CN115385934A - Sulbactam hapten as well as synthesis method and application thereof - Google Patents

Sulbactam hapten as well as synthesis method and application thereof Download PDF

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CN115385934A
CN115385934A CN202211319515.9A CN202211319515A CN115385934A CN 115385934 A CN115385934 A CN 115385934A CN 202211319515 A CN202211319515 A CN 202211319515A CN 115385934 A CN115385934 A CN 115385934A
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sulbactam
hapten
drying
acid
crude
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陈智卿
赵荣茂
巩玉洁
石强
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Beijing Nabai Bio Tech Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D499/86—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring with only atoms other than nitrogen atoms directly attached in position 6 and a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D499/04—Preparation
    • C07D499/08—Modification of a carboxyl radical directly attached in position 2, e.g. esterification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • 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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Abstract

The invention discloses a sulbactam hapten as well as a synthesis method and application thereof, wherein the chemical formula of the sulbactam hapten is as follows:
Figure 88040DEST_PATH_IMAGE002
. The invention adopts the sulbactam hapten, the synthesis method and the application thereof to realize the accurate detection of the sulbactam without cross reaction with other medicines.

Description

Sulbactam hapten as well as synthesis method and application thereof
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to a sulbactam hapten as well as a synthesis method and application thereof.
Background
At present, all dairy production enterprises strictly prohibit the addition of beta-lactamase in products, and the addition of beta-lactamase to milk and dairy products masks antibiotics. Beta-lactam antibiotics are the most widely used antibiotics in bovine milk production for the treatment of bovine mastitis and other bacterial infectious diseases. The milk in a certain period after the antibiotic medicine is used cannot be used as a raw material for human eating.
Due to the abuse of beta-lactam antibiotics, lawless persons can cover the overuse of antibiotics by decomposing the lactam antibiotics through adding beta-lactamase. However, detection of the residual content of β -lactamase in raw milk may lead to the appearance of antagonists, inhibitors of β -lactamase, which mask the residual content of antibiotics. At present, several beta-lactam derivatives are reported as beta-lactamase inhibitors, but have only clavulanic acid, sulbactam and tazobactam in clinical effect, and the sulbactam is more widely applied.
Sulbactam (sulbactam), with a molecular weight of 347, is a semi-synthetic penicillanic acid sulfone containing a 6-aminopenicilanic acid derived beta-lactam ring. Sulbactam is an irreversible inhibitor of a plurality of bacterial beta-lactamase and has an inhibiting effect on beta-lactamase produced by gram positive and negative bacteria (except pseudomonas aeruginosa).
When sulbactam is used in combination with certain penicillins or cephalosporins, the resulting synergy acts as an inhibitor of many beta-lactamase producing bacteria, thereby broadening the spectrum of activity of these antibiotics. In addition, it is also an inducer enzyme inhibitor that causes third-generation cephalosporin resistance. While sulbactam and these antibiotics have expanded the spectrum of activity, their effectiveness in treating a variety of gram-positive, anaerobic and gram-negative infections is also enhanced.
At present, the detection method of sulbactam mainly comprises high performance liquid chromatography tandem mass spectrometry, however, the detection operation of an instrument is complex, the cost is high, and the requirement of on-site scale rapid detection cannot be met. For the immunoassay method, the method has the advantages of low cost, high efficiency, high sensitivity and relatively low requirement on technical personnel, and is suitable for rapid detection of a large number of samples. Therefore, the high-efficiency and rapid detection of the sulbactam content can be realized by the immune method.
Disclosure of Invention
The invention aims to provide a sulbactam hapten, a synthesis method and application thereof, which can realize the accurate detection of sulbactam and have no cross reaction with other medicines.
In order to achieve the purpose, the invention provides sulbactam hapten which has the following chemical formula structure:
Figure 755871DEST_PATH_IMAGE001
。
a synthetic method of sulbactam hapten comprises the following steps:
s1, accurately weighing sulbactam acid, dissolving the sulbactam acid into dichloromethane, sequentially adding 4-DMAP and maleic anhydride, and reacting for 48 hours at 50 ℃;
s2, drying the mixed solution obtained in the step S1 through liquid quality detection, adding 5mL of water, adjusting the pH value of the solution by using 2moL/L HCl, extracting by using ethyl acetate, and combining organic phases;
s3, drying the organic phase obtained in the step S2 to obtain a crude hapten;
s4, purification: and (5) performing polarity purification on the crude product obtained in the step (S3) in a normal phase FLASH column.
Preferably, the steps are as follows:
s1, accurately weighing 200mg of sulbactam acid, dissolving the sulbactam acid into 4mL of dichloromethane, sequentially adding 62mg of 4-DMAP and 56mg of maleic anhydride, and reacting for 48 hours at 50 ℃;
s2, after liquid quality detection and drying treatment, adding 5mL of water, adjusting the pH value to 3 by using 2moL/L of HCL, extracting by using ethyl acetate, combining organic phases, and drying to obtain 230mg of crude hapten;
s3, purification: EA (1).
Therefore, the invention adopts the sulbactam hapten, the synthesis method and the application thereof to realize the accurate detection of the sulbactam without cross reaction with other medicines.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a LC profile of sulbactam hapten;
FIG. 2 is a MS profile of sulbactam hapten;
FIG. 3 is a schematic view of a colloidal gold detection reagent.
Detailed Description
The technical solution of the present invention is further illustrated by the accompanying drawings and examples.
All terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs unless specifically defined otherwise. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate.
The disclosures of the prior art documents cited in the present description are incorporated by reference in their entirety and are therefore part of the present disclosure.
Wherein, sulbactam has the following structure:
Figure 735328DEST_PATH_IMAGE002
the coupling of the sulbactam structural site is difficult, so the sulbactam acid is selected for coupling.
The structure of sulbactam acid is as follows:
Figure 328115DEST_PATH_IMAGE003
the structure of the sulbactam hapten is as follows:
Figure 785641DEST_PATH_IMAGE001
example one
The specific operation process of the synthesis of the sulbactam hapten is as follows:
s1, accurately weighing 200mg of sulbactam acid, dissolving the sulbactam acid into 4mL of dichloromethane, sequentially adding 62mg of 4-DMAP and 56mg of maleic anhydride, and reacting for 48 hours at 50 ℃.
And S2, sequentially carrying out liquid quality detection and drying treatment on the mixed solution obtained in the step S1, adding 5mL of water, adjusting the pH to 3 by using 2mol/L of HCL, extracting by using ethyl acetate, combining organic phases, and drying the organic phases to obtain 230mg of crude hapten.
S3, purification: EA (1) and normal phase FLASH were purified at this polarity to give 80mg pure product with a purity of 93.5%, as shown in fig. 1. The peak of molecular ion is m/z 337.20[ M + NH ] obtained from MS atlas 4 ] + I.e. molecular weight 319, with synthetic sulbactam hapten C 12 H 17 NO 7 The S molecular weights are consistent, as shown in FIG. 2.
The reaction route is as follows:
Figure 410133DEST_PATH_IMAGE005
example two
Coupling of a coated sulbactam antigen BSA carrier, which is performed as follows:
s1, weighing 10mg of pure hapten, dissolving the pure hapten in 1mL of DMF, slowly adding 21mgNHS and 58mgEDC, and stirring at room temperature overnight.
S2, weighing 60mg of BSA, dissolving the BSA into 1mL of 0.01mol/L PBS buffer solution with the pH value of 7.4, uniformly stirring, slowly dropwise adding the BSA into the activated hapten solution, and stirring for 3 hours at room temperature. Dialyzing with 0.01mol/L PBS (pH 7.2), changing water once during 2h, dialyzing for 48h, and storing at-20 deg.C.
The reaction route is as follows:
Figure 294912DEST_PATH_IMAGE006
Figure 374995DEST_PATH_IMAGE007
the same principle is used for coating the sulbactam antigen OVA carrier.
EXAMPLE III
Coupling of an immune sulbactam antigen BSA carrier, which is performed as follows:
s1, weighing 5mg of pure hapten, dissolving the pure hapten into 500 mu L of anhydrous methanol, dropwise adding 30 mu L of triethylamine, and stirring at room temperature for 30min;
s2, adding 60 mu L of isobutyl chloroformate, and stirring for 1h;
s3, weighing 30mgBSA, slowly adding the BSA into the solution obtained in the step S2, and stirring for 4 hours.
S4, dialyzing with 0.01mol/L PBS (pH 7.2), changing water once in the period of 2h, dialyzing for 48h, and storing at-20 ℃.
Example four
Preparation of monoclonal antibodies
Animal immunization: a Balb/c mouse of 6 weeks is taken as an immunized animal, the antigen of sulbactam and BSA is taken as immunogen, the immunizing dose is 80 mu g/mouse, the immunogen and an equal amount of Freund's complete adjuvant are mixed to prepare an emulsifier during first immunization, the emulsifier is injected into abdominal cavity at multiple points, the immunogen of the same dose and an equal amount of Freund's incomplete adjuvant are mixed and emulsified after three weeks, the boosting immunization is carried out once, the abdominal cavity boosting immunization is carried out once after four immunizations, and splenocytes are taken after 4 days.
Cell fusion and cloning: taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to a ratio of 4. Cloning the positive hole by using a micro-cloning method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.
Freezing and recovering cells: preparing hybridoma cells in logarithmic growth phase into 5 × 10 with frozen stock solution 6 And (3) subpackaging the cell suspension per mL into freezing tubes, storing for a long time in an ultra-low temperature refrigerator at-70 ℃, taking out the freezing tubes when recovering, immediately putting into a water bath at 37 ℃ for instant dissolution, centrifuging to remove freezing liquid, and transferring into a culture bottle for culture.
Preparation and purification of monoclonal antibody: injecting the hybridoma cells of Balb/c 8-week-old mice into the abdominal cavity by in vivo induction method to obtain 5 × 10 hybridoma cells 6 Ascites were collected 15 days later. Purifying ascites by affinity chromatography, subpackaging with small bottles, and storing at-20 deg.C.
EXAMPLE five
Application of sulbactam hapten and monoclonal antibody
1. The preparation of the sulbactam colloidal gold detection reagent comprises the following steps:
(1) Preparation of colloidal gold
1mL of chloroauric acid solution (mass percent) is taken, 99mL of ultrapure water is added to obtain chloroauric acid solution with the final concentration of 0.01 (mass percent), 1.6mL of trisodium citrate (mass percent) is taken to be rapidly added into the boiled chloroauric acid solution at one time after being heated to boil, the heating is continued until the solution is changed from light yellow to blue black and finally to bright red, the heating is continued for 5min after the color is stable, the cooling at room temperature is carried out, and pure water is supplemented to the original volume.
(2) Preparation of colloidal gold labeled sulbactam monoclonal antibody
Adjusting the pH value of the colloidal gold solution to 8.0, uniformly stirring by using a constant-speed stirrer, simultaneously dropwise adding the sulbactam monoclonal antibody, adding PEG with the equivalent antibody amount after 1 hour, fully reacting for 30min, adding BSA with the equivalent antibody amount, and continuously stirring for 30min after the addition. And centrifuging at 9000rpm for 30min to obtain a homogeneous gold-labeled antibody precipitate, and adding PBS for re-suspension to obtain the colloidal gold-labeled sulbactam monoclonal antibody.
(3) Preparation of colloidal gold detection reagent
As shown in fig. 3, the detection line T line formed by coating the sulbactam antigen BSA solution of example two on the nitrocellulose membrane, and the control line C line formed by spraying goat anti-mouse IgG. The detection line and the control line are parallel to each other, and the distance between the detection line and the control line is 0.5cm. And then, sequentially overlapping and adhering the sample pad, the detection line T sprayed with the sulbactam immune antigen and the nitrocellulose membrane and the absorbent paper of the control line C sprayed with the goat anti-mouse IgG on the bottom plate along the same direction.
Wherein, the detection line is close to the sample pad, the control line is close to the absorbent paper, and the stuck detection test paper is cut into test strips with equal width; adding colloidal gold labeled sulbactam monoclonal antibody into the reaction cup, and freeze-drying; and sealing, drying and storing the reaction cup and the test strip.
2. Sample detection
(1) Detection of sulbactam in the sample:
the method comprises the following steps: taking a fresh chicken tissue sample, mincing, extracting sulbactam residue by acetonitrile and acetone, removing fat by normal hexane, sampling, placing in a reaction cup, incubating for 10 minutes at room temperature, inserting a test strip, and incubating for 3 minutes at room temperature. And taking out the test strip, slightly scraping the test sponge pad, and judging the result. If the T line and the C line simultaneously display the purple red strip, the result is negative; if the color of the T line is lighter than that of the C line or the C line is colored and the T line is not colored, the result is positive; if the C line and the T line do not develop color, the detection reagent is invalid.
(2) And (3) detecting the sensitivity of the sulbactam colloidal gold detection reagent:
by a labeling experiment, the sensitivity of the sulbactam colloidal gold detection device can reach 50ppb, and the CV value is less than 15%.
(3) Specific detection of the sulbactam colloidal gold detection reagent:
in negative fish tissues, clavulanic acid, tetracycline, penicillin, cefapirin, erythromycin, lincomycin, tylosin, streptomycin, kanamycin, gentamicin and sulfadimidine are respectively added for 200ppb, and detection results are negative, which shows that sulbactam has excellent selectivity and has no cross reaction with the added medicines.
Therefore, the invention adopts the sulbactam hapten, the synthesis method and the application thereof to realize the accurate detection of sulbactam, and has no cross reaction with other medicines.
Finally, it should be noted that: the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.

Claims (3)

1. A sulbactam hapten, which has the following chemical formula structure:
Figure DEST_PATH_IMAGE001
。
2. a method for the synthesis of sulbactam hapten according to claim 1, comprising the steps of:
s1, accurately weighing sulbactam acid, dissolving the sulbactam acid into dichloromethane, sequentially adding 4-DMAP and maleic anhydride, and reacting for 48 hours at 50 ℃;
s2, drying the mixed solution obtained in the step S1 through liquid quality detection, adding 5mL of water, adjusting the pH value of the solution by using 2moL/L HCl, extracting by using ethyl acetate, and combining organic phases;
s3, drying the organic phase obtained in the step S2 to obtain a crude hapten;
s4, purification: and (5) performing polarity purification on the crude product obtained in the step (S3) in a normal phase FLASH column.
3. The method for synthesizing sulbactam hapten according to claim 2, characterized by comprising the following steps:
s1, accurately weighing 200mg of sulbactam acid, dissolving the sulbactam acid into 4mL of dichloromethane, sequentially adding 62mg of 4-DMAP and 56mg of maleic anhydride, and reacting for 48 hours at 50 ℃;
s2, after liquid quality detection and drying treatment, adding 5mL of water, adjusting the pH to 3 by using 2moL/L of HCL, extracting by using ethyl acetate, combining organic phases, and drying to obtain crude hapten 230mg;
s3, purification: the crude hapten was determined to be of PE: EA (1), normal phase FLASH was purified at this polarity to give 80mg pure product of 93.5% purity.
CN202211319515.9A 2022-10-26 2022-10-26 Sulbactam hapten as well as synthesis method and application thereof Pending CN115385934A (en)

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