CN116754760B - Method for coupling 2, 4-Dinitrophenol (DNP) with controlled cleavage of antibody - Google Patents
Method for coupling 2, 4-Dinitrophenol (DNP) with controlled cleavage of antibody Download PDFInfo
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- CN116754760B CN116754760B CN202310705253.8A CN202310705253A CN116754760B CN 116754760 B CN116754760 B CN 116754760B CN 202310705253 A CN202310705253 A CN 202310705253A CN 116754760 B CN116754760 B CN 116754760B
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- China
- Prior art keywords
- disulfide
- hydroxyethyl
- bis
- dnp
- dinitrophenol
- Prior art date
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Links
- 238000000034 method Methods 0.000 title claims abstract description 21
- 230000008878 coupling Effects 0.000 title claims abstract description 20
- 238000010168 coupling process Methods 0.000 title claims abstract description 20
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 20
- UFBJCMHMOXMLKC-UHFFFAOYSA-N 2,4-dinitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O UFBJCMHMOXMLKC-UHFFFAOYSA-N 0.000 title claims abstract description 18
- 238000003776 cleavage reaction Methods 0.000 title claims abstract description 10
- 230000007017 scission Effects 0.000 title claims abstract description 10
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 claims abstract description 43
- 150000002148 esters Chemical class 0.000 claims abstract description 43
- KYNFOMQIXZUKRK-UHFFFAOYSA-N 2,2'-dithiodiethanol Chemical compound OCCSSCCO KYNFOMQIXZUKRK-UHFFFAOYSA-N 0.000 claims abstract description 28
- -1 alcohol amine Chemical class 0.000 claims abstract description 28
- 239000002243 precursor Substances 0.000 claims abstract description 13
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 11
- UOULCEYHQNCFFH-UHFFFAOYSA-M sodium;hydroxymethanesulfonate Chemical compound [Na+].OCS([O-])(=O)=O UOULCEYHQNCFFH-UHFFFAOYSA-M 0.000 claims abstract description 9
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims abstract description 9
- VYZAHLCBVHPDDF-UHFFFAOYSA-N Dinitrochlorobenzene Chemical group [O-][N+](=O)C1=CC=C(Cl)C([N+]([O-])=O)=C1 VYZAHLCBVHPDDF-UHFFFAOYSA-N 0.000 claims abstract description 6
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000001590 oxidative effect Effects 0.000 claims abstract description 6
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 claims abstract description 4
- PBOPJYORIDJAFE-UHFFFAOYSA-N 2,4-dinitrobromobenzene Chemical compound [O-][N+](=O)C1=CC=C(Br)C([N+]([O-])=O)=C1 PBOPJYORIDJAFE-UHFFFAOYSA-N 0.000 claims abstract description 4
- FXMKXMJLXRTQSW-UHFFFAOYSA-N 2,4-dinitroiodobenzene Chemical compound [O-][N+](=O)C1=CC=C(I)C([N+]([O-])=O)=C1 FXMKXMJLXRTQSW-UHFFFAOYSA-N 0.000 claims abstract description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical group OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 21
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 10
- 239000003638 chemical reducing agent Substances 0.000 claims description 7
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 239000011230 binding agent Substances 0.000 claims description 6
- 239000003444 phase transfer catalyst Substances 0.000 claims description 6
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 claims description 6
- 229940031098 ethanolamine Drugs 0.000 claims description 5
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical group [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- HCUYBXPSSCRKRF-UHFFFAOYSA-N diphosgene Chemical compound ClC(=O)OC(Cl)(Cl)Cl HCUYBXPSSCRKRF-UHFFFAOYSA-N 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 claims description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N 1-butanol Substances CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 3
- GIVLNOBJINYUNK-UHFFFAOYSA-N 3-[bis(3-oxopropyl)phosphanyl]propanal hydrochloride Chemical compound Cl.C(=O)CCP(CCC=O)CCC=O GIVLNOBJINYUNK-UHFFFAOYSA-N 0.000 claims description 3
- BLFRQYKZFKYQLO-UHFFFAOYSA-N 4-aminobutan-1-ol Chemical compound NCCCCO BLFRQYKZFKYQLO-UHFFFAOYSA-N 0.000 claims description 3
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 3
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical compound ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 claims description 3
- WUGQZFFCHPXWKQ-UHFFFAOYSA-N Propanolamine Chemical compound NCCCO WUGQZFFCHPXWKQ-UHFFFAOYSA-N 0.000 claims description 3
- 238000003403 chloroformylation reaction Methods 0.000 claims description 3
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 claims description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 3
- XMYQHJDBLRZMLW-UHFFFAOYSA-N methanolamine Chemical group NCO XMYQHJDBLRZMLW-UHFFFAOYSA-N 0.000 claims description 3
- 229940087646 methanolamine Drugs 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- NAWZSHBMUXXTGV-UHFFFAOYSA-M triethyl(hexyl)azanium;bromide Chemical compound [Br-].CCCCCC[N+](CC)(CC)CC NAWZSHBMUXXTGV-UHFFFAOYSA-M 0.000 claims description 3
- FASLCARXKMYXDH-UHFFFAOYSA-M triethyl(octyl)azanium;bromide Chemical compound [Br-].CCCCCCCC[N+](CC)(CC)CC FASLCARXKMYXDH-UHFFFAOYSA-M 0.000 claims description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 35
- 238000006243 chemical reaction Methods 0.000 description 31
- 239000000243 solution Substances 0.000 description 22
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 230000003321 amplification Effects 0.000 description 12
- 238000003199 nucleic acid amplification method Methods 0.000 description 12
- 239000004793 Polystyrene Substances 0.000 description 11
- 229920002223 polystyrene Polymers 0.000 description 11
- 239000007810 chemical reaction solvent Substances 0.000 description 10
- 229960003180 glutathione Drugs 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000004005 microsphere Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 8
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 8
- 238000005481 NMR spectroscopy Methods 0.000 description 8
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 8
- 229910052739 hydrogen Inorganic materials 0.000 description 8
- 239000001257 hydrogen Substances 0.000 description 8
- 239000011325 microbead Substances 0.000 description 8
- 238000010898 silica gel chromatography Methods 0.000 description 8
- 238000001228 spectrum Methods 0.000 description 8
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 7
- 239000012099 Alexa Fluor family Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 7
- 125000003396 thiol group Chemical group [H]S* 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 150000003573 thiols Chemical class 0.000 description 5
- 241000283707 Capra Species 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000000386 microscopy Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 4
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 3
- 108010024636 Glutathione Proteins 0.000 description 3
- 239000007987 MES buffer Substances 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical group [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000007800 oxidant agent Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- HKNNAYPWWDWHFR-UHFFFAOYSA-N 1-sulfanylbutan-1-ol Chemical compound CCCC(O)S HKNNAYPWWDWHFR-UHFFFAOYSA-N 0.000 description 2
- PDXOPTFTOFXXSU-UHFFFAOYSA-N 1-sulfanylpentan-1-ol Chemical compound CCCCC(O)S PDXOPTFTOFXXSU-UHFFFAOYSA-N 0.000 description 2
- AEUVIXACNOXTBX-UHFFFAOYSA-N 1-sulfanylpropan-1-ol Chemical compound CCC(O)S AEUVIXACNOXTBX-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical group ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- LLIANSAISVOLHR-GBCQHVBFSA-N 5-[(3as,4s,6ar)-2-oxidanylidene-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LLIANSAISVOLHR-GBCQHVBFSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 238000006736 Huisgen cycloaddition reaction Methods 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 125000000304 alkynyl group Chemical group 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012650 click reaction Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229940050176 methyl chloride Drugs 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- NTTOTNSKUYCDAV-UHFFFAOYSA-N potassium hydride Chemical compound [KH] NTTOTNSKUYCDAV-UHFFFAOYSA-N 0.000 description 1
- 229910000105 potassium hydride Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- RVEZZJVBDQCTEF-UHFFFAOYSA-N sulfenic acid Chemical compound SO RVEZZJVBDQCTEF-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Abstract
The invention discloses a method for coupling 2, 4-Dinitrophenol (DNP) with controllable cleavage of an antibody, which comprises the steps of oxidizing mercaptoethanol to form bis (2-hydroxyethyl) disulfide; chloroformylating bis (2-hydroxyethyl) disulfide to obtain chloroformylated bis (2-hydroxyethyl) disulfide, and then reacting the chloroformylated bis (2-hydroxyethyl) disulfide with NHS to form bis (2-hydroxyethyl) disulfide-NHS ester; reacting a DNP precursor with an alcohol amine to form an amino-containing DNP, and then reacting the amino-containing DNP with bis (2-hydroxyethyl) disulfide-NHS ester to obtain DNP-bis (2-hydroxyethyl) disulfide-NHS ester, wherein the DNP precursor is 1-chloro-2, 4-dinitrobenzene, 1-bromo-2, 4-dinitrobenzene, 1-iodo-2, 4-dinitrobenzene, or 1-fluoro-2, 4-dinitrobenzene; and reacting the DNP-bis (2-hydroxyethyl) disulfide-NHS ester with an antibody to obtain a DNP-labeled antibody.
Description
Technical Field
The invention relates to a method for coupling 2, 4-Dinitrophenol (DNP) with controlled cleavage of an antibody.
Background
Immunological detection mainly uses the specificity of antibody-antigen to make immunological analysis on some markers to be detected in vivo, but the concentration of detected object is low, and some fluorescent groups need to be coupled for cascade amplification. The coupling is mainly performed by chemical coupling through some high active groups such as sulfhydryl, amino, hydroxyl, carboxyl and other groups which can be coupled on the antibody. But have some problems. Firstly, most of conjugates have poor water solubility, and most of the antibodies are in an aqueous solution environment, so that the conjugates cannot be effectively dissolved in the aqueous solution to be coupled with the antibodies; secondly, the conjugate has no activating group, further chemical modification is needed, and the chemical modification can have certain influence on the conjugate; thirdly, there is a problem of low coupling efficiency between the conjugate and the antibody. Thus, it is extremely important to establish a mature antibody conjugation process. It is desirable that the conjugate be efficiently conjugated to the antibody by a simple reaction. Thus, click reactions, such as the more classical click reactions-azido and alkynyl 1, 3-dipolar cycloaddition reactions, D-a reactions, thiol and thiol reactions, and thiol and alkene reactions, etc., are ideal methods for antibody coupling. The reaction has the characteristics of good biocompatibility, no biotoxicity, good water solubility, mild reaction conditions and almost no heating, so that the activity of the antibody is not affected, and the product can be directly used for immune reaction without special purification. Meanwhile, the method plays an important role in the aspects of polymer coupling, biosensing analysis, drug-antibody coupling and preparation and application of nano hydrogel.
Disclosure of Invention
The invention aims to provide a method for coupling 2, 4-Dinitrophenol (DNP) with controlled cleavage of an antibody.
The method for coupling 2, 4-Dinitrophenol (DNP) with controllable cleavage of an antibody comprises the following steps:
oxidizing the thiol to form bis (2-hydroxyethyl) disulfide;
chloroformylating bis (2-hydroxyethyl) disulfide to obtain chloroformylated bis (2-hydroxyethyl) disulfide, and then reacting the chloroformylated bis (2-hydroxyethyl) disulfide with NHS to form bis (2-hydroxyethyl) disulfide-NHS ester;
reacting a DNP precursor with an alcohol amine to form an amino-containing DNP, and then reacting the amino-containing DNP with bis (2-hydroxyethyl) disulfide-NHS ester to obtain DNP-bis (2-hydroxyethyl) disulfide-NHS ester, wherein the DNP precursor is 1-chloro-2, 4-dinitrobenzene, 1-bromo-2, 4-dinitrobenzene, 1-iodo-2, 4-dinitrobenzene, or 1-fluoro-2, 4-dinitrobenzene; and
reacting DNP-bis (2-hydroxyethyl) disulfide-NHS ester with an antibody to obtain DNP-labeled antibody.
In certain embodiments, the mercaptoalcohol is mercaptoethanol, mercaptopropanol, mercaptobutanol, or mercaptopentanol.
In certain embodiments, the DNP precursor is reacted with an alcohol amine under alkaline conditions in the presence of the phase transfer catalyst to form an amino group containing DNP.
In certain embodiments, the alcohol amine is methanol amine, ethanol amine, 3-amino-1-propanol, 4-amino-1-butanol, or 5-amino-1-butanol.
In certain embodiments, the phase transfer catalyst is tetrabutylammonium bromide, tetrabutylammonium fluoride, triethylhexylammonium bromide, or triethyloctylammonium bromide.
In certain embodiments, the chloroformylated bis (2-hydroxyethyl) disulfide is reacted with the NHS in the presence of an acid-binding agent to form the bis (2-hydroxyethyl) disulfide-NHS ester.
In certain embodiments, the acid binding agent is pyridine, triethylamine, or ethylenediamine.
In certain embodiments, the chloroformylating is performed with a chloroformylating reagent that is phosgene, diphosgene or triphosgene.
In certain embodiments, the DNP is dissociated from the DNP-labeled antibody in the presence of a reducing agent.
In certain embodiments, the reducing agent is GSH, dithiothreitol, dithioerythritol, tris (2-formylethyl) phosphine hydrochloride, or β -mercaptoethanol.
The method for coupling 2, 4-Dinitrophenol (DNP) with controllable cleavage of an antibody can couple DNP to the antibody and can dissociate DNP from the antibody under specific conditions. The prepared DNP-labeled antibodies have controllable cleavable chemical bonds. DNP-labeled antibodies use extremely biocompatible disulfide bonds as coupling sites for controlled cleavage, and can achieve reversible exchange between disulfide bonds and sulfhydryl groups in a reducing environment, such as GSH.
Drawings
FIG. 1 shows a nuclear magnetic resonance hydrogen spectrum of bis (2-hydroxyethyl) disulfide.
FIG. 2 shows a chloroformylation nuclear magnetic resonance hydrogen spectrum of bis (2-hydroxyethyl) disulfide.
FIG. 3 shows a nuclear magnetic resonance hydrogen spectrum of bis (2-hydroxyethyl) disulfide-NHS ester.
FIG. 4 shows a nuclear magnetic resonance hydrogen spectrum of 2, 4-nitrophenoxy ethylamine.
FIG. 5 shows a high performance liquid chromatogram of DNP-bis (2-hydroxyethyl) disulfide-NHS ester.
FIG. 6 shows a high performance liquid chromatogram of DNP-bis (2-hydroxyethyl) disulfide-NHS ester coupling to an antibody.
FIG. 7 shows microscopic images of DNP and Biotin labeled microbeads after fluorescent development, fluorescent amplification and reducing agent treatment.
Detailed Description
To achieve the above object, the present invention provides a method for controlled cleavage coupling of 2, 4-Dinitrophenol (DNP) to an antibody, comprising (1) construction of a reductive disulfide compound; (2) construction of a disulfide compound containing NHS ester; 3) Coupling DNP to one end of bis (2-hydroxyethyl) disulfide-NHS ester; and (4) labelling the DNP-bis (2-hydroxyethyl) disulfide-NHS ester to an antibody.
(1) Construction of a reducing disulfide Compound
The thiol is oxidized to form bis (2-hydroxyethyl) disulfide.
In the reaction solvent 1, mercapto alcohol is used as a precursor, and the mercapto group of the mercapto alcohol is oxidized into disulfide bond under the action of an oxidant, so that the reducible and cleavable disulfide compound, namely the bis (2-hydroxyethyl) disulfide, is constructed. After the reaction was completed, the reaction was quenched by sodium thiosulfate.
The mercapto alcohol is mercaptoethanol, mercaptopropanol, mercaptobutanol or mercaptopentanol, preferably mercaptoethanol. The oxidant is O 2 Hydrogen peroxide, elemental iodine or DMSO, preferably hydrogen peroxide. The mol ratio of the mercapto alcohol to the oxidant is 1:1-1: 1.5. the reaction solvent 1 is ethyl acetate, dichloromethane, acetonitrile, diethyl ether or tetrahydrofuran, preferably ethyl acetate. The reaction temperature is between minus 20 ℃ and room temperature; after the reaction, the mixture was separated and purified by silica gel column chromatography.
(2) Construction of NHS ester-containing disulfide Compounds
Bis (2-hydroxyethyl) disulfide is chloroformylated to obtain chloroformylated bis (2-hydroxyethyl) disulfide, and the chloroformylated bis (2-hydroxyethyl) disulfide is then reacted with N-hydroxysuccinimide (NHS) to form bis (2-hydroxyethyl) disulfide-N-hydroxysuccinimide ester.
Reacting bis (2-hydroxyethyl) disulfide with a chloroformylating reagent in a reaction solvent 2, chloroformylating hydroxyl groups on the bis (2-hydroxyethyl) disulfide to obtain bis (2-hydroxyethyl) disulfide chloroformate; reacting chloroformylated bis (2-hydroxyethyl) disulfide with NHS in the presence of an acid-binding agent to form bis (2-hydroxyethyl) disulfide-NHS ester, thereby constructing NHS ester capable of reacting with amino groups with high efficiency.
The chloroformylating reagent is phosgene, diphosgene, triphosgene, etc., preferably diphosgene. The mol ratio of the bis (2-hydroxyethyl) disulfide to the chloroformylating agent is 1:1-1:5. The reaction solvent 2 is dichloromethane, diethyl ether, or tetrahydrofuran, preferably diethyl ether. The reaction temperature is-20 ℃ to room temperature, preferably 0 ℃; after the reaction, separation and purification are carried out by silica gel column chromatography. The reaction mole ratio of the bis (2-hydroxyethyl) disulfide chloroformate to NHS is 1: 2-1:3; the reaction temperature is-20℃to 50℃and preferably 0 ℃. The acid binding agent is pyridine, triethylamine or ethylenediamine, preferably triethylamine. After the reaction, the mixture was separated and purified by silica gel column chromatography.
(3) Coupling DNP to one end of bis (2-hydroxyethyl) disulfide-NHS ester
Reacting the DNP precursor with an alcohol amine to form an amino-containing DNP, and then reacting the amino-containing DNP with bis (2-hydroxyethyl) disulfide-NHS ester to obtain DNP-bis (2-hydroxyethyl) disulfide-NHS ester.
Reacting the DNP precursor with an alcohol amine in the presence of the phase transfer catalyst in reaction solvent 3 under alkaline conditions to form an amino group containing DNP. Reacting the amino-containing DNP with bis (2-hydroxyethyl) disulfide-NHS ester in reaction solvent 4 to obtain DNP-bis (2-hydroxyethyl) disulfide-NHS ester. DNP-bis (2-hydroxyethyl) disulfide-NHS ester contains DNP at one end and disulfide of NHS ester at the other end.
The DNP precursor is 1-chloro-2, 4-dinitrobenzene, 1-bromo-2, 4-dinitrobenzene, 1-iodo-2, 4-dinitrobenzene, or 1-fluoro-2, 4-dinitrobenzene, preferably 1-chloro-2, 4-dinitrobenzene. The alcohol amine is methanol amine, ethanolamine, 3-amino-1-propanol, 4-amino-1-butanol or 5-amino-1-butanol, preferably ethanolamine; the reaction solvent 3 is toluene, tetrahydrofuran or N, N-dimethylformamide, preferably toluene. The base is sodium hydroxide, potassium hydride or sodium hydride, preferably potassium hydroxide; the phase transfer catalyst is tetrabutylammonium bromide, tetrabutylammonium fluoride, triethylhexylammonium bromide or triethyloctylammonium bromide, preferably tetrabutylammonium bromide. The reaction temperature for the reaction of the DNP precursor with the alcohol amine is from room temperature to 150 ℃, preferably 70 ℃. The molar ratio of amino-containing DNP to bis (2-hydroxyethyl) disulfide-NHS ester is 1:0.9 to 1:1.1, preferably 1:1. The reaction solvent 4 is dichloromethane, tetrahydrofuran, methanol, ethanol, ethyl acetate, or the like, preferably dichloromethane. The reaction time of the DNP containing amino group and the bis (2-hydroxyethyl) disulfide-NHS ester is 10 min-6 h, preferably 1h; the reaction temperature is from 0℃to room temperature, preferably room temperature. After the reaction, the mixture was separated and purified by silica gel column chromatography.
(4) DNP-bis (2-hydroxyethyl) disulfide-NHS ester labelling to antibodies
Reacting DNP-bis (2-hydroxyethyl) disulfide-NHS ester with an antibody to obtain DNP-labeled antibody.
DNP-bis (2-hydroxyethyl) disulfide-NHS ester was reacted with antibodies in reaction solvent 5. The molar ratio of DNP-bis (2-hydroxyethyl) disulfide-NHS ester to antibody is 1:1 to 10:1, preferably 1:1. The reaction solvent 5 is DMSO. The reaction temperature is 0 ℃ to room temperature, preferably room temperature; after the reaction, the mixture is centrifugally purified by an ultrafiltration centrifuge tube and washed for 3 to 5 times.
DNP on DNP-labeled antibodies may be cleaved from the antibody under reducing conditions. Among these, the reducing agents used are Glutathione (GSH), dithiothreitol, dithioerythritol, tris (2-formylethyl) phosphine hydrochloride, or beta-mercaptoethanol, preferably GSH.
The following examples are intended to illustrate the invention and are not intended to be limiting.
Example 1
(1) Synthesis of bis (2-hydroxyethyl) disulfide
Mercaptoethanol (10 g) is weighed and dissolved in 100ml of dichloromethane solution, 10ml of 30% hydrogen peroxide solution is slowly added dropwise, 0.01g of elemental iodine is added as an indicator for room temperature reaction, after the reaction is finished, the mixture is washed three times by saturated sodium thiosulfate solution and then three times by saturated saline solution, anhydrous sodium sulfate is dried overnight, the solvent is dried by spin, and the eluent is ethyl acetate. FIG. 1 shows a nuclear magnetic resonance hydrogen spectrum of bis (2-hydroxyethyl) disulfide.
(2) Synthesis of bis (2-hydroxyethyl) disulfide-NHS ester
Bis (2-hydroxyethyl) disulfide (1 g) was weighed out and dissolved in 10ml of dry dichloromethane solution and molecular sieves were dehydrated overnight. Triphosgene (1.9 g) is weighed and dissolved in dry 10ml of dichloromethane solution, bis (2-hydroxyethyl) disulfide solution is added dropwise at 0 ℃ for reaction for 12 hours, chloroformylated bis (2-hydroxyethyl) disulfide is obtained, after the reaction is finished, the solvent is dried by spin, and the solvent is separated and purified by silica gel column chromatography, wherein the eluent is dichloromethane. Chloroformylated bis (2-hydroxyethyl) disulfide (1 g) was weighed and dissolved in 10ml of a dry dichloromethane solution, 0.82g of NHS was weighed and dissolved in 10ml of an diethyl ether solution, and slowly added dropwise to the solution, and reacted in an ice bath, 1ml of triethylamine was slowly added dropwise, and after the reaction was completed, the solvent was dried by suction filtration, and purified by silica gel column chromatography (eluent was dichloromethane) to obtain bis (2-hydroxyethyl) disulfide-NHS ester. FIG. 2 shows a chloroformylation nuclear magnetic resonance hydrogen spectrum of bis (2-hydroxyethyl) disulfide. FIG. 3 shows a nuclear magnetic resonance hydrogen spectrum of bis (2-hydroxyethyl) disulfide-NHS ester.
(3) DNP coupling to one end of bis (2-hydroxyethyl) disulfide-NHS ester
1g of 1-chloro-2, 4-dinitrobenzene is weighed and dissolved in 20ml of acetonitrile solution, 0.28g of potassium hydroxide and 0.01g of tetrabutylammonium bromide are added, the mixture is heated to 50 ℃ for reaction for 1 hour, then 0.3g of ethanolamine is added, and the reaction is continued for 12 hours. After the reaction is finished, separating and purifying by silica gel column chromatography, eluting by a methylene dichloride/methanol mixed solvent to obtain 2, 4-nitrophenoxy ethylamine. The prepared 2, 4-nitrophenoxy ethylamine (0.5 g) and bis (2-hydroxyethyl) disulfide-NHS ester (0.96 g) are dissolved in 10ml of dry dichloromethane solution, reacted for 30min, the solvent is dried by spin, and the DNP-bis (2-hydroxyethyl) disulfide-NHS ester is obtained by silica gel column chromatography separation and purification (eluent is methyl chloride/methanol mixed solvent).
FIG. 4 shows a nuclear magnetic resonance hydrogen spectrum of 2, 4-nitrophenoxy ethylamine. FIG. 5 shows a high performance liquid chromatogram of DNP-bis (2-hydroxyethyl) disulfide-NHS ester.
(4) Labelling DNP-bis (2-hydroxyethyl) disulfide-NHS esters to antibodies
DNP-bis (2-hydroxyethyl) disulfide-NHS ester (0.01 g) was dissolved in 0.5ml DMSO to prepare a 40mM solution, the antibody was added in equimolar ratio with DNP-bis (2-hydroxyethyl) disulfide-NHS ester, incubated at room temperature for 20min, centrifuged in an amicon centrifuge tube, and washed 3 times with DMSO to obtain DNP-labeled goat anti-rabbit IgG antibody. FIG. 6 shows a high performance liquid chromatogram of DNP-bis (2-hydroxyethyl) disulfide-NHS ester coupling to an antibody.
Example 2
(1) Preparation of DNP and Biotin (Biotin) labeled polystyrene microspheres
Preparing morpholinoethanesulfonic acid buffer solution (MES, PH6.0, 0.1M) with ultrapure water, and preparing suspension of 5mg/mL with carboxyl polystyrene microsphere; then, 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution prepared by MES were added thereto so that the concentrations of EDC and NHS in the suspension system were 25mg/mL, and the suspension system was shaken for 1 hour.
10nmol oligonucleotide chain Dry powder (5' -NH) 2 AGATCGTACGCTATCAGTGCCAC-3') was dissolved in MES buffer and mixed with activated polystyrene microsphere suspension having a polystyrene microsphere content of 5mg, reacted for 4 hours at room temperature with shaking, and centrifuged, and the mixture was purified by using PBST buffer (8 mM Na 2 HPO 4 ，2mM KH 2 PO 4 After washing 10mM KCl,140mM NaCl,0.05% (V/V) Tween-20, pH 7.2-7.4 three times (5000 rpm,5 min), the mixture was resuspended in ultrapure water to obtain a bead solution in which the polystyrene microspheres were covalently bound to the oligonucleotide chains.
Experimental group: 0.1mg of polystyrene microbeads coupled with oligonucleotide chains was taken and 100. Mu.L of reaction mixture [40mM Tris-HCl (pH 7.5), 10mM MgCl was added 2 5mM DTT, 1.5. Mu.M DNP-11-ddATP, 0.5. Mu.M oligonucleotide strand (5'-TGTGGCACTGATAGCGTACGATCT-3'), 0.05 u/. Mu.LSequence version 2.0DNA polymerase]Incubation at 37℃for 5min, centrifugation and PBST washing three times (5000 rpm,5 min) gave a DNP-labeled polystyrene microsphere solution.
Control group: 0.1mg of polystyrene beads coupled with oligonucleotide chains was taken and 100. Mu.L of the reaction solution was addedThe mixture [40mM Tris-HCl (pH 7.5), 10mM MgCl ] 2 5mM DTT, 1.5. Mu.M Biotin-11-ddGTP, 0.5. Mu.M oligonucleotide strand (5'-CGTGGCACTGATAGCGTACGATCT-3'), 0.05 u/. Mu.LSequence version 2.0DNA polymerase]Incubation was carried out at 37℃for 5 minutes, centrifugation and PBST washing three times (5000 rpm,5 min) to obtain a solution of Biotin-labeled polystyrene microspheres.
(2) Fluorescent color development of microbeads
Experimental group: DNP-labeled polystyrene microspheres 0.1mg was used, 200. Mu.L of 5. Mu.g/mL Dinitrophenyl-KLH Polyclonal Antibody, alexa Fluor was added TM 488(Alexa Fluor TM 488 fluorescent group-labeled DNP antibody), 30min incubation at 37 ℃, centrifugation in pbst buffer (5000 rpm,3 min) wash 3 times, fluorescent microscopy photograph recording as in fig. 7A.
Control group: polystyrene microsphere 0.1mg marked by Biotin is taken, 200 mu L of 5 mu g/mL is added488-streptavidin conjugate (iFluor 488-labeled streptavidin conjugate), incubated at 37℃for 30min, centrifuged (5000 rpm,3 min), and PBST buffer washed 3 times, fluorescent microscopy photograph recorded as in FIG. 7D.
(3) Microbead fluorescent cascade amplification
DNP-labeled goat anti-rabbit IgG antibodies were prepared as secondary antibodies according to the procedure of example 1, and the labeled secondary antibody solution was diluted to 5. Mu.g/mL with PBST buffer; respectively taking the microbeads with fluorescent color obtained by the experimental group and the control group, adding 200 mu L of diluted secondary antibody solution, incubating for 30min at 37 ℃, and cleaning by PBST for 3 times; then 200. Mu.L of 5. Mu.g/mL Dinitrophenyl-KLH Polyclonal Antibody, alexa Fluor, were added separately TM 488(Alexa Fluor TM 488 fluorescent group-labeled DNP antibody), 30min incubation at 37 ℃,3 washes with pbst, fluorescent microscopy photographic recordings, as shown in fig. 7B (experimental group) and 7E (control group).
(4) Treatment with reducing agent to break disulfide bonds
10mM Glutathione (GSH) solution was prepared with ultrapure water, and 200. Mu.L of Glutathione (GSH) solution was added to the fluorescent cascade amplified microbeads obtained in the experimental group and the control group, respectively, incubated at 37℃for 30min, centrifuged, PBST washed 3 times, and recorded by fluorescent microscopy photographing, as shown in FIG. 7C (experimental group) and 7F (control group).
Principle of DNP-mediated fluorescence cascade amplification: dinitrophenyl-KLH Polyclonal Antibody, alexa Fluor TM 488 specific recognition DNP introduced with a fluorescent group, DNP-labeled goat anti-rabbit IgG antibody as a secondary antibody specifically recognizes Dinitrophenyl-KLH Polyclonal Antibody, alexa Fluor TM 488 again introduces a new DNP on cycle, and utilizes cascade identification of primary and secondary antibodies to realize cascade amplification of fluorescent signals.
As shown in fig. 7, the brightness of the microbeads after fluorescent cascade amplification in the experimental group (fig. 7B) is significantly improved compared with that before fluorescent amplification (fig. 7A); the brightness of the microbeads developed by biotin-labeled and streptavidin-coupled fluorescent groups under the same conditions is not greatly changed before and after the fluorescent cascade amplification (fig. 7D and 7E), which indicates that the effect of the fluorescent cascade amplification is not achieved. Two experiments showed that DNP-labeled secondary antibodies specifically recognized Alexa Fluor TM 488 fluorescent group-labeled DNP antibody and introduction of new DNP initiated fluorescence cascade amplification, indicating that DNP was successfully linked to the antibody and that the linkage of DNP did not affect the activity of the antibody.
As shown in fig. 7, the brightness of the beads after GSH treatment in the experimental group (fig. 7C) is obviously reduced to the level before the fluorescence cascade amplification, and the contrast group has no obvious change (fig. 7F), which indicates that the reductive environment breaks disulfide bonds, resulting in the detachment of DNP attached to the DNP-labeled goat anti-rabbit IgG antibody, resulting in the detachment of the fluorescence-labeled antibody for the subsequent cascade amplification, and the fluorescence of the beads is reduced.
Claims (9)
1. A method for controlled cleavage coupling of 2, 4-dinitrophenol to an antibody comprising:
oxidizing mercaptoethanol to form bis (2-hydroxyethyl) disulfide;
chloroformylating bis (2-hydroxyethyl) disulfide to obtain chloroformylated bis (2-hydroxyethyl) disulfide, and then reacting the chloroformylated bis (2-hydroxyethyl) disulfide with NHS to form bis (2-hydroxyethyl) disulfide-NHS ester;
reacting a 2, 4-dinitrophenol precursor with an alcohol amine to form an amino-containing 2, 4-dinitrophenol, and then reacting the amino-containing 2, 4-dinitrophenol with bis (2-hydroxyethyl) disulfide-NHS ester to obtain 2, 4-dinitrophenol-bis (2-hydroxyethyl) disulfide-NHS ester, wherein the 2, 4-dinitrophenol precursor is 1-chloro-2, 4-dinitrobenzene, 1-bromo-2, 4-dinitrobenzene, 1-iodo-2, 4-dinitrobenzene, or 1-fluoro-2, 4-dinitrobenzene; and
reacting 2, 4-dinitrophenol-bis (2-hydroxyethyl) disulfide-NHS ester with an antibody to obtain a 2, 4-dinitrophenol labeled antibody.
2. The process of claim 1 wherein the 2, 4-dinitrophenol precursor is reacted with an alcohol amine in the presence of a phase transfer catalyst under alkaline conditions to form an amino-containing 2, 4-dinitrophenol.
3. The method according to claim 2, wherein the alcohol amine is methanol amine, ethanol amine, 3-amino-1-propanol, 4-amino-1-butanol or 5-amino-1-butanol.
4. A process according to claim 2 or 3, wherein the phase transfer catalyst is tetrabutylammonium bromide, tetrabutylammonium fluoride, triethylhexylammonium bromide or triethyloctylammonium bromide.
5. The method of claim 1, wherein the chloroformylated bis (2-hydroxyethyl) disulfide is reacted with the NHS in the presence of an acid-binding agent to form the bis (2-hydroxyethyl) disulfide-NHS ester.
6. The method of claim 5, wherein the acid binding agent is pyridine, triethylamine or ethylenediamine.
7. The process according to claim 1, wherein the chloroformylation is carried out with a chloroformylating reagent which is phosgene, diphosgene or triphosgene.
8. The method of claim 1, wherein the 2, 4-dinitrophenol label is dissociated from the 2, 4-dinitrophenol labeled antibody in the presence of a reducing agent.
9. The method of claim 8, wherein the reducing agent is GSH, dithiothreitol, dithioerythritol, tris (2-formylethyl) phosphine hydrochloride, or β -mercaptoethanol.
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