compound ligand, and has hydrolase catalytic activity; in the artificial hydrolase, amino acid and azole compound ligand are combined with metal ions in a coordination form, and functional groups on the amino acid can form hydrogen bonds with nitrogen atoms or oxygen atoms which do not participate in coordination on the azole compound ligand.
The invention provides a preparation method of the metal organic framework artificial hydrolase, which comprises the following specific preparation steps:
firstly, dissolving a metal compound and an azole compound in a solvent together, and performing ultrasonic treatment until the metal compound and the azole compound are completely dissolved; then adding an aqueous solution of amino acid, and performing ultrasonic treatment until the solution is clear; the mixed solution was transferred to a teflon autoclave and reacted for a while with heating. After the reaction, cooling to room temperature, filtering to obtain powdery or crystalline products, and cleaning with N, N-dimethylformamide and methanol for 3 times respectively. The collected precipitated product was incubated overnight in methanol and filtered. And (5) drying the obtained precipitate in a drying oven to obtain the metal organic framework artificial hydrolase.
According to the present invention, different types of azole ligands can be used.
The azole compound ligand can be imidazoles, triazoles and tetrazoles.
The skilled artisan understands that the nitrogen atoms which remain after the MOF is assembled by coordination of the azole compound ligand and the metal ion and are not involved in coordination have a close relationship with the formation of hydrogen bonds.
The azole compound ligand can realize ordered coordination of MOF, and residual uncomplexed nitrogen atoms on the ligand can be used as acceptors for hydrogen bond formation. Therefore, the proper type of azole compound ligand is selected, so that the hydrogen bond mediated active center can be constructed efficiently, and the catalytic efficiency is improved remarkably.
According to the invention, different types of amino acid ligands may be used.
The amino acid ligand is an acidic amino acid, a neutral amino acid or a basic amino acid. The nature of the amino acid determines whether or not the metal ion coordinates successfully to form a MOF, and the uncoordinated amino acid does not enhance catalytic activity. Meanwhile, the length of the amino acid, the terminal functional group, and the configuration of the amino acid are all related to the catalytic efficiency of the hydrogen bond-mediated active center.
The present invention advantageously selects suitable amino acid ligands that can both stably coordinate to a metal to form a MOF, and whose functional groups at the amino acid end can also provide donors for hydrogen bond formation. The polarization of the hydrogen bond can make nucleophilic groups in the catalytic center have stronger attack action, and rapidly hydrolyze target substrates. For example, amino acids with hydroxyl groups at the ends, with nucleophilic oxygen, can allow for artificial hydrolytic enzyme designs with hydrogen bond mediated catalytic centers.
According to the present invention, different metal compounds may be used as the metal center. In some embodiments, the metal compound is a zinc source compound.
The zinc source compound is a metal salt, a metal oxide, or a metal complex. By varying the type of zinc source, the catalytic activity of the mimetic can be reasonably tuned. Determination of a suitable zinc source facilitates the preparation of artificial enzymes with high catalytic properties. The catalytic activity of artificial hydrolytic enzymes is highly affected by zinc salt anions, and different zinc salts have a larger influence on the MOF crystal structure.
In other embodiments, iron, cobalt, copper, nickel and manganese metal salts are used in place of zinc salts.
Therefore, the invention provides a preparation method for producing MOF artificial hydrolase with double catalytic active centers, wherein a metal center catalytic site formed by coordination of a metal center and an azole compound ligand can simulate a Lewis acid catalytic center of metalloprotease at the same time, and a hydrogen bond mediated catalytic center assembled by amino acid and the azole compound ligand can simulate a catalytic triplet in an enzyme molecule. The assembly mode of the double ligands and the metal successfully simulates the metal Lewis acid site and the hydrogen bond mediated active site of the similar catalytic triplet in the natural hydrolase to realize the collaborative catalysis of the double catalytic centers, so that the MOF-based artificial hydrolase has excellent catalytic activity, high stability and good repeated use performance in an extreme operating environment, and provides a new technology for biomimetic catalysis, environmental repair and biosensors under the industrial scale condition.
The key technical problem to be solved in the construction of the artificial enzyme is how to precisely control the chemical environment of the second coordination sphere of the active site of the artificial enzyme hydrolase to improve the catalytic activity. The organic ligand is selected based on the coordination environment around the active site of the bionic natural enzyme. Based on the zinc metal center of the natural carboxypeptidase and the secondary amino acid ligand environment, the invention selects proper amino acid and azole compounds as organic ligands to realize double-ligand metal-organic framework synthesis, namely, hydroxyl groups in the amino acid and nitrogen in the azole compounds form hydrogen bonds, oxygen in the hydroxyl groups are activated to form oxygen anion active groups to construct hydrogen bond mediated active sites, and double-site synergistic catalysis is realized with metal Lewis active sites. Meanwhile, the reasonable collocation of the amino acid ligand and the azole compound ligand, the adjustment of the synthesis proportion of the double ligand and the metal compound, the regulation and control of the reaction temperature and other processes further realize the reasonable design and accurate regulation and control of the metal organic framework artificial hydrolase secondary coordination sphere microenvironment.
Compared with the prior art, the invention has the following advantages:
(1) Compared with the trial-and-error synthesis mode, the product of the invention realizes 3-21 times of catalytic activity improvement based on the accurate regulation and control of the artificial enzyme bionic secondary ligand environment.
(2) The reasonable collocation of the double ligand and the metal successfully imitates the active center of the natural enzyme, and realizes the construction and the synergistic catalysis of double active sites.
(3) The metal organic frame artificial hydrolase synthesized by the method has excellent catalytic activity and stability, can adapt to various complex severe reaction environments, and realizes the efficient degradation of amide compounds.
The amide compound comprises ochratoxin A, ochratoxin B, chlorpropham, acetaminophen, N-acetyl-L-phenylalanine, N-Boc-L-phenylalanine, N-acetyl-L-cysteine, L-alanyl-L-tyrosine, N-acetyl-L-histidine and N-acetyl-m-toluidine, glutamic acid-serine dipeptide, glutamic acid-aspartic acid dipeptide, glutamic acid-threonine dipeptide, glutamic acid-lysine dipeptide, glutamic acid-glutamic acid dipeptide, glutamic acid-arginine dipeptide, glutamic acid-serine dipeptide, metazachlor, acetochlor, metolachlor, dichlormid, boscalid, cycloxaprin, metalaxyl, tebufenpyrad, flubendiamide, chlorantraniliprole, N- (2, 6-diethylphenyl) -N-butoxymethyl chloroacetamide, fluopyram, acrylamide, bovine serum protein, casein, whey protein, and myoglobin.
Drawings
FIG. 1 shows the hydrogen nuclear magnetic resonance spectrum of ZAF synthesized in example 1 1 H NRM), where b is a partial enlargement of a.
Fig. 2 shows a Scanning Electron Microscope (SEM) image of the surface morphology of ZAF synthesized in example 1.
FIG. 3 shows the X-ray diffraction XRD (a) and Fourier infrared spectrum FT-IR (b) of ZAF synthesized in example 1.
Fig. 4 shows the X-ray diffraction (XRD) pattern of ZAF (amino acid) synthesized in example 2.
FIG. 5 shows the hydrogen nuclear magnetic resonance spectrum of ZAF (serine) synthesized in example 2 1 H NRM), wherein b and c are aA partial enlarged view.
FIG. 6 shows a Fourier infrared spectrum (FT-IR) of ZAF (serine) synthesized in example 2.
Fig. 7 shows X-ray diffraction (XRD) patterns of different ZAFs (amino acids).
Fig. 8 shows different Zn: azole compound ligand: x-ray diffraction (XRD) pattern of ZAF (amino acid) prepared according to the synthesis ratio of amino acid.
Figure 9 shows the X-ray diffraction (XRD) patterns of different molar amounts of modified ZAF synthesized X-amino acids @ ZAF.
Fig. 10 shows the X-ray diffraction (XRD) patterns of different ZAFs (serine) (azoles).
Fig. 11 shows Scanning Electron Microscopy (SEM) images of different ZAFs (serine) (azoles).
Fig. 12 shows the ZAF versus ZAF (amino acid) catalytic activity.
FIG. 13 shows the catalytic activity of different serine to zinc molar amounts in xserine @ ZAF.
Fig. 14 shows the effect of different temperatures of ZAF and ZAF (serine) on catalytic activity.
Fig. 15 shows a comparison of catalytic activity of ZAF (serine) reactions and incubations at 80 ℃.
FIG. 16 shows the effect of different concentrations of guanidine hydrochloride on ZAF (serine) catalytic activity.
Fig. 17 shows the catalytic efficiency of ZAF (serine) compared to native carboxypeptidase hydrolysis of ochratoxin a.
Detailed Description
The present invention provides a method for preparing a metal organic framework artificial hydrolase with double catalytic active centers. And extends into most MOF metals. Other compositions of MOFs useful in the present invention are not particularly limited so long as they can be prepared by preparing a dual ligand MOF, having both lewis acidic metal active centers and hydrogen bond-mediated active centers formed by the azole and amino acid.
The preparation method of the metal organic frame artificial hydrolase takes amino acid, azole compound ligand and metal compound as raw materials, and assembles and coordinates metal ions with the amino acid and the azole compound ligand under the solution condition by a solvothermal method to form the metal organic frame artificial hydrolase with a stable structure.
The MOF-based artificial hydrolase according to the present invention comprises a metal compound having at least one metal compound, at least two organic ligands. The two organic ligands are of different types, namely azole compound ligand and amino acid ligand.
As used herein, "metal compound" means a chemical moiety containing at least one atom or ion of at least one zinc source compound, iron source compound, cobalt source compound, copper source compound, nickel source compound and manganese source compound. Thus, the expression of metal compounds includes, for example, salts, oxides and complexes.
Suitable metal source compounds forming part of the MOF artificial enzyme may be selected from the group consisting of metal salts, metal oxides, metal complexes of zinc, iron, cobalt, copper, nickel or manganese in combination with different anionic groups, and combinations thereof. The zinc source compound is selected from, but not limited to, zinc sulfate (ZnSO) 4 ) Zinc acetate (Zn (OAc) 2 ) Zinc oxide (ZnO), zinc nitrate (Zn (NO) 3 ) 2 ) Zinc chloride (ZnCl) 2 ) Zinc perchlorate (Zn (ClO) 4 ) 2 ) Zinc carbonate (ZnCO) 3 ) Zinc dihydrogen phosphate (ZnH) 2 PO 4 ) Zinc hydroxide (Zn (OH) 2 ) Zinc citrate, zinc phosphate, zinc lactate, zinc phthalocyanine, zinc oxalate, zinc methacrylate, zinc borate, diisopropyl zinc, diethyl zinc, zinc fluoroborate, disodium zinc ethylenediamine tetraacetate. The iron source compound is selected from, but not limited to, ferric chloride (FeCl) 3 ) Ferrous chloride (FeCl) 2 ) Ferric nitrate (Fe (NO) 3 ) 3 ) Ferric sulfate (Fe) 2 (SO 4 ) 3 ). The cobalt source compound is selected from but not limited to zirconium chloride (CoCl) 2 ) Cobalt bromide (CoBr) 2 ) Cobalt iodide (CoI) 2 ) Cobalt hydroxide (Co (OH) 2 ) Cobalt carbonate (CoCO) 3 ) Cobalt nitrate (Co (NO) 3 ) 2 ) Cobalt sulfate (CoSO) 4 ). The copper source compound is selected from, but not limited to, copper acetate (Cu (OAc) 2 ) Copper sulfate (CuSO) 4 ) Copper chloride (CuCl) 2 ) Copper nitrate (Cu (NO) 3 ) 2 ). The nickel source compound is selected from, but not limited to, nickel chloride (NiCl) 2 ) Nickel sulfate (NiSO) 4 ) Nickel nitrate (Ni (NO) 3 ) 2 ) Nickel bromide (NiBr) 2 ) Nickel hydroxide (Ni (OH) 2 ). The manganese source compound is selected from, but not limited to, manganese chloride (MnCl) 2 ) Manganese acetate (Mn (OAc) 2 ) Manganese sulfate (MnSO) 4 ) Manganese nitrate (Mn (NO) 3 ) 2 ). The chromium source compound is selected from, but not limited to, chromium nitrate (Cr (NO 3 ) 3 ) Chromium chloride (CrCl) 3 ) Chromium sulfate (Cr) 2 (SO 4 ) 3 ) Chromium acetate (Cr (OAc) 3 )。
The azole ligand organic ligands suitable for the purposes of the present invention may be selected from, but are not limited to, imidazole, 2-methylimidazole, 2-nitroimidazole, 2-bromo-1H-imidazole, imidazole-2-carboxylic acid ethyl ester, 2-mercaptoimidazole, 2-propylimidazole, 2-ethylimidazole, 2-butylimidazole, 1H-imidazole-4-carboxylic acid, 2-methyl-5-nitroimidazole, brominated 1-octyl-3-methylimidazole, imidazole-4, 5-dicarboxylic acid, 4-phenylimidazole-5-amino-4-imidazole carboxamide, imidazole-4-carboxylic acid methyl ester, 4-nitroimidazole, 4- (hydroxymethyl) imidazole, 4-chloroimidazole, 4-methyl-5-hydroxymethylimidazole, 4-imidazolecarboxaldehyde, 4-imidazolecarboxylic acid ethyl ester, 2-imidazolecarboxaldehyde, benzimidazole, 5-amino-2-methylbenzimidazole, 2- (methylthiobenzimidazole), 2-chloro-4-fluoro-1H-benzimidazole, 2-mercapto, 5-aminobenzimidazole, 5-methylbenzimidazole, 5, 6-dimethylbenzimidazole, 2-methyl-2-imidazole, 2-fluoro-1H-benzimidazole, 2-mercapto-benzimidazole, 5-methylbenzimidazole, piperidine-carboxylic acid ethyl-2-imidazole, 4-methyl-imidazole, 5-carboxybenzimidazole, 1H-benzimidazole-2-sulfonic acid, 6-nitrobenzimidazole, benzimidazole-7-carboxylic acid methyl ester, 4,5,6, 7-tetrahydro-1H-benzimidazole-5-carboxylic acid, 4-aminobenzimidazole, 5-methoxybenzimidazole, 6-bromo-1H-benzimidazole-4-carboxylic acid, 1,2, 4-triazole, 3-amino-5-methyl-4H-1, 2, 4-triazole, 3-amino-5-methylsulfanyl-1H-1, 2, 4-triazole, 3, 5-dimethyl-1, 2, 4-triazole, 1-bromo-1H-1, 2, 4-triazole-3-carboxylic acid ethyl ester, ethyl-1H-1, 2, 4-triazol-5-ylacetic acid ester, 1-methyl-1, 2, 4-triazole, 3-chloro-1, 2, 4-triazole, 1,2, 3-nitro-2H-1, 2, 3-benzotriazole, 1-hydroxybenzotriazole, 5-methylsulfanyl-1H-2, 4-benzotriazole, 7-bromo-1H-1, 2, 4-triazole-3-carboxylic acid ethyl-1H-1, 2, 4-triazole, 1-methyl-1, 2, 4-triazole, 3-chloro-1, 2, 3-triazole, 1, 3-hydroxy-2, 5-benzotriazole, 7-hydroxy-5-hydroxy-2, 7-2-triazole, 7-carboxylic acid ethyl-1, 6-bromo-1, 4-triazole, 1H-tetrazole, 1H-tetrazole-5-acetic acid, 5- (4-pyridyl) -1H-tetrazole, 5- (3-pyridyl) -1H-tetrazole, 1H-tetrazole-5-acetic acid ethyl ester, 1-methyl-1H-tetrazole, 5- (ethylsulfanyl) -1H-tetrazole, 5- (4-nitrophenyl) -1H-tetrazole, 5- (2-pyridyl) -1H-tetrazole, 5-chloromethyl-1H-tetrazole, 5- (4-carboxyphenyl) -1H-tetrazole, 8-chlorotetrazole [1,5-A ] pyridine, 5-propyl-1H-tetrazole, 6-bromotetrazolo [1,5-a ] pyridine, 3- (1H-tetrazole) benzaldehyde, 3-tetrazol-1-yl-phenol, 5- (3-methoxyphenyl) -1H-tetrazole, 2-methyl-5- (1H-tetrazole-5-yl) aniline, 5- (4-hydroxyphenyl) -1H-tetrazole, 5- (2-methylphenyl) -1H-tetrazole, 5-methylphenyl) -1H-tetrazole, 3-methyl-5-yl-4-phenyl-tetrazole, 3-methyl-1H-tetrazole, 3-methyl-4-yl-phenyl-4-tetrazole, 1H-methyl-4-yl-tetrazole, methyl 4- (2H-1, 2,3, 4-tetrazol-5-yl) benzoate, 5- (3-nitrophenyl) -2H-tetrazole, 1H-tetrazol-1-acetamide.
Amino acid ligands suitable for the purposes of the present invention may be selected from the group consisting of L-tyrosine, L-hydroxyproline, L-hydroxylysine, L-serine, L-glutamic acid, 4-hydroxy-L-glutamic acid, 3-hydroxy-L-glutamic acid, L-aspartic acid, S-2-hydroxyethyl-L-cysteine, S-hydroxy-L-cysteine, L-threo-3-hydroxyaspartic acid, N (5) -hydroxy-L-arginine, L-threonine, L-homoserine, N- (t-butoxycarbonyl) -L-homoserine, N-benzyloxycarbonyl-L-homoserine, 2-methyl-L-serine, N-acetyl-L-serine, L-isoserine, L-6-hydroxynorleucine, 3-hydroxy-L-leucine, N-Boc-3-hydroxy-L-valine, L-BATE-hydroxyvaline, 5-hydroxy-L-tryptophan, glycyl-L-serine, glycyl-L-threonine, L-glycyl-L-serine, glycyl-L-tyrosine.
In some embodiments, solvents that may be used in synthesizing MOFs include at least one of N, N-dimethylformamide, N-dimethylacetamide, water, methanol, ethanol, terephthalic acid, diethylformamide, toluene, chlorobenzene, or a mixed solution thereof. The MOF artificial enzyme is prepared by adopting a solvothermal or hydrothermal method in a synthesis mode, and the solvent adopts a single solvent or a mixed solvent, so long as MOF can be formed, and the type of the solvent is not limited.
In hydrothermal or solvothermal synthesis, solvents play a critical role. The effect of the solvent on the metal-organic framework compound structure is mainly represented by the solubility of the reactant in the solvent, the polarity of the solvent itself, the coordination ability of the solvent and the templating agent effect. In the porous metal-organic framework compound, in order to avoid the interpenetration phenomenon caused by the fact that the space is too large, solvent molecules can be used as guest molecules to be filled in holes of the compound, and can also be used as templates, and the size and the shape of the volume of the solvent molecules can be used for inducing the formation of the metal-organic framework compound with different structures and functions.
In some embodiments, MOFs that are not easily formed with stable structures at 4, 20, 30, 40, 50, 60, 70, 80 ℃ require extended reaction times if MOF powders are obtained. And at 90, 100, 120, 130, 140, 160, 180, 200, 220 ℃, high-yield and high-quality MOF artificial hydrolytic enzyme can be obtained by controlling the reaction time for 12-168 hours. Therefore, the synthesis temperature of the MOF of the invention is controlled between 90 and 220 ℃.
In some embodiments, the methods according to the invention are used to synthesize MOFs using solvothermal methods. Depending on the type of MOF precursor used, suitable solvents and synthesis temperatures are chosen, and solvothermal synthesis of MOFs is known to take a long time, so that the degree of crystallization is analysed according to the XRD pattern of the synthesized MOFs, with a reaction time between 12 and 168 hours.
In some embodiments, a zinc azole backbone (ZAF) is used as an artificial hydrolase to mimic the active center of a natural metalloprotease. Since imidazole, whose enzyme active center is histidine, and carboxylic acid of amino acid generate stable coordination structure based on zinc coordination, the coordination of azole and zinc just mimics this structure. In addition, ZAF has high thermal stability and chemical stability, can stably keep the original state in water, so that the ZAF can well catalyze target substrates in water and can be recycled. The artificial enzyme which can be prepared by the invention can be a zinc imidazole skeleton, a zinc triazole skeleton and a zinc tetrazole skeleton, and the ligand can be imidazole, triazole and tetrazole.
The MOF artificial enzyme prepared by the invention contains micropores, mesopores or micro-mesopores which coexist, and the specific surface area is calculated based on Brunauer Emmet and Teller method (BET) fitting.
The micropores refer to MOFs with the pore size smaller than 2nm measured by a nitrogen adsorption method, the pore size of the mesoporous Kong Zhi is between 2 and 50nm, and the coexistence of the micropores and the mesopores refers to MOFs with two pore sizes.
In some embodiments, dual active site MOF artificial hydrolases are prepared using a coordination pattern of the azole ligand and the amino acid dual ligand with a metal salt (zinc). The molar amount of metal salt used, organic ligand may include the following ranges: between about 0.01mmol and 8mmol of metal salt; between about 0.01mmol and 8mmol of azole ligand; between about 0.01mmol and 8mmol of amino acid ligand.
According to the method of the invention, the MOF artificial hydrolase is prepared, and the artificial enzyme with target activity can be obtained only by adjusting the proportion of the organic ligand according to the proportion of the active part. Metal salt: azole compounds: the molar ratio of amino acids is in the following range: 1 to 16:1 to 16: 1-16.
Different MOF precursor ratios have a direct effect on the crystallinity and yield of MOFs, and meanwhile, due to the difference of different types of coordination capacities, the effect brought by precursors with different ratios is the content of active centers, so that the difference of the catalytic activity of artificial enzymes is caused.
Example 1
The synthesis of ZAF precursor includes the following steps:
zinc nitrate hexahydrate (1 mmol,297.5 mg) and benzotriazole (1 mmol,117.35 mg) were dissolved in a mixture of 30mL of N, N-dimethylformamide and 10mL of deionized water and sonicated for 5min. The mixture was then transferred to a teflon high pressure reactor and heated at 140 ℃ for 24h. After cooling to room temperature, the solid was collected by filtration. The powder was washed 3 times with DMF and methanol and dried at 60℃under vacuum.
The ZAF crystal properties were confirmed by NMR, SEM, PXRD and FT-IR measurements, and the coordination structure of ZAF was confirmed, as shown in FIGS. 1 to 3. The nuclear magnetic resonance data of ZAF in FIG. 1 indicate that ZAF contains benzotriazole ligands. The scanning electron microscope pictures of the ZAF in FIG. 2 show that the ZAF has an irregular morphology, with individual particle sizes of about 200nm. The X-ray powder diffraction of ZAF in fig. 3 a shows that the ZAF has a diffraction peak with high intensity and a good crystal structure. The Fourier infrared spectrum of ZAF in FIG. 3 b shows 550cm -1 Zn-N bond characteristic peak of 1278cm -1 ,1226cm -1 And 1170cm -1 The characteristic peaks of (2) represent the vibration of-N-or-n=n-in the benzotriazole, indicating successful coordination of the benzotriazole with zinc ions.
Example 2
The synthesis of ZAF (amino acid) comprises the following operation steps:
in order to simulate the active center structures of two natural hydrolases, amino acids are introduced to construct a catalytic microenvironment and a catalytic center. Zinc nitrate hexahydrate (1 mmol,297.5 mg) and benzotriazole (1 mmol,117.4 mg) were sonicated in 30mL DMF and then 10mL of aqueous amino acids (1 mmol) of serine, homoserine, threonine, glutamic acid, aspartic acid, etc. were added, respectively, and these amino acid types were listed above, and the synthesized product was designated as ZAF (amino acid), the specific name being determined by the amino acid type, such as ZAF (serine), ZAF (homoserine) … …. The mixture was heated at 140℃for 24h. After cooling to room temperature, the pale white powder was collected by centrifugation and washed three times with DMF and methanol. The collected precipitate was incubated overnight in methanol and then dried under vacuum at 120℃for 12h. The collected powder was used for catalytic hydrolysis of hippuric acid-L-phenylalanine to evaluate the catalytic activity.
After the introduction of serine, the formed ZAF (serine) crystals were confirmed to have peaks similar to those of ZAF using powder XRD, as shown in fig. 4. Furthermore, serine was successfully introduced into the ZAF framework using nuclear magnetic resonance hydrogen spectroscopy and fourier infrared spectroscopy, as shown in fig. 5 and 6. XRD of some other ZAFs (amino acids) was also examined, and as shown in fig. 7, the crystal diffraction peaks of some ZAFs (amino acids) were different after the introduction of the amino acids due to the difference in the nature and coordination ability of the different amino acids.
Example 3
To investigate the effect of zinc ions of different origins on the catalytic activity of synthetic artificial enzymes, MOF artificial hydrolases were prepared by varying the type of metal salt. 20 zinc salts, zinc oxides, zinc hydroxides or zinc complexes of different origins (these zinc source compound species are listed above) were dissolved in 10mL DMF and each zinc source compound was in separate experiments for a total of 20 experiments. Benzotriazole (1 mmol,117.4 mg) was then sonicated in 20mL DMF and an aqueous solution of the optimal amino acid of example 2 (100 mM,10 mL) was added. The two were mixed and sonicated for 3 minutes and the mixture was heated at 140 ℃ for 24h. After cooling to room temperature, the solid was collected by filtration. The powder was washed 3 times with DMF and methanol and dried at 60℃under vacuum. The hydrolytic catalytic activity was evaluated by hydrolyzing the model substrate hippuric acid-L-phenylalanine.
Example 4
In order to optimize the catalytic activity of the artificial hydrolase, the influence of different reactant ratios on the catalytic activity was investigated. The group with the best catalytic activity in example 3 was selected, and its synthesis process was consistent, only the zinc ions were changed: azole compounds: the molar ratio of amino acids is set to 1:1:1,4:4:3,2:2:1,4:3:4,4:3:3,4:4:2,2:1:2,4:2:3,2:1:1, 9 total groups. The obtained powder was evaluated for its hydrolysis catalytic activity by hydrolyzing the model substrate hippuric acid-L-phenylalanine.
Characterization of the crystal characteristic peaks of artificial hydrolases synthesized in different ratios using powder XRD, fig. 8 shows that changing the ratio of synthetic raw materials changes the structure of the crystal from a certain level, in which zinc ions: azole compounds: the molar ratio of amino acid is 4:3:4,4:3:3,2:1:2 and 2:1:1, the characteristic peak of ZAF becomes a low diffraction peak or disappears, indicating that the change in the ratio of the raw materials causes a partial change in the structure, and the selection of an appropriate ratio is very important for synthesizing the catalytic activity of artificial hydrolase.
Example 5
For synthesizing metal organic frames by solvothermal method, the selection of synthesis time is also very important. The group with the best catalytic activity in example 4 was selected, the synthesis procedure was identical, and only the synthesis time was changed. After reactions 12, 24, 48, 72, 96, 120h at 140 ℃ white powder or white crystals were obtained, respectively. The hydrolytic catalytic activity was evaluated by hydrolyzing the model substrate hippuric acid-L-phenylalanine.
Example 6
Amino acid @ ZAF
The amino acid @ ZAF was prepared by a two-step process using ZAF as the precursor of MOF, post-modified amino acids. L-serine, L-glutamic acid, L-homoserine, L-threonine, L-aspartic acid, L-tyrosine, 2-methyl-L-serine, L-isoserine, L-hydroxyproline, 4-hydroxy-L-glutamic acid, N-methyl-L-serine, N-acetyl-L-serine, L-isoserine, L-6-hydroxynorleucine, 3-hydroxy-L-leucine, 5-hydroxy-L-tryptophan, S-2-hydroxyethyl-L-cysteine, S-hydroxy-L-cysteine, N-Boc-3-hydroxy-L-valine, L-hydroxylysine were introduced into ZAF dispersion, and the precursor MOF was modified in solution using the above 20 amino acid solutions and two-times coordinated to form a double catalytic center, with each amino acid in a total of 20 experiments. First, 100mg of ZAF synthesized in example 1 was dispersed in 15mL of DMF, and then each amino acid (0.3 mmol,5 mL) was introduced into the above ZAF suspension. Transferring the mixture into a Teflon high-pressure reaction kettle, and reacting for 24 hours at 120 ℃. Naturally cooling to room temperature, filtering to obtain precipitate, washing with DMF and methanol for 3 times, oven drying at 60deg.C overnight, and collecting powder for catalytic hydrolysis of hippuric acid-L-phenylalanine to evaluate catalytic activity.
Example 7
x amino acid @ ZAF
In order to study the influence of the catalytic center of the constructed amino acid with different molar contents on the catalytic activity of the artificial hydrolase, the amino acid @ ZIF with the best activity in example 3 is selected, and x amino acid @ ZAF is prepared by adjusting the molar amount of the different amino acids, wherein x represents the molar amount (mmol/g) of the amino acid added per g of zinc ions in the synthesis process. First, different molar amounts of amino acid were dissolved in 5mL of deionized water and then added to DMF suspension containing 100mg ZAF. The mixture was transferred to a teflon autoclave and reacted for 24 hours at 120 ℃. Naturally cooling to room temperature, filtering to obtain precipitate, washing with DMF and methanol for 3 times, oven drying at 60deg.C overnight, and collecting powder. The amount of amino acid contained in the powder is obtained by inductively coupled plasma emission spectroscopy (ICP-OES), elemental Analysis (EA), photoelectron spectroscopy (XPS), and the like.
The peaks of the crystalline forms of xserine@ZAF synthesized by modifying ZAF with different molar amounts of serine were determined by powder XRD, while the peaks of ZAF (serine) were aligned. As shown in FIG. 9, the characteristic peaks of serine @ ZAF and ZAF synthesized by post-modified serine are consistent, and have good crystal structures, which indicate that the introduction of amino acid does not cause defects and changes of the parent ZAF structure. As the serine content is improved, the position of the XRD characteristic peak is not changed obviously, the structure of the parent ZAF is not changed by introducing amino acid, and the post-modification method is proved to be the same as the one-step synthesis method, and the crystal form structure of the parent ZAF is not influenced by introducing amino acid.
Example 8
To investigate the influence of different azole ligands and functional groups on the catalytic activity of artificial hydrolase, ZAF (serine) with the best activity in example 2 was selected, and based on this, the azole ligand was replaced to prepare ZAF (serine) (X), X representing the azole ligand. The best amino acid from example 2, the best zinc source from example 3, and the best synthesis ratio from example 4 were selected. Different azole ligands (1 mmol) and zinc salts (1 mmol) were dissolved by sonication in 30mL DMF and an aqueous solution of serine (100 mM,10 mL) with optimal catalytic activity as in example 2 was added. The mixture was heated at 140℃for 24h. After cooling to room temperature, the solid was collected by filtration. The powder was washed 3 times with DMF and methanol and dried at 60℃under vacuum. The hydrolytic catalytic activity was evaluated by hydrolyzing the model substrate hippuric acid-L-phenylalanine.
Powder XRD characterizes the crystal characteristics of a portion of azole ligands (fig. 10), SEM-characterized surface morphology (fig. 11), different azole ligands have different solubilities and functional groups, and also have different coordination abilities with metals, thus also causing differences in their crystal structure, synthesis yield and catalytic activity. The specific embodiment is ZAF (serine) (5-methylbenzotriazole), ZAF (serine) (1, 2, 4-triazole) is of an amorphous structure, ZAF (serine) (benzotriazole), ZAF (serine) (5-carboxybenzotriazole), ZAF (serine) (1-hydroxybenzotriazole) and ZAF (serine) (benzimidazole) have better crystal structure, wherein ZAF (serine) (5-methylbenzotriazole) and ZAF (serine) (1-hydroxybenzotriazole) show more regular morphology, and the rest concentration is irregular particle aggregation.
Example 9
In order to explore the versatility of constructing metallo-organic framework artificial enzymes with active sites, M-ZAF is prepared by replacing metal centers, M is different metals (Fe, co, cu, ni, mn), the synthesis method refers to ZAF, and only the types of metal salt compounds are changed: copper, iron, cobalt, nickel, manganese metal salt compound (1 mmol) and benzotriazole (1 mmol) were uniformly dissolved in a mixture of 30mL of N, N-dimethylformamide and 10mL of deionized water, and sonicated for 5min. The mixture was then transferred to a teflon reactor and heated at 140 ℃ for 24h. After cooling to room temperature, the solid was collected by filtration. The powder was washed 3 times with DMF and methanol and dried at 60℃under vacuum to give M-ZAF as a solid powder.
Example 10
In order to explore the versatility of constructing multienzyme active metallo-organic framework artificial enzymes by different metal centers, M-ZAF (serine) is prepared by replacing the metal centers, M is different metals (Fe, co, cu, ni, mn), and the synthesis method refers to ZAF (amino acid). The active center structures of two natural hydrolases are simulated, and the catalytic microenvironment and the catalytic center are constructed by introducing amino acid. Iron, cobalt, copper, nickel, manganese metal salt compound (1 mmol) and benzotriazole (1 mmol,117.4 mg) were dissolved in 30mL DMF, and then 10mL of aqueous serine (1 mmol) was added, respectively. The mixture was heated at 140℃for 24h. After cooling to room temperature, the pale white powder was collected by centrifugation and washed three times with DMF and methanol. The collected precipitate was incubated overnight in methanol and then dried under vacuum at 120℃for 12h, designated M-ZAF (serine). The collected powder was used for catalytic hydrolysis of hippuric acid-L-phenylalanine to evaluate the catalytic activity.
Comparative example 1
Comparison of catalytic activity of pure ZAF and ZAF (amino acid) introduced with amino acid.
A batch of pure ZAF and amino acid-introduced ZAF (amino acids) was prepared according to the standard procedure described in example 1 and example 2 herein. 6mg of ZAF and ZAF (amino acid) were weighed and added to 3mL of Tris-HCl solution (pH 6.0) containing 1mM hippuric acid-L-phenylalanine, and reacted at 40℃for 40min. The product hippuric acid concentration is detected by HPLC, and the catalytic activity is evaluated and compared. As shown in fig. 12, the introduction of amino acids to prepare ZAF (amino acids) enhanced catalytic activity to a different extent than pure ZAF, and also showed that the selection of amino acids had an important influence on catalytic activity. The catalytic activity of ZAF (serine), ZAF (homoserine) and ZAF (threonine) is 2-3 times that of ZAF, which also proves that the active site mediated by hydrogen bond formed by hydroxyl in amino acid and nitrogen of azole ligand and the metal Lewis acid site synergistically catalyze to enhance the hydrolysis efficiency.
Comparative example 2
To confirm that the difference in ZAF and ZAF (amino acid) catalytic activity derives from the hydrogen bond-mediated catalytic active sites conferred by the amino acids. Amino acids were introduced into ZAF and serine @ ZAF was synthesized by the secondary high Wen Xiushi. The hydroxyl at the tail end of serine can form an effective hydrogen bond with the azole compound, and the oxygen of the hydroxyl becomes nucleophilic oxygen anion through the activation of the hydrogen bond, so that a hydrogen bond-mediated active site is constructed. As shown in fig. 13, the catalytic activity was also significantly improved with the increase of the introduced amount of serine in ZAF, which proves that the reason for the improvement of the catalytic activity is that the introduction of serine can obtain more excellent catalytic activity than ZAF because of the hydrogen bond-mediated active site constructed by the amino acid introduction.
Comparative example 3
To confirm the role of its hydrogen bonds in the catalytic sites, the effect of ZAF and ZAF (amino acid) on catalytic activity was compared under different temperature conditions and different concentrations of guanidine hydrochloride conditions to destroy the hydrogen bonds. 6mg of ZAF and ZAF (amino acids) were added to 3mL of Tris-HCl solution (pH 6.0) containing 1mM hippuric acid-L-phenylalanine, respectively, and the reaction temperatures were set at 20, 30, 40, 50, 60, 70, 80℃for 40min. In the same experimental operation, guanidine hydrochloride with different concentrations is added into the reaction system.
As shown in FIG. 14, the catalytic activity of ZAF does not change much in the temperature range of 20-80 ℃, indicating that ZAF has higher thermal stability. Whereas for ZAF (serine) the hydrolysis reaction rate of HPPA drops sharply (70%) when the temperature exceeds 70 ℃, indicating that the enhancement of activity by the addition of serine is lost under the complete structure of ZAF (serine). The activity of ZAF (serine) decreases sharply, whereas the catalytic activities of pure ZAF and pure ZAF (serine) are the same, probably due to the absence of hydrogen bonds between serine and BTA at high temperatures. In addition, ZAF (serine) was incubated at 80℃for 1h, and after cooling, the catalytic activity was re-examined. As shown in fig. 15, the high temperature reaction at 80 ℃ caused the hydrogen bond to break, resulting in the catalytic activity to drop close to that of ZAF, and after incubation and cooling, the catalytic activity was restored to the same as that of ZAF (serine), indicating that the hydrogen bond in ZAF (serine) has good reversibility. Similar phenomena were also observed when ZAF (serine) and ZAF were exposed to guanidine hydrochloride (gdnchi) treatment (fig. 16). As the concentration of GdnHCl increases, the rate of ZAF (serine) -catalyzed HPPA hydrolysis reaction gradually decreases to 3.07. Mu.M.min -1 Similar to pure ZAF. Whereas ZAF also shows similar catalytic activity in the presence of gdn hcl.
Comparative example 4
ZAF (amino acid) compared with the catalytic Activity of native carboxypeptidase A
In practical application, ochratoxin A with high toxicity and wide pollution range is used as a hydrolysis substrate. Ochratoxin A (OTA) (250. Mu.g.mL) -1 40. Mu.L) of methanol solution was slowly added to 1.96mL of Tris-HCl solution (pH 6.0) with stirring to give a final concentration of 5. Mu.g/mL of OTA. To 2mL of the substrate solution, 2mg of ZAF (serine) and an equal mass of carboxypeptidase A protein were added, and the reaction was darkened with stirring at 40℃for a period of time. After the reaction, methanol in the same amount is added into the substrate solution immediately, the catalyst is removed by centrifugal filtration, and the reaction is stopped. The concentration of the OTA remaining in the solution was measured by fluorescence spectroscopy, and the degradation efficiency of OTA was calculated.
Fig. 17 shows that as ochratoxin a concentration increases, the initial rate of ZAF (serine) is greater than that of native carboxypeptidase a, indicating that ZAF (serine) exhibits more excellent catalytic activity than native carboxypeptidase a.
The above examples merely represent several embodiments of the present invention, and the description thereof is more specific and detailed, but the technical scope thereof is not limited to the above embodiments. Various modifications and implementations of the invention will be apparent to those skilled in the art without departing from the spirit of the invention, and these modifications and implementations are intended to be within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. The metal organic frame artificial hydrolase is characterized in that the artificial hydrolase is a metal organic frame material containing amino acid and azole compound ligand, and has hydrolase catalytic activity; in the artificial hydrolase, amino acid and azole compound ligand are combined with metal ions in a coordination form, and functional groups on the amino acid can form hydrogen bonds with nitrogen atoms or oxygen atoms which do not participate in coordination on the azole compound ligand.
2. A method for preparing a metal organic framework artificial hydrolase according to claim 1, comprising: amino acid and azole compounds are used as organic ligands, metal compounds are used as metal centers, and metal ions are assembled and coordinated with the amino acid and azole compound ligands under the condition of solution by a solvothermal method to form the metal organic framework artificial hydrolase with stable structure.
3. The process according to claim 2, wherein the azole ligand is selected from the group consisting of imidazole, 2-methylimidazole, 2-nitroimidazole, 2-bromo-1H-imidazole, imidazole-2-carboxylic acid ethyl ester, 2-mercaptoimidazole, 2-propylimidazole, 2-ethylimidazole, 2-butylimidazole, 1H-imidazole-4-carboxylic acid, 2-methyl-5-nitroimidazole, brominated 1-octyl-3-methylimidazole, imidazole-4, 5-dicarboxylic acid, 4-phenylimidazole-5-amino-4-imidazole carboxamide, imidazole-4-carboxylic acid methyl ester, 4-nitroimidazole, 4- (hydroxymethyl) imidazole, 4-chloroimidazole, 4-methyl-5-hydroxymethylimidazole, 4-imidazolecarboxaldehyde, 4-imidazolecarboxethyl, 2-imidazolecarboxaldehyde, benzimidazole, 5-amino-2-methylbenzimidazole, 2- (methylthiobenzimidazole), 2-chloro-4-fluoro-1H-benzimidazole, 2-mercaptobenzimidazole, 5-aminobenzimidazole, 5-methylbenzimidazole, 5, 6-methylbenzimidazole, 2-chloro-4-fluoro-1H-benzimidazole, 2-mercaptobenzimidazole, 5-methylbenzimidazole, 2- (methylthiobenzimidazole) and piperidine-2-methyl-4-carboxylic acid methyl-4-methylimidazole, 4-methylbenzimidazole, 5-carboxybenzimidazole, 1H-benzimidazole-2-sulfonic acid, 6-nitrobenzimidazole, benzimidazole-7-carboxylic acid methyl ester, 4,5,6, 7-tetrahydro-1H-benzimidazole-5-carboxylic acid, 4-aminobenzimidazole, 5-methoxybenzimidazole, 6-bromo-1H-benzimidazole-4-carboxylic acid, 1,2, 4-triazole, 3-amino-5-methyl-4H-1, 2, 4-triazole, 3-amino-5-methylsulfanyl-1H-1, 2, 4-triazole, 3, 5-dimethyl-1, 2, 4-triazole, 1-bromo-1H-1, 2, 4-triazole-3-carboxylic acid ethyl ester, ethyl-1H-1, 2, 4-triazol-5-ylacetic acid, 1-methyl-1, 2, 4-triazole, 3-chloro-1, 2, 4-triazole, 1, 3-nitro-2H-1, 2, 3-triazole, benzotriazole, 1-hydroxy-benzotriazole, 5-hydroxy-benzotriazole, 7-bromo-1H-1, 2, 4-triazol-carboxylic acid ethyl-1H-1, 2, 4-triazol-5-carboxylic acid ethyl-1, 2, 4-hydroxy-benzotriazole, 6-bromo-5-carboxylic acid ethyl-4-hydroxy-benzotriazole, 6-hydroxy-1, 5-hydroxy-benzotriazole, 1H-1,2, 3-benzotriazol-5-ylboronic acid, 1H-tetrazole-5-acetic acid, 5- (4-pyridyl) -1H-tetrazole, 5- (3-pyridyl) -1H-tetrazole, 1H-tetrazole-5-acetic acid ethyl ester, 1-methyl-1H-tetrazole, 5- (ethylsulfanyl) -1H-tetrazole, 5- (4-nitrophenyl) -1H-tetrazole, 5- (2-pyridyl) -1H-tetrazole, 5-chloromethyl-1H-tetrazole, 5- (4-carboxyphenyl) -1H-tetrazole, 8-chlorotetrazole [1,5-A ] pyridine, 5-propyl-1H-tetrazole, 6-bromotetrazolo [1,5-a ] pyridine, 3- (1H-tetrazole) benzaldehyde, 3-tetrazol-1-yl-phenol, 5- (3-methoxyphenyl) -1H-tetrazole, 2-methyl-5- (1H-tetrazole-5-yl) aniline, 5- (2-chlorophenyl) -1H-tetrazole, 5- (4-carboxyphenyl) -1H-tetrazole, 5-methylphenyl) -1H-tetrazole, 2-methylphenyl-propionic acid, 3-methoxy-5- (5-methyl-tetrazol-1-yl) -aniline, methyl 4- (2H-1, 2,3, 4-tetrazol-5-yl) benzoate, 5- (3-nitrophenyl) -2H-tetrazol, 1H-tetrazol-1-acetamide.
4. The method according to claim 2, wherein the amino acid ligand is selected from the group consisting of L-tyrosine, L-hydroxyproline, L-hydroxylysine, L-serine, L-glutamic acid, 4-hydroxy-L-glutamic acid, 3-hydroxy-L-glutamic acid, L-aspartic acid, S-2-hydroxyethyl-L-cysteine, S-hydroxy-L-cysteine, L-threo-3-hydroxyaspartic acid, N (5) -hydroxy-L-arginine, L-threonine, L-homoserine, N- (t-butoxycarbonyl) -L-homoserine, N-benzyloxycarbonyl-L-homoserine, 2-methyl-L-serine, N-acetyl-L-serine, L-isoserine, L-6-hydroxynorleucine, 3-hydroxy-L-leucine, N-Boc-3-hydroxy-L-valine, L-BATE-hydroxyvaline, 5-hydroxy-L-tryptophan, glycyl-L-serine, glycyl-L-tyrosine, and glycyl-L-threonine.
5. The method of claim 2, wherein the metal compound comprises a compound containing at least one atom or ion of at least one zinc source compound, iron source compound, cobalt source compound, copper source compound, nickel source compound, and manganese source compound, or a combination of a plurality of compounds; the zinc source compound is selected from zinc sulfate, zinc acetate, zinc oxide, zinc nitrate, zinc chloride, zinc perchlorate, zinc carbonate, zinc dihydrogen phosphate, zinc hydroxide, zinc citrate, zinc phosphate, zinc lactate, zinc phthalocyanine, zinc oxalate, zinc methacrylate, zinc borate, diisopropyl zinc, diethyl zinc, zinc fluoroborate, disodium zinc ethylenediamine tetraacetate; the iron source compound is selected from ferric chloride, ferrous chloride, ferric nitrate and ferric sulfate; the cobalt source compound is selected from zirconium chloride, cobalt bromide, cobalt iodide, cobalt hydroxide, cobalt carbonate, cobalt nitrate and cobalt sulfate; the copper source compound is selected from copper acetate, copper sulfate, copper chloride and copper nitrate; the nickel source compound is selected from nickel chloride, nickel sulfate, nickel nitrate, nickel bromide and nickel hydroxide; the manganese source compound is selected from manganese chloride, manganese acetate, manganese sulfate and manganese nitrate; the chromium source compound is selected from chromium nitrate, chromium chloride, chromium sulfate, and chromium acetate.
6. The method according to claim 2, wherein the solvent used for synthesizing the metal organic framework artificial hydrolase is at least one selected from the group consisting of N, N-dimethylformamide, N-dimethylacetamide, water, methanol, ethanol, terephthalic acid, diethylformamide, toluene, chlorobenzene, and a mixed solution thereof.
7. The preparation method according to claim 2, wherein the synthesis temperature of the metal organic framework artificial hydrolase is controlled to be 90-220 ℃ and the reaction time is controlled to be 12-168 h.
8. The method of claim 2, wherein the metal compound: azole compounds: the molar ratio of the amino acid is 1-16: 1 to 16:1 to 16.
9. Use of the metal organic framework artificial hydrolase according to claim 1 for hydrolyzing an amide bond in an amide compound.
10. The use according to claim 9, wherein said amide compound comprises ochratoxin a, ochratoxin B, chlorpropham, paracetamol, N-acetyl-L-phenylalanine, N-Boc-L-phenylalanine, N-acetyl-L-cysteine, L-alanyl-L-tyrosine, N-acetyl-L-histidine and N-acetyl-m-toluidine, glutamic acid-serine dipeptide, glutamic acid-aspartic acid dipeptide, glutamic acid-threonine dipeptide, glutamic acid-lysine dipeptide, glutamic acid-glutamic acid dipeptide, glutamic acid-arginine dipeptide, glutamic acid-serine dipeptide, alachlor, acetochlor, isopropamide, dichlormid, cyproconazole, metalaxyl, tebufenpyrad, tolylamide, flubendiamide, N- (2, 6-diethylphenyl) -N-butoxymethylacetamide, fluoxalactam, acrylamide, serum albumin, casein, whey protein.
CN202310881834.7A 2023-07-18 2023-07-18 Metal organic framework artificial hydrolase and preparation method and application thereof Pending CN117106189A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115611813A (en) * 2022-11-09 2023-01-17 天津大学 Preparation method and application of artificial enzyme with carboxylesterase and carbonic anhydrase double-enzyme activities

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115611813A (en) * 2022-11-09 2023-01-17 天津大学 Preparation method and application of artificial enzyme with carboxylesterase and carbonic anhydrase double-enzyme activities

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