CN114539171B - MAC type multi-cluster tandem linear hapten and artificial antigen as well as preparation methods and applications thereof - Google Patents
MAC type multi-cluster tandem linear hapten and artificial antigen as well as preparation methods and applications thereof Download PDFInfo
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- C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
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Abstract
The invention discloses a MAC multi-cluster tandem linear hapten and an artificial antigen containing 3-methyl-quinoxaline-2-carboxylic acid (MQCA), amantadine (AMD) and chloramphenicol amine (CAPA) characteristic skeleton structures, and a preparation method and application thereof. The hapten is an artificial antigen prepared by sequentially connecting 3-methyl-quinoxaline-2-carboxylic acid, amantadine and chloramphenicol amine 3-drug framework structures with amino alkyl carboxylic acid and alkyl diacid with different lengths to form a multi-cluster series linear hapten, and coupling the hapten with carrier protein. The polyclonal serum titer obtained by adopting the artificial antigen is between 8 and 16K, and the inhibition rate of hapten MAC-4C and MAC-6C is between 51 and 85 percent, which shows that the multi-cluster tandem linear hapten and the artificial antigen thereof prepared by the invention have good immunogenicity and can be used for preparing monoclonal antibodies, single-chain antibodies and nano antibodies subsequently.
Description
Technical Field
The invention relates to the technical field of antigen-antibody detection, in particular to a MAC multi-cluster tandem linear hapten and an artificial antigen containing 3-methyl-quinoxaline-2-carboxylic acid (MQCA), amantadine (AMD) and chloramphenicol amine (CAPA) characteristic skeleton structures, and a preparation method and application thereof.
Background
At present, the problem of livestock and poultry product safety is still serious due to abuse of veterinary drugs such as antibiotics and the like. Among them, the forbidden antibiotics such as amantadine, olaquindox, chloramphenicol are common, and cause great potential harm to physical and mental health of consumers. At present, veterinary drug residue analysis of animal products is mainly performed in a mode of combining a large-scale precise instrument confirmation technology and an immunoassay screening technology, wherein the instrument confirmation technology method mainly adopts a chromatography-mass spectrometry technology, but has the defects of complex sample pretreatment operation, time consumption, high cost, unsuitability for field detection and the like. The immunoassay method has the advantages of consistent sensitivity with the conventional instrument analysis, suitability for on-site screening, simplicity, rapidness, low cost, simple pretreatment and the like. However, at present, the detection of pesticide and veterinary drug residues in foods is mainly a single-residue immunoassay method, and for example, in Chinese patent, immunoassay methods for amantadine, olaquindox and chloramphenicol independently are disclosed. However, in view of diversity of veterinary drug types and universality and complexity of compound use thereof, the problem of multi-residue of veterinary drugs is very prominent, and only single-residue immunodetection technology is developed but cannot meet actual detection requirements, so how to realize multi-residue immunodetection of non-common structural antibiotics is a new development direction. However, related hapten, antigen and antibody for simultaneously detecting non-common structure small molecule drugs including amantadine, olaquindox and chloramphenicol are lacking.
Disclosure of Invention
The invention provides the MAC type multi-cluster tandem linear hapten which can realize the identification of the organic micromolecule medicaments with a non-shared structure in order to overcome the defects and the shortages in the prior art. The 3 antibiotic drug molecular skeleton structural units of the olaquindox, the amantadine and the chloramphenicol are linked into a multi-cluster tandem linear hapten through proper linear coupling spacer arms.
The second object of the invention is to provide a preparation method of the MAC type multi-cluster tandem linear hapten.
A third object of the present invention is to provide the use of said MAC-type multi-cluster tandem linear hapten.
The fourth object of the invention is to provide a MAC type multi-cluster tandem linear artificial antigen.
The fifth object of the invention is to provide a method for preparing the MAC type multi-cluster tandem linear artificial antigen.
A sixth object of the present invention is to provide the use of the MAC-type multi-cluster tandem linear artificial antigen.
A seventh object of the present invention is to provide a multi-cluster tandem linear antigen-specific antibody.
The above object of the present invention is achieved by the following technical solutions:
a MAC type multi-cluster tandem linear hapten containing 3-methyl-quinoxaline-2-carboxylic acid (MQCA), amantadine (AMD) and chloramphenicol amine (CAPA) skeleton structural units has a molecular structure shown in a formula I:
wherein R is 1 And R is 2 Respectively selected from alkyl groups with 2-5 carbon atoms, namely R 1 And R is 2 Selected from any one of ethyl to amyl groups respectively.
The MAC type multi-cluster tandem linear hapten is prepared by connecting skeleton structural units of Amantadine (AMD), olaquindox Metabolite (MQCA) and Chloramphenicol (CAPA) through amino acid dendrimers of different spacer arms.
Preferably, said R 1 Selected from propyl or pentyl, R 2 Selected from ethyl or butyl.
Further preferably, the MAC multi-cluster tandem linear hapten has the structural formula shown as MAC-4C or MAC-6C:
the preparation method of the MAC type multi-cluster tandem linear hapten comprises the following steps:
s1, respectively preparing olaquindox metabolites, amantadine and chloramphenicol intermediates: methyl acetoacetate is used as a raw material to obtain a olaquindox metabolite intermediate shown in a formula II through NBS bromination, 4-nitroo-phenylenediamine cyclization, nitro reduction, amino protecting group feeding and ester bond hydrolysis; the method comprises the steps of taking adamantane as a raw material, connecting an amino acid arm protected by tert-butoxycarbonyl on 1 amino group after nitration, nitroreduction, aminoacylation and hydrolysis, then connecting fluorenylmethoxycarbonyl on the other 1 amino group, and finally removing Boc protecting group to obtain an amantadine intermediate shown in a formula III; hydrolyzing and removing dichloroacetyl of chloramphenicol, and coupling through an acylation condensation reaction to obtain a chloramphenicol intermediate shown in a formula IV;
wherein R is 1 And R is 2 Respectively selected from alkyl groups with 2-5 carbon atoms, namely R 1 And R is 2 Selected from any of ethyl to pentyl groups respectivelyMeaning one;
s2, condensing and coupling the amantadine intermediate shown in the formula III with the carboxyl of the olaquindox metabolite intermediate shown in the formula II through the amino thereof, removing the Fmoc protecting group on the amino of the amantadine intermediate, condensing and coupling the amino with the carboxyl of the chloramphenicol intermediate shown in the formula IV to obtain an intermediate, and removing the TMB protecting group on the amino terminal of the olaquindox metabolite intermediate to obtain the MAC type multi-cluster tandem linear hapten shown in the formula I.
Preferably, said R 1 Selected from propyl or pentyl, R 2 Selected from ethyl or butyl.
The invention also provides application of the MAC type multi-cluster tandem linear hapten in preparation of an MAC type multi-cluster tandem linear artificial antigen.
A MAC type multi-cluster tandem linear artificial antigen is obtained by amino coupling carrier protein of hapten shown in formula I, and has a molecular structure shown in formula V:
wherein R is 1 And R is 2 Respectively selected from alkyl groups with 2-5 carbon atoms, namely R 1 And R is 2 Selected from any one of ethyl to amyl groups respectively.
Preferably, said R 1 Selected from propyl or pentyl, R 2 Selected from ethyl or butyl.
Further preferably, the MAC multi-cluster tandem linear artificial antigen has the structural formula VIII or IX as follows:
preferably, the carrier protein is Lactoferrin (LF), keyhole Limpet Hemocyanin (KLH) or Ovalbumin (OVA), wherein LF, KLH are used for the preparation of immunogens and OVA are used for the preparation of coating precursors.
The preparation method of the MAC type multi-cluster tandem linear artificial antigen comprises the step of coupling carrier protein on amino groups of the MAC type multi-cluster tandem linear hapten shown in a formula I.
Preferably, the coupling is by diazotisation.
As a preferred embodiment, the preparation method of the MAC multi-cluster tandem linear artificial antigen comprises the following steps: dissolving MAC type multi-cluster tandem linear hapten shown in formula I in 2ml of water, adjusting pH to 2.0 with 0.1M HCl, adding 10mg/ml NaNO 2 About 350. Mu.l, and the reaction was carried out at 4℃for 30 minutes in the absence of light, which was designated as solution A. 10mg of carrier protein was weighed and dissolved in 4mL of CBS buffer solution (0.01M, pH 9.0), and the solution was stirred and dissolved to prepare solution B. Under the magnetic stirring, the solution A is sucked and added into the solution B drop by drop, and the reaction is carried out for 12 hours under the magnetic stirring at the temperature of 4 ℃. The reaction was dialyzed against PBS at 4℃for 3 days, with 2 changes of dialysate per day. The immunogen can be obtained and frozen in a refrigerator at the temperature of minus 20 ℃ for standby.
The invention also provides application of the MAC type multi-cluster tandem line artificial antigen in preparation of monoclonal/polyclonal antibodies, single-chain antibodies or nano antibodies.
A polyclonal antibody is prepared by immunizing mice with MAC type multi-cluster serial linear artificial antigen shown in formula V.
Preferably, the polyclonal antibody uses a carrier protein as Lactoferrin (LF) or a MAC type multi-cluster tandem line artificial antigen of Keyhole Limpet Hemocyanin (KLH) as an immunogen, a female Balb/C mouse of proper age is selected for immunization, the titer and the inhibition rate of antiserum are measured after the 4 th immunization and the 5 th immunization, and the serum of the mouse is collected after the performance is stable, so that the polyclonal antibody is obtained.
The invention also provides an immune detection method for simultaneously detecting amantadine, olaquindox and chloramphenicol, which adopts indirect ELISA, takes any one of the artificial antigens as a coating antigen, and adopts the polyclonal antibody to detect a sample to be detected.
The invention has the following beneficial effects:
the invention provides a MAC type multi-cluster serial linear hapten, the structural general formula of which is shown in formula I, wherein 3 drug skeleton structures of Amantadine (AMD), olaquindox Metabolite (MQCA) and Chloramphenicol (CAPA) are respectively connected in sequence by adopting amino alkyl carboxylic acids or alkyl diacid with different lengths to form the multi-cluster serial linear hapten, artificial antigen is prepared based on the hapten coupling carrier protein, immune animal is tested for immune response effect, the potency of the induced multi-antiserum under 1 mug/mL coating antigen is between 8 and 16K, and the inhibition ratio of the induced multi-antiserum to the 1 mug/mL MAC type multi-cluster serial linear hapten is between 51 and 85 percent, so that the multi-cluster serial linear hapten and the artificial antigen prepared by the invention have good immunogenicity and can be used for preparing monoclonal antibodies, single-chain antibodies and nanoantibodies subsequently. The invention provides a new thought for enriching the multi-component recognition core reagent antibody of the micromolecular organic matters, lays a foundation for innovating the multi-residue analysis method of antibiotics, and has good application prospect.
Drawings
FIG. 1 is an ultraviolet scanning pattern of a MAC type multi-cluster tandem linear hapten, an artificial antigen and a carrier protein.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 preparation of MAC Multi-Cluster tandem Linear hapten
1. A preparation method of a MAC-4C hapten comprises the following steps:
s1, preparation of intermediate 3
Methyl acetoacetate, compound 1 (50 g,431 mmol) was added to water (1L), N-bromosuccinimide (i.e., NBS) (88 g,494 mmol) was added at 70℃and stirred at room temperature for half an hour until the system was transparent. Subsequently 4-nitroo-phenylenediamine, compound 2 (63 g,412 mmol), was added and stirring continued at 70℃for 8 hours, after completion of the reaction monitored by liquid-mass spectrometry (i.e. LC-MS), cooled to room temperature and quenched with PE: EA=1:1 organic solvent extraction (500 mL. Times.6). The organic phases were combined, dried and spun-dried. The crude product was isolated and purified by column chromatography (PE: ea=10:1) to give intermediate 3 (14 g, yield: 13.8%,57 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 248.1。
S2, preparation of intermediate 4
Intermediate 3 (14 g,57 mmol) was dissolved in tetrahydrofuran (i.e., THF) (100 mL) and methanol (100 mL) and zinc powder (40 g,634 mmol) was added. Ammonium chloride (38 g,710 mmol) was added in portions at 0deg.C and the reaction was continued to stir at 0deg.C for half an hour, after completion of the reaction monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction solution was filtered through celite, and the filter cake was washed with a mixed solvent of dichloromethane and methanol. The filtrate was concentrated. The residue was separated and purified by column chromatography (PE: ea=100:0 to 50:100) to give intermediate 4 (7 g, yield: 56.14%,32 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 218.1。 1 H NMR(400MHz,DMSO)δ7.72(d,J＝9.0Hz,1H),7.32(dd,J＝9.0,2.5Hz,1H),6.90(d,J＝2.4Hz,1H),6.11(s,2H),3.93(s,3H),2.68(s,3H)。
S3, preparation of intermediate 5
Intermediate 4 (7 g,32 mmol) and 2, 4-dimethoxybenzaldehyde (i.e., DMB) (10.2 g,61.4 mmol) were dissolved in dichloromethane (i.e., DCM) (100 mL) at room temperature, sodium triacetoxyborohydride (i.e., STAB) (20.5 g,96.7 mmol) was added and the reaction was stirred at room temperature overnight. After completion of the reaction was monitored by liquid-mass spectrometry (i.e., LC-MS), water (100 mL) was added to the reaction solution, extracted with EA (100 ml×5), and the organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by column chromatography (PE: ea=100:0 to 65:100) to give intermediate 5 (8.5 g, yield: 72.2%,23.1 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 368.0。 1 H NMR(400MHz,DMSO)δ7.74(t,J＝11.0Hz,1H),7.43(dt,J＝27.5,13.8Hz,1H),7.17(d,J＝8.3Hz,1H),7.05(t,J＝5.6Hz,1H),6.63(dd,J＝16.4,2.4Hz,2H),6.47(dd,J＝8.3,2.4Hz,1H),4.28(d,J＝5.6Hz,2H),3.91(s,3H),3.85(s,3H),3.74(s,3H),2.69(s,3H)。
S4, preparation of intermediate 6
Intermediate 5 (8.5 g,23.1 mmol) and NaOH (1.85 g,46.2 mmol) were dissolved in THF (20 mL) and water (10 mL). The reaction solution was stirred at room temperature for 1 hour. After completion of the reaction by liquid-mass spectrometry (i.e., LC-MS), the pH was adjusted to weak acidity with concentrated hydrochloric acid, followed by extraction with DCM (100 ml×8), and the organic layer was washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. After filtration, intermediate 6 (7.4 g, yield: 90.5%,20.9 mmol) was concentrated to give an orange solid, namely, a olaquindox metabolite (3-methyl-quinoxaline-2-carboxylic acid) intermediate. MS (ESI) M/z (M+H) + 354.1。 1 H NMR(400MHz,DMSO)δ7.70(d,J＝9.1Hz,1H),7.40(dd,J＝9.1,2.4Hz,1H),7.17(d,J＝8.3Hz,1H),7.00(s,1H),6.62(dd,J＝16.2,2.4Hz,2H),6.47(dd,J＝8.4,2.3Hz,1H),4.27(d,J＝5.6Hz,2H),3.85(s,3H),3.73(s,4H),2.66(s,3H)。
S5, preparation of intermediate 8
Amantadine, compound 7 (75 g, 553mmol), was slowly added to stirred fuming nitric acid (345 g) at 0deg.C and the reaction was continued with stirring at 0deg.C for 1 hour. Acetonitrile (55.5 g,1.37 mol) was then added dropwise at 0℃and stirring was continued at 0℃for 1 hour. Finally concentrated sulfuric acid (705 g) was slowly added at 0℃followed by stirring overnight at room temperature. After completion of the reaction, which was monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction solution was poured into ice (about 1 kg), pH was adjusted to alkaline with sodium carbonate solid, and a yellow viscous substance was precipitated. The water was decanted, EA (about 100 mL) was added to agglomerate the yellow viscous material, and filtered. The yellow precipitate was washed with EA followed by water to give intermediate 8 (80 g) as a white solidA body. MS (ESI) M/z (M+H) + 251.1。 1 H NMR(400MHz,DMSO)δ7.36(s,2H),2.11(d,J＝10.5Hz,2H),2.10–1.98(m,2H),1.91–1.74(m,8H),1.73(s,6H),1.49(s,2H)。
S6, preparation of intermediate 9
Intermediate 8 (70 g) was reacted in HCl (10%, 500 mL) with stirring at 110℃for 2 days, after completion of the reaction monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction mixture was concentrated under reduced pressure to give intermediate 9 (63 g,264 mmol) as a white solid. MS (ESI) M/z (M+H) + 167.1。 1 H NMR(400MHz,DMSO)δ8.40(s,6H),2.33-2.20(m,2H),2.02(s,2H),1.84-1.66(m,8H),1.52(s,2H)。
S7, preparation of intermediate 11
Boc-protected 4-aminobutyric acid, compound 10 (5.66 g,27.8 mmol), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (12.7 g,33.4 mmol) and N, N-Diisopropylethylamine (DIEA) (10.8 g,83.6 mmol) were dissolved in 50mL of dimethyl sulfoxide (DMSO) and the reaction stirred at room temperature for 1 h. The above liquid was slowly dropped into a solution of intermediate 9 (10 g,41.8 mmol) and DIEA (10.8 g,83.6 mmol) in DMSO (100 mL) while stirring, and after the reaction was continued to be completed at room temperature with stirring for half an hour, after monitoring the reaction completion by a liquid-mass spectrometer (i.e., LC-MS), fluorenylmethoxycarbonyl chloride (Fmoc-Cl) (21.63 g,83.6 mmol) and DIEA (10.8 g,83.6 mmol) were directly added to the reaction solution, and the reaction was continued to be stirred for 1 hour with stirring, after monitoring the reaction completion by a liquid-mass spectrometer (i.e., LC-MS), water (300 mL) was added, extracted with EA (100 ml×5), and the organic layer was washed with saturated brine (100 mL) and dried with anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by column chromatography (PE: ea=100:0 to 35:65) to give intermediate 11 (6.9 g, yield: 43.2%,12 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 574.3。 1 H NMR(400MHz,DMSO)δ7.88(d,J＝7.5Hz,1H),7.71(d,J＝7.4Hz,1H),7.48–7.36(m,1H),7.33(td,J＝7.4,1.1Hz,1H),7.11(s,1H),6.76(s,1H),4.18(s,1H),2.94–2.79(m,1H),2.11(s,2H),2.03–1.93(m,3H),1.92–1.61(m,4H),1.54(dt,J＝14.3,7.2Hz,2H),1.37(s,5H)。
S8 preparation of intermediate 12
Intermediate 11 (6.9 g,12 mmol) was dissolved in 4M dioxane hydrochloride solution (50 mL) and the reaction stirred at room temperature for 1 hour. Thin Layer Chromatography (TLC) showed the reaction was complete. The reaction was concentrated to give intermediate 12 (7 g) as a pale yellow oily liquid. MS (ESI) M/z (M+H) + 474.2。 1 H NMR(400MHz,DMSO)δ7.89(d,J＝7.5Hz,6H),7.71(d,J＝7.4Hz,2H),7.52(s,1H),7.41(t,J＝7.2Hz,3H),7.33(td,J＝7.4,1.1Hz,2H),7.12(s,1H),4.20(d,J＝14.9Hz,3H),2.74(dd,J＝14.1,6.4Hz,2H),2.17–2.03(m,6H),1.78(ddd,J＝25.2,22.1,15.0Hz,12H),1.51(s,2H)。
S9, preparation of intermediate 13
Intermediate 6 (3.5 g,9.9 mmol), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (i.e., HATU) (4.5 g,11.8 mmol) and N, N-diisopropylethylamine (i.e., DIEA) (3.8 g,30 mmol) were dissolved in 20mL of dichloromethane and the reaction stirred at room temperature for 1 hour. The above liquid was added to a stirring solution of intermediate 12 (6 g) in DCM (100 mL) and the reaction was continued at room temperature for half an hour, after completion of the reaction by liquid-mass spectrometry (i.e., LC-MS), water (100 mL) was added, extracted with DCM (100 mL. Times.3), and the organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was separated and purified by column chromatography (PE: ea=100:0 to 0:100) to give intermediate 13 (6.9 g, yield: 85.9%,8.5 mmol) as an orange solid. MS (ESI) M/z (M+H) + 809.2。 1 H NMR(400MHz,CDCl3)δ8.24(t,J＝6.1Hz,1H),7.81–7.70(m,3H),7.56(t,J＝12.1Hz,2H),7.39(t,J＝7.3Hz,2H),7.34–7.28(m,2H),7.24(d,J＝8.3Hz,1H),7.18(dd,J＝9.1,2.6Hz,1H),6.96(d,J＝2.5Hz,1H),6.51(d,J＝2.3Hz,1H),6.46(dd,J＝8.2,2.3Hz,1H),4.38(d,J＝5.5Hz,2H),4.19(t,J＝6.1Hz,1H),3.84(d,J＝11.5Hz,3H),3.80(s,3H),3.54(q,J＝6.5Hz,2H),3.14(dt,J＝12.8,6.4Hz,1H),3.04(d,J＝8.3Hz,3H),2.80(s,6H),2.21(t,J＝6.9Hz,4H),1.95(dt,J＝30.2,8.9Hz,9H),1.45(t,J＝5.6Hz,6H)。
S10, preparation of intermediate 15
Chloramphenicol, compound 14 (25 g,77.6 mmol) was stirred in HCl (1M, 500 mL) at 100deg.C overnight. LC-MS showed the reaction was complete. The reaction mixture was cooled to room temperature, and dichloroacetic acid was removed by extraction with Ethyl Acetate (EA) (500 mL. Times.3). The remaining aqueous phase was spin-dried to give chloramphenicol hydrolysate chloramphenicol amine (CAPA) as intermediate 15 (15 g, yield: 91.1%,70.7 mmol) as a white solid.
S11, preparation of intermediate 17
Intermediate 15 (1 g,4.03 mmol), succinic anhydride, compound 16 (402 mg,4.02 mmol) and DIEA (1 g,8.04 mmol) were dissolved in 10ml of n, n-Dimethylformamide (DMF) at room temperature and stirred overnight. LC-MS showed the reaction was complete. The reaction solution was filtered and purified by reverse phase column (formic acid FA, water: acetonitrile=100:0 to 85:15) to obtain intermediate 17 (1 g, yield: 80%,3.2 mmol) as a white solid. MS (ESI) M/z (M+H) +313.0.
S12, preparation of intermediate 18
Intermediate 13 (1 g,1.2 mmol) was dissolved in 20mL ethanol (EtOH), diethylamine (5 mL) was added and the reaction was continued with stirring at room temperatureAnd 5 hours. TLC showed the reaction was complete. The reaction was concentrated and dried twice with EtOH. The residue was dissolved with Ethyl Acetate (EA), filtered through a small amount of silica gel (200-300 mesh), and after washing the impurities with EA, the silica gel was washed with methanol to which diethylamine was added. The obtained filtrate was dried by spinning, and taken twice with ethanol to obtain intermediate 18 (650 mg, yield: 92.4%,1.11 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 587.3。1H NMR(400MHz,DMSO)δ8.67(t,J＝5.9Hz,1H),7.70(d,J＝9.1Hz,1H),7.44–7.32(m,2H),7.15(d,J＝8.3Hz,1H),6.99(t,J＝5.5Hz,1H),6.64(dd,J＝20.6,2.3Hz,2H),6.47(dd,J＝8.3,2.4Hz,1H),4.27(d,J＝5.7Hz,2H),3.85(s,4H),3.73(s,4H),3.26–3.18(m,5H),2.72–2.67(m,4H),2.11–2.03(m,4H),1.82–1.64(m,9H),1.46(d,J＝19.2Hz,7H)。
S13, preparation of intermediate 19
Intermediate 18 (650 mg,1.11 mmol), intermediate 17 (348 mg,1.11 mmol) and DIEA (428 mg,3.33 mmol) were dissolved in DMF (20 mL), HATU (506 mg,1.33 mmol) was added and the reaction stirred at room temperature for 1 hour. LC-MS showed the reaction was complete. Water (50 mL) was added, the mixture was extracted with EA (50 mL. Times.3), and the organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by reverse phase column (FA, water: acetonitrile=100:0 to 50:50) to give intermediate 19 (527 mg, yield: 53.9%,0.599 mmol) as an orange solid. MS (ESI) M/z (M+H) + 881.3。
Preparation of S14.MAC-4C
Intermediate 19 (227 mg,0.599 mmol) was dissolved in DCM (10 mL), 1mL trifluoroacetic acid (TFA) was added and the reaction was continued with stirring at room temperature for half an hour. TLC showed the reaction was complete. The reaction solution was concentrated and passed through a high-efficiency reverse phase column to prepare MAC-4C (200 mg, yield: 45.7%,0.273 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 731.1。1H NMR(400MHz,DMSO)δ8.67(t,J＝5.8Hz,1H),8.16(d,J＝8.8Hz,2H),7.68(d,J＝9.0Hz,1H),7.57(d,J＝8.7Hz,2H),7.50(d,J＝9.1Hz,1H),7.37(s,1H),7.30(s,1H),7.25(dd,J＝9.0,2.5Hz,1H),6.92(d,J＝2.4Hz,1H),6.03(s,2H),5.76(d,J＝4.8Hz,1H),5.03–4.95(m,1H),5.00–4.77(t,1H),3.94(d,J＝8.0Hz,1H),3.56–3.48(m,1H),3.30–3.22(m,3H),2.69(s,3H),2.18(dd,J＝13.9,7.4Hz,2H),2.14–2.03(m,8H),1.78(dt,J＝14.4,9.2Hz,10H),1.48(s,2H)。
2. A preparation method of a MAC-6C hapten comprises the following steps:
S1-S6 are the same as the preparation process of the MAC-4C hapten;
s7, preparation of intermediate 21
Boc protected aminocaproic acid, compound 20 (5.66 g,27.8 mmol), HATU (12.7 g,33.4 mmol) and DIEA (10.8 g,83.6 mmol) were dissolved in DMSO (50 mL) and the reaction stirred at room temperature for 1 h. The above liquid was slowly added dropwise to a stirring solution of intermediate 9 (10 g,41.8 mmol) and DIEA (10.8 g,83.6 mmol) in DMSO (100 mL) and the reaction was continued with stirring at room temperature for half an hour. LC-MS showed that the reaction was complete. Fmoc-Cl (21.63 g,83.6 mmol) and DIEA (10.8 g,83.6 mmol) were added directly to the reaction solution and the reaction was continued with stirring for 1 hour. LC-MS showed the reaction was complete. Water (300 mL) was added, the mixture was extracted with EA (100 mL. Times.5), and the organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by column chromatography (PE: ea=100:0 to 35:65) to give intermediate 21 (8.2 g, yield: 48.9%,13.6 mmol) as a pale yellow solid. MS (ESI) M/z (M+H-100) + 502.1。 1 H NMR(400MHz,DMSO)δ7.88(d,J＝7.5Hz,2H),7.70(s,2H),7.41(s,2H),7.34(dd,J＝7.4,1.0Hz,3H),7.10(s,1H),6.74(t,J＝5.3Hz,1H),4.18(s,3H),4.03(q,J＝7.1Hz,1H),3.00–2.79(m,2H),2.11(s,4H),1.90–1.70(m,8H),1.36(s,16H),1.18(t,J＝7.1Hz,4H)。
S8, preparation of intermediate 22
Intermediate 21 (8.2 g,13.6 mmol) was dissolved in 4M dioxane hydrochloride (50 mL) and the reaction was continued with stirring at room temperature for 1 hour, the Boc protecting group was removed, and after TLC showed completion of the reaction, the reaction was concentrated to give intermediate 22 (8.12 g) as a pale yellow oily liquid. MS (ESI) M/z (M+H) + 502.3。 1 H NMR(400MHz,DMSO)δ7.37(dt,J＝32.7,7.4Hz,1H),7.37(dt,J＝32.7,7.4Hz,1H),4.18(s,1H),3.57(s,2H),2.75–2.71(m,0H),2.69(s,1H),2.52–2.48(m,1H),2.12(s,1H),2.01(t,J＝7.3Hz,0H),1.83(dd,J＝36.3,23.0Hz,1H),1.50(dtd,J＝22.5,15.0,7.5Hz,1H),1.33–1.17(m,1H)。
S9 preparation of intermediate 23
Intermediate 6 (1.8 g,5.1 mmol), HATU (2.3 g,6.1 mmol) and DIEA (1.9 g,15.3 mmol) were dissolved in DCM (20 mL) and the reaction stirred at room temperature for an additional 1 h. The above liquid was added to a stirring solution of intermediate 22 (2.6 g,5.1 mmol) in DCM (100 mL) and the reaction was continued with stirring at room temperature for half an hour. LC-MS showed that the reaction was complete. Water (100 mL) was added, the mixture was extracted with DCM (100 mL. Times.3), and the organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was separated and purified by column chromatography (PE: ea=100:0 to 0:100) to give intermediate 23 (3.4 g, yield: 80.4%,4.1 mmol) as an orange solid. MS (ESI) M/z (M+H) + 837.2。 1 H NMR(400MHz,DMSO)δ8.65(s,1H),7.88(d,J＝7.4Hz,2H),7.70(dd,J＝8.2,4.0Hz,3H),7.40(dd,J＝13.7,4.9Hz,3H),7.32(td,J＝7.4,1.0Hz,3H),7.15(d,J＝8.3Hz,1H),6.98(s,1H),6.63(dd,J＝20.1,2.4Hz,2H),6.46(dd,J＝8.4,2.4Hz,1H),5.76(s,1H),4.27(d,J＝5.6Hz,2H),4.18(s,3H),3.84(s,3H),3.73(s,3H),3.28–3.21(m,2H),2.69(d,J＝1.4Hz,4H),2.08(d,J＝17.2Hz,3H),1.79(d,J＝20.5Hz,7H),1.58–1.41(m,7H),1.28(dd,J＝19.1,12.1Hz,7H)。
S10, preparation of intermediate 24
Intermediate 23 (3.0 g,3.6 mmol) was dissolved in EtOH (20 mL), diethylamine (5 mL) was added and the reaction stirred at room temperature for 5 hours. TLC showed the reaction was complete. The reaction was concentrated and dried twice with EtOH. The residue was dissolved with EA, filtered through a small amount of silica gel (200-300 mesh), and after washing the impurities with EA, the silica gel was washed with methanol to which diethylamine was added. The obtained filtrate was dried by spinning, and taken twice with ethanol to obtain intermediate 24 (2.2 g, yield: 97.2%,3.5 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 615.3。 1 H NMR(400MHz,DMSO)δ8.65(t,J＝5.9Hz,1H),7.70(d,J＝9.1Hz,1H),7.39(dd,J＝9.1,2.5Hz,1H),7.25(s,1H),7.15(d,J＝8.4Hz,1H),6.98(t,J＝5.7Hz,1H),6.63(dd,J＝17.9,2.4Hz,2H),6.47(dd,J＝8.3,2.4Hz,1H),5.32(t,J＝4.7Hz,0H),4.39–4.19(m,3H),3.85(s,3H),3.73(s,3H),3.55–3.40(m,4H),2.69(s,3H),1.74(d,J＝16.2Hz,6H),1.56–1.44(m,5H),0.85(t,J＝6.7Hz,1H)。
S11 preparation of intermediate 26
Monomethyl adipate, compound 25 (2.19 g,13.7 mmol), HATU (7.81 g,20.6 mmol) and DIEA (8.85 g,68.5 mmol) were dissolved in DMF (50 mL) and the reaction stirred at room temperature for 1 h. Intermediate 15 (2.9 g,13.7 mmol) was added to the solution and the reaction was continued with stirring at room temperature for half an hour. LC-MS showed the reaction was complete. The filtered liquid was separated and purified by reverse phase column chromatography (FA, water: acetonitrile=100:0 to 90:10) to give intermediate 26 (3.2 g, yield: 65.7%,9.0 mmol) as a white solid. MS (ESI) M/z (M+H) + 355.2。
S12 preparation of intermediate 27
Intermediate 26 (1.6 g,4.5 mmol) and LiOH (0.4 g,9 mmol) were dissolved in THF (20 mL) and water (10 mL) and stirring continued at room temperatureThe mixture was stirred and reacted for 1 hour. LC-MS showed the reaction was complete. The reaction solution was adjusted to pH neutral with concentrated hydrochloric acid, and after removal of THF by distillation under reduced pressure, intermediate 27 (1.8 g) was obtained as a white solid by lyophilization without further purification. MS (ESI) M/z (M-H) - 339.1。
S13 preparation of intermediate 28
Intermediate 24 (2.2 g,2.93 mmol), intermediate 27 (1.1 g,3.22 mmol) and DIEA (1.13 g,8.79 mmol) were dissolved in DMF (20 mL), HATU (1.34 g,3.52 mmol) was added and the reaction stirred at room temperature for 1 hour. LCMS showed complete reaction. Water (50 mL) was added, the mixture was extracted with EA (50 mL. Times.3), and the organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was isolated and purified by reverse phase column (FA, water: acetonitrile=100:0 to 50:50) to give 28 (1 g, yield: 35.1%,1.0 mmol) as an orange solid. MS (ESI) M/z (M+H) + 937.2。 1 H NMR(400MHz,DMSO)δ8.66(s,1H),8.38(s,1H),8.15(d,J＝8.7Hz,2H),7.70(d,J＝9.2Hz,1H),7.57(d,J＝8.7Hz,2H),7.50–7.34(m,2H),7.21(dd,J＝43.5,15.5Hz,4H),6.98(d,J＝5.2Hz,1H),6.63(dd,J＝18.0,2.2Hz,3H),6.56–6.43(m,1H),5.32(s,1H),5.01(s,1H),4.84(s,1H),4.27(d,J＝5.5Hz,2H),3.97(s,1H),3.85(s,3H),3.73(s,3H),3.51(s,2H),2.68(s,4H),2.11(s,2H),1.91–1.71(m,11H),1.63–1.39(m,8H)。
Preparation of S14.MAC-6C
Intermediate 28 (1 g,1.06 mmol) was dissolved in DCM (20 mL), TFA (2 mL) was added and the reaction stirred at room temperature for 5min. TLC showed the reaction was complete. The reaction solution was concentrated and passed through a high-efficiency reverse phase column to prepare MAC-6C (350 mg, yield: 44.5%,0.44 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 787.4。 1 H NMR(400MHz,DMSO)δ8.65(t,J＝5.8Hz,1H),8.15(d,J＝8.8Hz,2H),7.68(d,J＝9.0Hz,1H),7.57(d,J＝8.7Hz,2H),7.44(d,J＝9.2Hz,1H),7.29(s,1H),7.25(dd,J＝9.0,2.5Hz,1H),7.23(s,1H),6.91(d,J＝2.4Hz,1H),6.02(s,2H),5.80(d,J＝4.8Hz,1H),5.01(dd,J＝4.6,2.4Hz,1H),4.82(dd,J＝6.3,4.7Hz,1H),3.98(d,J＝8.8Hz,1H),3.54(dd,J＝15.8,9.2Hz,1H),3.30–3.22(m,3H),2.68(s,3H),2.11(s,2H),2.07–1.93(m,6H),1.89(t,J＝6.7Hz,2H),1.86–1.74(m,8H),1.59–1.43(m,6H),1.35–1.17(m,6H)。
Example 2 preparation of MAC Multi-Cluster tandem Linear Artificial antigen
The embodiment provides a preparation method of a MAC type multi-cluster tandem linear artificial antigen, which mainly comprises the synthesis of an immunogen and a coating antigen. The immunogen is different from the coating antigen in the preparation of carrier types, and the carrier used by the immunogen is bovine Lactoferrin (LF) and Keyhole Limpet Hemocyanin (KLH); the coating antigen adopts Ovalbumin (OVA) as carrier protein. A method for preparing immunogen. The synthetic immunogen/coating antigen method adopted by the invention is a diazotization method. The method comprises the following specific steps:
weighing 22.09mg of MAC-4C (23.79 mg of MAC-6C) and dissolving in 1ml of water, adjusting pH to 2.0 with 0.1M HCl, and adding 10mg/ml NaNO 2 About 350. Mu.l, and the reaction was carried out at 4℃for 30 minutes in the absence of light, which was designated as solution A. 10mg of carrier protein was weighed and dissolved in 4mL of CBS buffer solution (0.01M, pH 9.0), and the solution was stirred and dissolved to prepare solution B. Under the magnetic stirring, the solution A is sucked and added into the solution B drop by drop, and the reaction is carried out for 12 hours under the magnetic stirring at the temperature of 4 ℃. The reaction was dialyzed against PBS at 4℃for 3 days, with 2 changes of dialysate per day. The immunogen can be obtained and frozen in a refrigerator at the temperature of minus 20 ℃ for standby. The hapten prepared in example 1 and the artificial antigen prepared in example 2 are respectively identified by ultraviolet wavelength scanning (150-400 nm), and the results are shown in figure 1, and compared with the carrier protein and the hapten, the antigen coupling is successful as shown in the results, wherein the characteristic absorption peak and peak shape are changed after coupling.
EXAMPLE 3 preparation of polyclonal serum with multiple clusters in series Linear Artificial antigen specificity
Animal immunization: using healthy 6-week-old Balb/c female mice as experimental animals, the two immunogens prepared in example 2 were each mixed and emulsified with equal amounts of adjuvant (first complete Freund's adjuvant followed by incomplete Freund's adjuvant), and subcutaneously injected into the neck, back and abdominal cavities of the mice at an immunization dose of 0.5mL (containing 0.5mg immunogen). The first immunization is carried out by emulsifying 0.5mL of complete Freund's adjuvant with antigen for immunization, emulsifying 0.5mL of incomplete Freund's adjuvant with antigen for 4 weeks for boosting, then immunization is carried out once every 2 weeks, a small amount of blood is taken from tail vein for antibody quality identification, after the antibody is stable, the best-performance mouse is selected for collecting serum, the temperature bath is carried out for 30min at 37 ℃, the room temperature is centrifuged for 20min, the supernatant is taken out and split-packed in a centrifuge tube, and the supernatant is stored at-20 ℃ for use.
Evaluation of antiserum effects: the coating antigen MAC-4C-OVA or MAC-6C-OVA prepared in example 2 is taken as a detection antibody, the serum of the mice is measured by adopting an indirect competition ELISA method, and the potency and the inhibition rate of each antiserum are comprehensively considered to evaluate the serum. The specific operation steps are as follows:
1) And (3) wrapping the plate: the coating antigen MAC-4℃ -OVA or MAC-6C-OVA was diluted to 1000ng/mL with 0.05M carbonate buffer (pH 9.6) and coated at 100. Mu.L/well overnight at 4 ℃; removing the coating liquid, washing with PBST for 2 times, adding 120 μl of sealing liquid (5% skimmed milk) into each hole, and sealing at 37deg.C for 3 hr; discarding the sealing liquid, drying at 37 ℃ for 60min, and sealing the sealing bag at 4 ℃ for standby to obtain the packaged ELISA plate.
2) Serum titers and inhibition assays: and (3) the ELISA plate packaged in the step (1) is provided with the titer column: 50 mu L of PBS and 50 mu L of serum diluted in a gradient multiple are added to each well respectively; inhibition column: mu.L of diluted 1000ng/mL drug (MAC-4C or MAC-6C) and 50. Mu.L of serum diluted in a gradient multiple were added to each well and 2 groups were performed in parallel. Incubating at 37deg.C for 40min, washing with PBST for five times, drying the liquid in the hole, adding enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted at 1:5000, incubating at 37deg.C for 30min, washing with PBST for five times, drying the liquid in the hole, adding 100 μl TMB substrate solution, and developing at 37deg.C in dark for 10min; mu.L of stop solution (10% H) was added 2 SO 4 ) Terminating the reaction; the absorbance at 450nm was read with a microplate reader.
3) Experimental results
The results of ELISA-based measurement of the serum titers and inhibition ratios of mice are shown in Table 1, wherein the titers are between 8 and 16K, and the inhibition ratios of the MAC-4C and the MAC-6C at the concentration of 1 mug/mL are between 51 and 85%.
TABLE 1 mouse antiserum Effect
The results show that the MAC type multi-cluster tandem linear hapten and the artificial antigen thereof prepared by the invention have good immunogenicity and can be used for preparing monoclonal antibodies, single-chain antibodies and nano antibodies subsequently.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. A MAC type multi-cluster tandem linear hapten sequentially containing 3-methyl-quinoxaline-2-carboxylic acid, amantadine and chloramphenicol skeleton structural units is characterized by having a molecular structure shown in a formula I:
wherein R is 1 And R is 2 Respectively selected from alkylene groups with 2-5 carbon atoms.
2. The method for preparing the MAC type multi-cluster tandem linear hapten, which is characterized by comprising the following steps:
s1, respectively preparing a olaquindox metabolite intermediate, an amantadine intermediate and a chloramphenicol intermediate: methyl acetoacetate is used as a raw material to obtain a olaquindox metabolite intermediate shown in a formula II through NBS bromination, 4-nitroo-phenylenediamine cyclization, nitro reduction, amino protecting group feeding and ester bond hydrolysis; the method comprises the steps of taking adamantane as a raw material, connecting an amino acid arm protected by tert-butoxycarbonyl on 1 amino group after nitration, nitroreduction, aminoacylation and hydrolysis, connecting fluorenylmethoxycarbonyl on the other 1 amino group, and finally removing Boc protecting group to obtain an amantadine intermediate shown in a formula III; hydrolyzing and removing dichloroacetyl of chloramphenicol, and coupling through an acylation condensation reaction to obtain a chloramphenicol intermediate shown in a formula IV;
wherein R is 1 And R is 2 Respectively selected from alkylene with 2-5 carbon atoms;
s2, condensing and coupling the amantadine intermediate shown in the formula III with the carboxyl of the olaquindox metabolite intermediate shown in the formula II through the amino thereof, removing Fmoc protecting group on the amino of the amantadine intermediate, condensing and coupling the amino with the carboxyl of the chloramphenicol intermediate shown in the formula IV to obtain an intermediate, and removing protecting group on the amino at the end of the olaquindox metabolite intermediate to obtain the MAC multi-cluster tandem linear hapten shown in the formula I.
3. A MAC type multi-cluster tandem linear artificial antigen, which is obtained from the amino-coupled carrier protein of the hapten as defined in claim 1, and has a molecular structure represented by formula V:
wherein R is 1 And R is 2 Respectively selected from alkylene groups with 2-5 carbon atoms.
4. The MAC-type multi-cluster tandem linear artificial antigen of claim 3, wherein the carrier protein is lactoferrin, keyhole limpet hemocyanin, or ovalbumin.
5. The method for preparing the MAC type multi-cluster tandem linear artificial antigen according to claim 3 or 4, which is characterized in that the carrier protein is coupled on the amino group of the MAC type multi-cluster tandem linear hapten according to claim 1.
6. The method of claim 5, wherein the coupling is by diazotisation.
7. Use of the MAC-type multi-cluster tandem linear artificial antigen according to claim 3 or 4 for preparing monoclonal/polyclonal antibodies, single chain antibodies or nanobodies.
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