CN115947700A - Novel amidase chromogenic substrate and synthesis method and application thereof - Google Patents
Novel amidase chromogenic substrate and synthesis method and application thereof Download PDFInfo
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- CN115947700A CN115947700A CN202310019875.5A CN202310019875A CN115947700A CN 115947700 A CN115947700 A CN 115947700A CN 202310019875 A CN202310019875 A CN 202310019875A CN 115947700 A CN115947700 A CN 115947700A
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- chromogenic substrate
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- aminothiazoly loximate
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- 102000005922 amidase Human genes 0.000 title claims abstract description 46
- 108700023418 Amidases Proteins 0.000 title claims abstract description 45
- 239000003593 chromogenic compound Substances 0.000 title claims abstract description 32
- 238000001308 synthesis method Methods 0.000 title description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 34
- 238000006243 chemical reaction Methods 0.000 claims abstract description 32
- 102000004190 Enzymes Human genes 0.000 claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- KZZWQCKYLNIOBT-UHFFFAOYSA-N 5-amino-2-nitrobenzoic acid Chemical compound NC1=CC=C([N+]([O-])=O)C(C(O)=O)=C1 KZZWQCKYLNIOBT-UHFFFAOYSA-N 0.000 claims abstract description 19
- 230000000694 effects Effects 0.000 claims abstract description 19
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 16
- 239000000460 chlorine Substances 0.000 claims abstract description 16
- 229910052801 chlorine Inorganic materials 0.000 claims abstract description 16
- 239000012153 distilled water Substances 0.000 claims abstract description 13
- 238000003756 stirring Methods 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 150000007530 organic bases Chemical class 0.000 claims abstract description 10
- 239000003960 organic solvent Substances 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 10
- 239000002904 solvent Substances 0.000 claims abstract description 10
- 238000001704 evaporation Methods 0.000 claims abstract description 8
- 239000007787 solid Substances 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 7
- 150000007529 inorganic bases Chemical class 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 6
- 239000002535 acidifier Substances 0.000 claims abstract description 5
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 4
- 238000001914 filtration Methods 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 37
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 35
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 claims description 16
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 12
- 229930186147 Cephalosporin Natural products 0.000 claims description 11
- 229940124587 cephalosporin Drugs 0.000 claims description 11
- 102000004157 Hydrolases Human genes 0.000 claims description 9
- 108090000604 Hydrolases Proteins 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- -1 (E) -5- (2- (2-aminothiazole-4-yl) -2- (methoxyimino) acetamido) -2-nitrobenzoic acid Chemical compound 0.000 claims description 4
- 150000001263 acyl chlorides Chemical class 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- KYVBNYUBXIEUFW-UHFFFAOYSA-N 1,1,3,3-tetramethylguanidine Chemical compound CN(C)C(=N)N(C)C KYVBNYUBXIEUFW-UHFFFAOYSA-N 0.000 claims description 3
- 239000012359 Methanesulfonyl chloride Substances 0.000 claims description 3
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- QARBMVPHQWIHKH-UHFFFAOYSA-N methanesulfonyl chloride Chemical compound CS(Cl)(=O)=O QARBMVPHQWIHKH-UHFFFAOYSA-N 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims 7
- 230000037361 pathway Effects 0.000 claims 1
- 238000002390 rotary evaporation Methods 0.000 abstract description 5
- 230000008020 evaporation Effects 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 238000011002 quantification Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000010189 synthetic method Methods 0.000 abstract 2
- 230000001376 precipitating effect Effects 0.000 abstract 1
- 239000000047 product Substances 0.000 description 15
- 239000006228 supernatant Substances 0.000 description 11
- 239000000758 substrate Substances 0.000 description 9
- 108010073038 Penicillin Amidase Proteins 0.000 description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 6
- 150000001408 amides Chemical class 0.000 description 5
- 238000001291 vacuum drying Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 150000001780 cephalosporins Chemical class 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- QHVQEQRGDKOHHC-UHFFFAOYSA-N 2-nitro-5-[(2-phenylacetyl)amino]benzoic acid Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(NC(=O)CC=2C=CC=CC=2)=C1 QHVQEQRGDKOHHC-UHFFFAOYSA-N 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000011942 biocatalyst Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 235000019253 formic acid Nutrition 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 2
- 102000004092 Amidohydrolases Human genes 0.000 description 2
- 108090000531 Amidohydrolases Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 108010024026 Nitrile hydratase Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- WLJVXDMOQOGPHL-UHFFFAOYSA-N phenylacetic acid Chemical compound OC(=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-UHFFFAOYSA-N 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000000707 stereoselective effect Effects 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 108010048241 acetamidase Proteins 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000008430 aromatic amides Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- GPRBEKHLDVQUJE-VINNURBNSA-N cefotaxime Chemical compound N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C(O)=O)=O)C(=O)/C(=N/OC)C1=CSC(N)=N1 GPRBEKHLDVQUJE-VINNURBNSA-N 0.000 description 1
- 229960004261 cefotaxime Drugs 0.000 description 1
- 238000005557 chiral recognition Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- CSCPPACGZOOCGX-WFGJKAKNSA-N deuterated acetone Substances [2H]C([2H])([2H])C(=O)C([2H])([2H])[2H] CSCPPACGZOOCGX-WFGJKAKNSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000012434 nucleophilic reagent Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229960003424 phenylacetic acid Drugs 0.000 description 1
- 239000003279 phenylacetic acid Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Images
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a novel amidase chromogenic substrate, a synthetic method and application thereof, wherein the synthetic method comprises the following steps: step S1, adding a compound of aminothiazoly loximate with a structure I and an organic solvent into a round-bottom flask, slowly dropwise adding a chlorine acylating agent, and after the addition, performing temperature control reaction to obtain a yellow clear solution II containing aminothiazoly loximate; and S2, placing the reaction liquid in a rotary evaporator, removing the solvent by evaporation to generate yellow liquid flow, adding 5-amino-2-nitrobenzoic acid and acetone into the yellow liquid flow, fully stirring, controlling the temperature, dropwise adding organic base or inorganic base, continuing to perform heat preservation reaction, removing the solvent by rotary evaporation after the reaction is finished, adding a proper amount of distilled water, fully stirring, adjusting the pH value by using an acidifier, precipitating yellow solid, uniformly mixing, centrifuging, and obtaining a target product III by conventional filtration, washing and drying. The invention has the advantages of high sensitivity and good accuracy in enzyme activity quantification and screening; no terminator is needed, and the method has the advantages of simple steps, mild reaction and the like.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a novel amidase chromogenic substrate as well as a synthesis method and application thereof.
Background
Amidases (Amidases, EC 3.5.1.4) are important biocatalysts that catalyze the hydrolysis of the C-N bond of amides to produce the corresponding carboxylic acids and ammonia. The nature of the enzymatic reaction is to catalyze the transfer of an acyl group from its donor (substrate amide) to an acceptor (e.g., co-substrate water). Therefore, when nucleophilic reagent such as hydroxylamine or hydrazine which is stronger than water exists in the system as a receptor, hydroxamic acid or acyl hydrazine can be generated, the substrate spectrum of the amidase is very wide, the amidase can efficiently catalyze the hydrolysis of various non-natural aliphatic, heterocyclic and aromatic amides, and the amidase has the advantages of high chemoselectivity, regioselectivity, stereoselectivity, no need of coenzyme and the like. Numerous studies have shown that the chiral recognition behavior is dependent on the amide hydrolysis step in the stereoselective biotransformation of nitriles coupled to Nitrile hydratase (EC 4.2.1.84). Amidase enantioselective biocatalysis shows great potential in the synthesis of chiral carboxylic acid, hydroxamic acid and amide derivatives with complex structures, and becomes an important tool enzyme for chiral biocatalysis. For a particular biocatalytic process, the discovery of a suitable, highly efficient biocatalyst, especially a stereoselective biocatalyst, is the most critical step. The traditional screening methods of gas chromatography and high performance liquid chromatography are time-consuming and labor-consuming for determining the activity and stereoselectivity of a large number of samples one by one, so that the development of a rapid and accurate screening method of cephalosporin amidase is particularly urgent.
At present, the enzyme activity determination method of cephalosporin antibiotic hydrolase such as Penicillin G Acylase (PGA) has been reported in documents, penicillin G Acylase (PGA) determines that 2-nitro-5-phenylacetamidobenzoic acid (NIPAB) as a living substrate is hydrolyzed to generate phenylacetic acid and 5-amino-2-nitrobenzoic acid (ANBA), and a spectrophotometer can be used for detecting at a wavelength of 405 nm. However, the prior NIPAB method is based on that the enzyme reaction is zero-order reaction within a certain time, ethanol is commonly used as a terminator, and the reaction termination effect and impurities will affect the detection result, thereby affecting the accuracy and efficiency of screening.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a novel amidase chromogenic substrate and a synthesis method and application thereof, and the specific technical scheme is as follows:
the invention provides a synthesis method of a novel amidase chromogenic substrate, which comprises the following steps:
step S1, firstly, adding a compound of aminothiazoly loximate with a structure I and an organic solvent into a round-bottom flask, then slowly dropwise adding a chlorine acylating agent at the temperature of 0-5 ℃, and reacting for 1-2 hours at the controlled temperature after the addition is finished to obtain a yellow clear solution II containing the aminothiazoly loximate;
and S2, firstly, placing the reaction liquid obtained in the step S1 in a rotary evaporator to evaporate and remove the solvent to generate yellow liquid flow, then adding 5-amino-2-nitrobenzoic acid and acetone into the yellow liquid flow containing the aminothiazoly loximate acyl chloride, fully stirring, then controlling the temperature to be 0-5 ℃, dropwise adding organic base or inorganic base, continuing to perform heat preservation reaction for 1-3 hours, rotationally evaporating to remove the solvent after the reaction is finished, adding a proper amount of distilled water, fully stirring, adjusting the pH to be 2-3 by using an acidifier, separating out yellow solid, uniformly mixing and centrifuging, and finally obtaining the target product III through conventional filtration, washing and drying.
As a preferred technical scheme of the invention, the organic solvent in the step 1 is one or a mixture of ethyl acetate, dichloromethane, acetone and toluene.
As a preferable technical scheme of the invention, the chlorine acylating agent in the step 1 is one of oxalyl chloride, thionyl chloride and methanesulfonyl chloride.
As a preferred technical solution of the present invention, in the step 1, the mass ratio of the aminothiazoly loximate to the organic solvent is 1: (15-35) with the unit of g/mL; the molar ratio of the aminothiazoly loximate to the chlorine acylating agent is 1: (1-1.6).
As a preferred technical solution of the present invention, in the step 1, the chlorine acylating agent needs to be dissolved in N, N-dimethylformamide at a temperature of 0 to 5 ℃, and the molar ratio of the chlorine acylating agent to the N, N-dimethylformamide is 1: (1.1-1.5).
As a preferred technical solution of the present invention, in the step 2, the molar ratio of the aminothiazoly loximate acyl chloride to the 5-amino-2-nitrobenzoic acid is 1: (0.8-1.2); the mol ratio of the aminothiazoly loximate to the organic base is 1: (1.2-1.6).
As a preferred technical scheme of the invention, in the step 2, the organic base is one or more of triethylamine, tetramethylguanidine and diethylamine; the inorganic base is one or more of sodium hydroxide, sodium carbonate and sodium bicarbonate.
The invention also provides a novel amidase chromogenic substrate which is named as (E) -5- (2- (2-aminothiazole-4-yl) -2- (methoxyimino) acetamido) -2-nitrobenzoic acid, and the molecular formula of the novel amidase chromogenic substrate is C 13 H 11 N 5 O 6 S, molecular weight of 365.32, structural formula as follows:
the invention also provides application of the novel amidase chromogenic substrate in a screening way of the third generation cephalosporin amide hydrolase.
As a preferred technical scheme of the invention, the enzyme activity determination method of the third generation cephalosporin amide hydrolase is as follows:
formulating ANBA-OD 405 Standard curve: using 50mmol/L phosphate buffer solution (pH7.0) to perform gradient dilution (0, 4, 8, 12, 16, 20, 24, 28, 32 mg/L) on ANBA, taking the concentration gradient as an abscissa, and taking OD value measured under the condition of 405nm as an ordinate to draw a standard curve;
wherein the enzyme activity is defined as that the amidase quantity required for decomposing 1 mu mol of novel amidase chromogenic substrate in 1min is one enzyme activity unit under the condition of 37 ℃;
the value of the crude enzyme activity of amidase from different strains U is calculated with reference to the following formula: u = OD 405/ T is the reaction time (min), V is the volume of the enzyme solution sample (mL), and k is the slope of the standard curve.
The beneficial effects of the invention are:
1. compared with 2-nitro-5-phenylacetamidobenzoic acid (NIPAB), the novel activity-measuring substrate and the probe substrate designed based on the improved NIPAB method have the advantages that the chromogenic product ANBA after reaction can be measured at a position of 405nm by utilizing a spectrophotometer, and the enzyme activity quantification and screening has high sensitivity and good accuracy;
2. compared with the terminator ethanol in the NIPAB method, the method has the advantages of simple steps, mild reaction and the like;
3. the invention effectively solves the problem of substrate interference, and the substrate is free from impurity generation after reaction, thereby having no influence on the detection result;
4. the invention provides reference and reference for the screening of the third generation cephalosporin amide hydrolase.
Drawings
FIG. 1 shows a NMR hydrogen spectrum of a novel amidase chromogenic substrate of the invention;
FIG. 2 shows a Fourier infrared spectrum of a novel amidase chromogenic substrate of the invention;
FIG. 3 shows ESI-MS high resolution mass spectra of novel amidase chromogenic substrates of the present invention;
FIG. 4 shows a MALDI mass spectrometry analysis of the novel amidase chromogenic substrate of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides a novel amidase chromogenic substrate and a synthesis method thereof, and solves the problem that the prior NIPAB as a substrate affects the detection result due to reaction termination effect and impurities, thereby affecting the accuracy and efficiency of screening.
The technical scheme in the embodiment of the invention is to obtain the novel amidase chromogenic substrate, and the synthetic route is as follows:
for better understanding of the above technical solutions, the following detailed descriptions will be provided in conjunction with the drawings and the detailed description of the embodiments.
A synthesis method of a novel amidase chromogenic substrate comprises the following steps:
step S1, firstly, adding a compound aminothiazoly loximate with a structure I and an organic solvent into a round-bottom flask, then slowly dropwise adding a chlorine acylating agent at the temperature of 0-5 ℃, and after the addition, reacting at the temperature of 1-2 hours to obtain a yellow clear solution II containing the aminothiazoly loximate;
and S2, firstly, placing the reaction liquid obtained in the step S1 in a rotary evaporator to evaporate and remove the solvent to generate yellow liquid flow, then adding 5-amino-2-nitrobenzoic acid and acetone into the yellow liquid flow containing the aminothiazoly loximate, fully stirring, then controlling the temperature to be 0-5 ℃, dropwise adding organic base or inorganic base, continuously preserving the temperature for reaction for 1-3 hours, rotationally evaporating to remove the solvent after the reaction is finished, adding a proper amount of distilled water, fully stirring, adjusting the pH to be 2-3 by using an acidifying agent, separating out yellow solid, uniformly mixing and centrifuging, and finally obtaining the target product III through conventional filtration, washing and drying.
Preferably, the organic solvent in step 1 is one or more of ethyl acetate, dichloromethane, acetone and toluene.
Preferably, the chlorine acylating agent in the step 1 is one of oxalyl chloride, thionyl chloride and methanesulfonyl chloride.
Preferably, in the step 1, the ratio of the mass of the aminothiazoly loximate to the volume of the organic solvent is 1: (15-35) with the unit of g/mL; the mol ratio of the aminothiazoly loximate to the chlorine acylating agent is 1: (1-1.6).
Preferably, in the step 1, the chlorine acylating agent is dissolved in the N, N-dimethylformamide at a controlled temperature of 0-5 ℃, and the molar ratio of the chlorine acylating agent to the N, N-dimethylformamide is 1: (1.1-1.5).
Preferably, in the step 2, the molar ratio of the aminothiazoly loximate acyl chloride to the 5-amino-2-nitrobenzoic acid is 1: (0.8-1.2); the mol ratio of the aminothiazoly loximate to the organic base is 1: (1.2-1.6).
Preferably, in the step 2, the organic base is one or more of triethylamine, tetramethylguanidine and diethylamine; the inorganic base is one or more of sodium hydroxide, sodium carbonate and sodium bicarbonate.
Preferably, the acidifying agent in step 2 is a weak organic acid, and the weak organic acid is formic acid or acetic acid.
Preferably, in the step 2, the reaction product is washed with distilled water for 1 to 2 times; and drying the reaction product by adopting a vacuum drying oven for 48 hours.
A novel amidase chromogenic substrate is named as (E) -5- (2- (2-aminothiazole-4-yl) -2- (methoxyimino) acetamido) -2-nitrobenzoic acid, and the molecular formula is C 13 H 11 N 5 O 6 S, molecular weight of 365.32, structural formula as follows:
the application of a novel amidase chromogenic substrate in a third generation cephalosporin amide hydrolase screening way.
Preferably, the enzyme activity of the third generation cephalosporin amide hydrolase is determined as follows:
formulating ANBA-OD 405 Standard curve: using 50mmol/L phosphate buffer solution (pH7.0) to perform gradient dilution (0, 4, 8, 12, 16, 20, 24, 28, 32 mg/L) on ANBA, taking the concentration gradient as an abscissa, and taking OD value measured under the condition of 405nm as an ordinate to draw a standard curve;
wherein, the enzyme activity is defined as that the amidase quantity required for decomposing 1 mu mol of novel amidase chromogenic substrate in 1min is one enzyme activity unit at the temperature of 37 ℃;
the value of the crude enzyme activity of amidase from different strains U is calculated with reference to the following formula: u = OD 405/ T × V × k, T is the reaction time (min), V isVolume of enzyme sample (mL), k is the slope of the standard curve.
Description of the drawings: as the ainothiazoly loximate is a precursor compound of the third-generation cephalosporin cefotaxime, the characteristics of the precursor compound are utilized to judge that the novel product amidase strain can be screened at present, but the hydrolase capable of hydrolyzing the third-generation cephalosporin is required to be further screened by whole genome analysis.
The invention screens and enriches the bacterial strain of amidase by taking the novel amidase chromogenic substrate as a unique carbon source or nitrogen source, and the enzyme activity is determined by the improved NIPAB enzyme activity determination method.
The above terms explain: ANBA: 5-amino-2-nitrobenzoic acid; the NIPAB: 2-nitro-5-phenylacetamidobenzoic acid; PGA: penicillin G acylase; DMF: n, N-dimethylformamide; SOCl 2 : thionyl chloride.
The specific embodiment of the invention is as follows:
example 1.0g of aminothiazoly loximate is added into 33mL of ethyl acetate, 1.09ml of a mixed solution of N, N-dimethylformamide and 0.99mL of thionyl chloride (exothermic reaction) is slowly added dropwise into a separating funnel at the temperature of 0 ℃, after the addition, the mixture is kept warm and reacts for 2h to obtain a yellow clear solution containing aminothiazoly loximate, a rotary evaporator is used for evaporation for 15min to generate a yellow fluid liquid, a solution of 1.09g of 5-amino-2-nitrobenzoic acid dissolved in 45mL of acetone is directly added and stirred for 2h, the solution becomes clear, the reaction state is monitored by TLC (acetone: water = 9:1), the solution is concentrated to 8mL by rotary evaporation, 32mL of distilled water is added and stirred magnetically overnight, the reaction solution is transferred into a centrifuge tube the next day, centrifuged for 10min at 10000r, a supernatant is poured out, the supernatant is washed with distilled water and then dried in a vacuum drying oven at the temperature of 40 ℃ for two days, and finally the target yellow product 1.469g is obtained, and the yield is 73%.
Example 2 adding 1.0g of aminothiazoly loximate to 33mL of dichloromethane, slowly adding a mixed solution of 1.09ml of N, N-dimethylformamide and 0.99mL of thionyl chloride (exothermic reaction) dropwise in a separating funnel under the condition of controlling the temperature to 0 ℃, keeping the temperature for 2h after the addition to obtain a yellow clear solution containing aminothiazoly loximate, evaporating for 15min by a rotary evaporator to generate a yellow fluid, directly adding a solution of 1.09g of 5-amino-2-nitrobenzoic acid dissolved in 45mL of acetone, stirring for 2h to clarify the solution, monitoring the reaction state by TLC (acetone: water = 9:1), concentrating to 8mL by rotary evaporation, adding 32mL of distilled water, magnetically stirring overnight, transferring the reaction solution into a centrifuge tube the next day, centrifuging for 10min under 10000r, pouring out the supernatant, washing with distilled water, putting into a vacuum drying oven at 40 ℃ for two days to finally obtain 1.205g of the target yellow product with the yield of 60%.
Example 3 adding 0.3g of aminothiazoly loximate into 10mL of ethyl acetate, slowly adding 0.33mL of a mixed solution of N, N-dimethylformamide and 0.30mL of thionyl chloride (reaction is exothermic) dropwise at the temperature of 0 ℃, after finishing the reaction for 2h, obtaining an organic solution containing aminothiazoly loximate, evaporating for 15min by a rotary evaporator to generate a dark yellow liquid, directly adding a solution of 0.33g of 5-amino-2-nitrobenzoic acid dissolved in 15mL of acetone, stirring for 3h, clarifying the solution, monitoring the reaction state by TLC (acetone: water = 9:1), adding 0.50mL of triethylamine as a catalyst into the reaction solution at the temperature of 0 ℃ to precipitate a yellow solid, and after finishing the reaction for 2h. And (3) taking the supernatant, removing the solvent by rotary evaporation, adding 10mL of distilled water, placing the mixture in a constant-temperature water bath kettle at 60 ℃ for 30min to obtain a clear yellow solution, adjusting the pH value of the solution to be 2-3 by using formic acid to separate out a yellow solid, centrifuging the solution at 10000r for 10min, pouring out the supernatant, washing the supernatant by using distilled water, and then placing the supernatant into a vacuum drying oven to dry the solution for two days at 40 ℃ to obtain 0.372g of a target yellow product finally, wherein the yield is 62%.
Example 4 adding 0.3g of aminothiazoly loximate into 10mL of ethyl acetate, slowly dropping 0.33ml of a mixed solution of N, N-dimethylformamide and 0.30mL of thionyl chloride (exothermic reaction) while controlling the temperature to 0 ℃, after finishing the reaction for 2h, obtaining an organic solution containing aminothiazoly loximate, evaporating for 15min by a rotary evaporator to generate a dark yellow liquid, directly adding a solution of 0.33g of 5-amino-2-nitrobenzoic acid dissolved in 15mL of acetone, stirring for 3h, clarifying the solution, monitoring the reaction state by TLC (acetone: water = 9:1), controlling the temperature of the reaction solution to 0 ℃, adding 0.55mL of a sodium bicarbonate solution as a catalyst (yellow solid is precipitated), and after finishing the reaction for 2h. And (3) taking the supernatant, removing the solvent by rotary evaporation, adding 10mL of distilled water, placing the mixture in a constant-temperature water bath kettle at 60 ℃ for 30min to obtain a clear yellow solution, adjusting the pH value of the solution to be 2-3 by using formic acid to separate out a yellow solid, centrifuging the solution at 10000r for 10min, pouring out the supernatant, washing the supernatant by using distilled water, and then placing the supernatant into a vacuum drying oven to dry the solution for two days at 40 ℃ to obtain 0.372g of a target yellow product finally, wherein the yield is 55%.
The performance test analysis of the invention is as follows:
1. as shown in fig. 1, the target product III after drying was subjected to nmr hydrogen spectroscopy, and the target product III was dissolved in deuterated acetone (C) 3 D 6 O); thus, the target product III can be verified to have the number of 11H;
2. as shown in FIG. 2, the dried target product III was subjected to Fourier transform infrared spectroscopy because the amide-based compound was present at 3150 and 3390cm -1 The characteristic of 'double peaks' appears, the absorption peak intensity is m (medium intensity absorption), and the absorption peak belongs to an N-H stretching vibration peak, 1720cm -1 Is C = O stretching vibration peak of aromatic carboxylic acid and bending vibration peak of N-H, 1570cm -1 Is the oscillation peak of benzene ring skeleton, 1550cm -1 Is the nitro group stretching vibration peak; thus, the chemical structure of the target product III can be verified;
3. as shown in FIGS. 3 and 4, ESI-MS and MALDI high resolution mass spectrometry of the dried target product III was performed, in which [ M + H ] was observed at 366.05001M/z] + The ion peak and the latter not only show hydrogenation peaks, but also show [ M + Na ] at 388.03224 and 404.00586M/z respectively] + Ion peak and [ M + K ]] + An ion peak; this allows the molecular weight of the target product III to be verified.
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (10)
1. A method for synthesizing a novel amidase chromogenic substrate is characterized by comprising the following steps:
step S1, firstly, adding a compound aminothiazoly loximate with a structure I and an organic solvent into a round-bottom flask, then slowly dropwise adding a chlorine acylating agent at the temperature of 0-5 ℃, and after the addition, reacting at the temperature of 1-2 hours to obtain a yellow clear solution II containing the aminothiazoly loximate;
and S2, firstly, placing the reaction liquid obtained in the step S1 in a rotary evaporator to evaporate and remove the solvent to generate yellow liquid flow, then adding 5-amino-2-nitrobenzoic acid and acetone into the yellow liquid flow containing the aminothiazoly loximate, fully stirring, then controlling the temperature to be 0-5 ℃, dropwise adding organic base or inorganic base, continuously preserving the temperature for reaction for 1-3 hours, rotationally evaporating to remove the solvent after the reaction is finished, adding a proper amount of distilled water, fully stirring, adjusting the pH to be 2-3 by using an acidifying agent, separating out yellow solid, uniformly mixing and centrifuging, and finally obtaining the target product III through conventional filtration, washing and drying.
2. The method for synthesizing the novel amidase chromogenic substrate according to claim 1, wherein the method comprises the following steps: the organic solvent in the step 1 is one or a mixture of ethyl acetate, dichloromethane, acetone and toluene.
3. The method for synthesizing the novel amidase chromogenic substrate according to claim 1, wherein the method comprises the following steps: the chlorine acylating agent in the step 1 is one of oxalyl chloride, thionyl chloride and methanesulfonyl chloride.
4. The method for synthesizing a novel amidase chromogenic substrate according to claim 1, wherein: in the step 1, the volume ratio of the mass of the aminothiazoly loximate to the volume of the organic solvent is 1: (15-35) with the unit of g/mL; the molar ratio of the aminothiazoly loximate to the chlorine acylating agent is 1: (1-1.6).
5. The method for synthesizing a novel amidase chromogenic substrate according to claim 1, wherein: in the step 1, the chlorine acylating agent is dissolved in the N, N-dimethylformamide at the temperature of 0-5 ℃, and the molar ratio of the chlorine acylating agent to the N, N-dimethylformamide is 1: (1.1-1.5).
6. The method for synthesizing a novel amidase chromogenic substrate according to claim 1, wherein: in the step 2, the mol ratio of the aminothiazoly loximate acyl chloride to the 5-amino-2-nitrobenzoic acid is 1: (0.8-1.2); the molar ratio of the aminothiazoly loximate to the organic base is 1: (1.2-1.6).
7. The method for synthesizing a novel amidase chromogenic substrate according to claim 1, wherein: in the step 2, the organic base is one or more of triethylamine, tetramethylguanidine and diethylamine; the inorganic base is one or more of sodium hydroxide, sodium carbonate and sodium bicarbonate.
8. A novel amidase chromogenic substrate synthesized according to the method of any one of claims 1 to 7, characterized in that: the novel amidase chromogenic substrate is named as (E) -5- (2- (2-aminothiazole-4-yl) -2- (methoxyimino) acetamido) -2-nitrobenzoic acid, and the molecular formula is C 13 H 11 N 5 O 6 S, molecular weight is 365.32, structural formula is as follows:
9. use of a novel amidase chromogenic substrate according to claim 8 in the screening pathway for third generation cephalosporin amide hydrolases.
10. The use according to claim 9, wherein the third generation cephalosporin amide hydrolase is assayed by the following method:
formulating ANBA-OD 405 Standard curve: using 50mmol/L phosphate buffer solution (pH7.0) to perform gradient dilution (0, 4, 8, 12, 16, 20, 24, 28, 32 mg/L) on ANBA, taking the concentration gradient as an abscissa, and taking OD value measured under 405nm as an ordinate to draw a standard curve;
wherein the enzyme activity is defined as that the amidase quantity required for decomposing 1 mu mol of novel amidase chromogenic substrate in 1min is one enzyme activity unit under the condition of 37 ℃;
the value U of the amidase activity of the crude enzyme solution from the different strains was calculated with reference to the following formula: u shape＝OD 405/ T is the reaction time (min), V is the volume of the enzyme solution sample (mL), and k is the slope of the standard curve.
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| US4965193A (en) * | 1984-08-06 | 1990-10-23 | Washington Research Foundation | Detection of microbial beta-lactamase |
| CN101201356A (en) * | 2007-11-13 | 2008-06-18 | 华东理工大学 | Method for rapidly measuring activity of penicillin acidated enzyme |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4965193A (en) * | 1984-08-06 | 1990-10-23 | Washington Research Foundation | Detection of microbial beta-lactamase |
| CN101201356A (en) * | 2007-11-13 | 2008-06-18 | 华东理工大学 | Method for rapidly measuring activity of penicillin acidated enzyme |
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| MARKO GOLICNIK: ""Alternative algebraic rate-integration approach for progress-curve analysis of enzyme kinetics"", 《ENG. LIFE SCI.》, vol. 12, no. 1, pages 104 - 108 * |
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| WYNAND B. L. ALKEMA, ET AL.: ""The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase"", 《EUR. J. BIOCHEM.》, vol. 269, pages 2093, XP002527253, DOI: 10.1046/J.1432-1033.2002.02857.X * |
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