GB2081257A - Biologically Interesting Compounds Labeled with Chlorotriazinyl-aminofluorescein - Google Patents
Biologically Interesting Compounds Labeled with Chlorotriazinyl-aminofluorescein Download PDFInfo
- Publication number
- GB2081257A GB2081257A GB8118754A GB8118754A GB2081257A GB 2081257 A GB2081257 A GB 2081257A GB 8118754 A GB8118754 A GB 8118754A GB 8118754 A GB8118754 A GB 8118754A GB 2081257 A GB2081257 A GB 2081257A
- Authority
- GB
- United Kingdom
- Prior art keywords
- compound according
- dtaf
- amino
- added
- aminofluorescein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 35
- GZAJOEGTZDUSKS-UHFFFAOYSA-N 5-aminofluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(N)=CC=C21 GZAJOEGTZDUSKS-UHFFFAOYSA-N 0.000 claims abstract description 9
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical group C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 5
- 125000002924 primary amino group Chemical class [H]N([H])* 0.000 claims abstract description 4
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 6
- 229930182566 Gentamicin Natural products 0.000 claims description 6
- 229960002518 gentamicin Drugs 0.000 claims description 6
- LOUPRKONTZGTKE-LHHVKLHASA-N quinidine Chemical group C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@H]2[C@@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-LHHVKLHASA-N 0.000 claims description 6
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N Theophylline Natural products O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 claims description 5
- 229960000278 theophylline Drugs 0.000 claims description 4
- JNDZQWRVBINENC-UHFFFAOYSA-N 4-acetamido-n-[2-(ethylamino)ethyl]benzamide Chemical group CCNCCNC(=O)C1=CC=C(NC(C)=O)C=C1 JNDZQWRVBINENC-UHFFFAOYSA-N 0.000 claims description 3
- MNEGCHZAKQMXCP-UHFFFAOYSA-N 5-amino-2-ethylpentanoic acid Chemical group CCC(C(O)=O)CCCN MNEGCHZAKQMXCP-UHFFFAOYSA-N 0.000 claims description 3
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical group IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 claims description 3
- 229960004821 amikacin Drugs 0.000 claims description 3
- LKCWBDHBTVXHDL-RMDFUYIESA-N amikacin Chemical compound O([C@@H]1[C@@H](N)C[C@H]([C@@H]([C@H]1O)O[C@@H]1[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O1)O)NC(=O)[C@@H](O)CCN)[C@H]1O[C@H](CN)[C@@H](O)[C@H](O)[C@H]1O LKCWBDHBTVXHDL-RMDFUYIESA-N 0.000 claims description 3
- 150000001721 carbon Chemical group 0.000 claims description 3
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 claims description 3
- 229960001404 quinidine Drugs 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical group IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 claims description 2
- 239000002647 aminoglycoside antibiotic agent Substances 0.000 claims description 2
- 229960001767 dextrothyroxine Drugs 0.000 claims description 2
- 229960002695 phenobarbital Drugs 0.000 claims description 2
- 150000003335 secondary amines Chemical class 0.000 claims description 2
- 125000001424 substituent group Chemical group 0.000 claims description 2
- 229940126575 aminoglycoside Drugs 0.000 claims 3
- 239000007850 fluorescent dye Substances 0.000 claims 2
- MHQFHNSBKDXJBM-UHFFFAOYSA-N 4-acetamido-n-(2-aminoethyl)benzamide Chemical group CC(=O)NC1=CC=C(C(=O)NCCN)C=C1 MHQFHNSBKDXJBM-UHFFFAOYSA-N 0.000 claims 1
- RNQFIALWYREPBF-UHFFFAOYSA-N 5-amino-2-propylpentanoic acid Chemical group CCCC(C(O)=O)CCCN RNQFIALWYREPBF-UHFFFAOYSA-N 0.000 claims 1
- 229940126574 aminoglycoside antibiotic Drugs 0.000 claims 1
- 210000002966 serum Anatomy 0.000 abstract description 5
- 210000001124 body fluid Anatomy 0.000 abstract description 2
- 239000010839 body fluid Substances 0.000 abstract description 2
- 125000000467 secondary amino group Chemical class [H]N([*:1])[*:2] 0.000 abstract description 2
- 125000004432 carbon atom Chemical group C* 0.000 abstract 1
- 238000001514 detection method Methods 0.000 abstract 1
- 210000002381 plasma Anatomy 0.000 abstract 1
- 210000003296 saliva Anatomy 0.000 abstract 1
- 210000002700 urine Anatomy 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 78
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 40
- HWQQCFPHXPNXHC-UHFFFAOYSA-N 6-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound C=1C(O)=CC=C2C=1OC1=CC(O)=CC=C1C2(C1=CC=2)OC(=O)C1=CC=2NC1=NC(Cl)=NC(Cl)=N1 HWQQCFPHXPNXHC-UHFFFAOYSA-N 0.000 description 37
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 28
- 239000000872 buffer Substances 0.000 description 24
- 239000000243 solution Substances 0.000 description 19
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 238000000034 method Methods 0.000 description 13
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- 239000002904 solvent Substances 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 11
- NIJJYAXOARWZEE-UHFFFAOYSA-N di-n-propyl-acetic acid Natural products CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 description 11
- 230000010287 polarization Effects 0.000 description 11
- 238000004809 thin layer chromatography Methods 0.000 description 11
- MSRILKIQRXUYCT-UHFFFAOYSA-M valproate semisodium Chemical compound [Na+].CCCC(C(O)=O)CCC.CCCC(C([O-])=O)CCC MSRILKIQRXUYCT-UHFFFAOYSA-M 0.000 description 11
- 229960000604 valproic acid Drugs 0.000 description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000003556 assay Methods 0.000 description 9
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 8
- 239000000700 radioactive tracer Substances 0.000 description 8
- 239000000741 silica gel Substances 0.000 description 8
- 229910002027 silica gel Inorganic materials 0.000 description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 7
- 150000001412 amines Chemical class 0.000 description 7
- 239000013078 crystal Substances 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 229910052938 sodium sulfate Inorganic materials 0.000 description 7
- 235000011152 sodium sulphate Nutrition 0.000 description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 238000002875 fluorescence polarization Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 239000007795 chemical reaction product Substances 0.000 description 5
- 238000003018 immunoassay Methods 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 235000019198 oils Nutrition 0.000 description 5
- VGCXGMAHQTYDJK-UHFFFAOYSA-N Chloroacetyl chloride Chemical compound ClCC(Cl)=O VGCXGMAHQTYDJK-UHFFFAOYSA-N 0.000 description 4
- MZRVEZGGRBJDDB-UHFFFAOYSA-N N-Butyllithium Chemical compound [Li]CCCC MZRVEZGGRBJDDB-UHFFFAOYSA-N 0.000 description 4
- KEECCEWTUVWFCV-UHFFFAOYSA-N N-acetylprocainamide Chemical compound CCN(CC)CCNC(=O)C1=CC=C(NC(C)=O)C=C1 KEECCEWTUVWFCV-UHFFFAOYSA-N 0.000 description 4
- 239000013060 biological fluid Substances 0.000 description 4
- -1 desimprmine Chemical compound 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 239000000908 ammonium hydroxide Substances 0.000 description 3
- 239000012470 diluted sample Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 108010074605 gamma-Globulins Proteins 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 235000009518 sodium iodide Nutrition 0.000 description 3
- QCXJEYYXVJIFCE-UHFFFAOYSA-N 4-acetamidobenzoic acid Chemical compound CC(=O)NC1=CC=C(C(O)=O)C=C1 QCXJEYYXVJIFCE-UHFFFAOYSA-N 0.000 description 2
- BMVWCPGVLSILMU-UHFFFAOYSA-N 5,6-dihydrodibenzo[2,1-b:2',1'-f][7]annulen-11-one Chemical compound C1CC2=CC=CC=C2C(=O)C2=CC=CC=C21 BMVWCPGVLSILMU-UHFFFAOYSA-N 0.000 description 2
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- 239000005977 Ethylene Substances 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
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- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 description 1
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- 229910019142 PO4 Inorganic materials 0.000 description 1
- CXOFVDLJLONNDW-UHFFFAOYSA-N Phenytoin Chemical compound N1C(=O)NC(=O)C1(C=1C=CC=CC=1)C1=CC=CC=C1 CXOFVDLJLONNDW-UHFFFAOYSA-N 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- RDHPKYGYEGBMSE-UHFFFAOYSA-N bromoethane Chemical compound CCBr RDHPKYGYEGBMSE-UHFFFAOYSA-N 0.000 description 1
- 229960000623 carbamazepine Drugs 0.000 description 1
- FFGPTBGBLSHEPO-UHFFFAOYSA-N carbamazepine Chemical compound C1=CC2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 FFGPTBGBLSHEPO-UHFFFAOYSA-N 0.000 description 1
- RBHJBMIOOPYDBQ-UHFFFAOYSA-N carbon dioxide;propan-2-one Chemical compound O=C=O.CC(C)=O RBHJBMIOOPYDBQ-UHFFFAOYSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- XAEWZDYWZHIUCT-UHFFFAOYSA-N desipramine hydrochloride Chemical compound [H+].[Cl-].C1CC2=CC=CC=C2N(CCCNC)C2=CC=CC=C21 XAEWZDYWZHIUCT-UHFFFAOYSA-N 0.000 description 1
- 229960003829 desipramine hydrochloride Drugs 0.000 description 1
- 229960005156 digoxin Drugs 0.000 description 1
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 1
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 1
- 229960001066 disopyramide Drugs 0.000 description 1
- UVTNFZQICZKOEM-UHFFFAOYSA-N disopyramide Chemical compound C=1C=CC=NC=1C(C(N)=O)(CCN(C(C)C)C(C)C)C1=CC=CC=C1 UVTNFZQICZKOEM-UHFFFAOYSA-N 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229950008325 levothyroxine Drugs 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229960005181 morphine Drugs 0.000 description 1
- SCZVXVGZMZRGRU-UHFFFAOYSA-N n'-ethylethane-1,2-diamine Chemical compound CCNCCN SCZVXVGZMZRGRU-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229960001158 nortriptyline Drugs 0.000 description 1
- 239000010502 orange oil Substances 0.000 description 1
- 229960005489 paracetamol Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- DDBREPKUVSBGFI-UHFFFAOYSA-N phenobarbital Chemical compound C=1C=CC=CC=1C1(CC)C(=O)NC(=O)NC1=O DDBREPKUVSBGFI-UHFFFAOYSA-N 0.000 description 1
- 229960002036 phenytoin Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- JVXZJEUVAOVXIX-UHFFFAOYSA-N propan-2-one;pyridine Chemical compound CC(C)=O.C1=CC=NC=C1 JVXZJEUVAOVXIX-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- KVCGISUBCHHTDD-UHFFFAOYSA-M sodium;4-methylbenzenesulfonate Chemical compound [Na+].CC1=CC=C(S([O-])(=O)=O)C=C1 KVCGISUBCHHTDD-UHFFFAOYSA-M 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/22—Cyclohexane rings, substituted by nitrogen atoms
- C07H15/222—Cyclohexane rings substituted by at least two nitrogen atoms
- C07H15/226—Cyclohexane rings substituted by at least two nitrogen atoms with at least two saccharide radicals directly attached to the cyclohexane rings
- C07H15/234—Cyclohexane rings substituted by at least two nitrogen atoms with at least two saccharide radicals directly attached to the cyclohexane rings attached to non-adjacent ring carbon atoms of the cyclohexane rings, e.g. kanamycins, tobramycin, nebramycin, gentamicin A2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
- C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
- C07D473/06—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3
- C07D473/08—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3 with methyl radicals in positions 1 and 3, e.g. theophylline
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- 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
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Plural Heterocyclic Compounds (AREA)
- Saccharide Compounds (AREA)
Abstract
Fluorescent derivatives of biologically interesting compounds which permit the detection and determination of said compounds in body fluids such as serum, plasma, saliva and urine, are characterized by the formula: <IMAGE> wherein X is a biologically interesting moiety having a molecular weight of less than 2,000 and having at least one primary or secondary amine or hydroxyl group by which it attaches to a carbon atom of the triazine ring; and Z is 4-aminofluorescein or 5- aminofluorescein attached through the 4-amino or 5-amino, respectively to the 2-position of the triazine ring.
Description
SPECIFICATION
Biologically Interesting Compounds Labeled with Dichloritriazinyl-aminofluorescein
This disclosure relates generally to fluorescent compounds which have proved particularly useful in a variety of immunofluorescence procedures to detect and determine quantities of small biologically interesting molecules in body fluids. The low molecular weight substances which are desired to be detected or monitored include therapeutically administered drugs, drugs having a known potential for abuse, and various other compounds which are of interest in diagnosing diaease or monitoring thereapy.
While the claimed compounds can be utilized in a variety of immunofluorescence techniques; they are particularly useful in a procedure called fluorescence polarization immunoassay. A fluorescence polarization immunoassay combines the specificity of an immunoassay with the speed and convenience of a homogeneous method. When a fluorescent conjugate is bound to an antibody, linearly polarized light which is used to excite the fluorescent conjugate remains highly polarized upon emission because the fluorophore is constrained from rotating between the time the light is absorbed and emitted. On the other hand, when the fluorescent conjugate is not bound by antibody, its rotation is much faster, molecules are more randomly oriented, and the emitted light is depolarized.
Fluorescence polarization, then, provides a direct measure of bound and free fluorescent conjugate in a competitive binding immunoassay. Fluorescent conjugate competes for antibody binding sites with the compound of interest in the patient sample. The greater the concentration of the compound being assayed, the larger the fraction of fluorescent conjugate unbound by antibody, and therefore, a greater degree of depolarized light is emitted. The precise relationship between the concentration of analyte and polarization is established by generating a standard curve. Calibrators with known amounts of analyte are read and the polarization recorded. When an unknown is read, its concentration is estimated by interpolation between standards.
Fluorescent conjugates of derivatives of low molecular weight compounds are not unique. U.S.
Patent 3,998,943 (1976) specifically describes the preparation of fluorescently labeled insulin using fluorescein isothiocyanate (FITC) and fluorescently-labeied morphine using 4-amino-fluorescein hydrochloride.
The fluorescent derivatives of the present invention are labeled with dichlorotriazinylaminofluorescein which is commonly referred to as DTAF. DTAF is the product of the reaction of aminofluorescein and cyanuric chloride. In an article by Blakeslee, et al, Jounal of
Immunological Methods, 13, 305-320, (1976), DTAF was described as an effective reagent for conjugating fluorescein to immunoglobulins (IgG) having molecular weights of at least 160,000.
The present invention discloses and claims the use of DTAF to impart fluorescence to a wide variety of biologically interesting compounds which, when labeled, can be characterized by the formula:
wherein X is a biologically interesting moiety having a molecular weight of less than 2,000 and having at least one reactive substituent selected from the group consisting of a primary or secondary amine or hydroxyl group by which it attaches to a carbon atom of the triazine ring; and Z is selected from the group consisting of 4-amino fluorescein and 5-aminofluorescein attached through the 4-amino or 5amino, respectively to the number 2 carbon of the triazine ring.
DTAF can be readily prepared according to the method adopted by Blakeslee, et al (supra). DTAF
Isomer I is prepared from 5-amino fluorescein; Isomer II is derived from 4-amino fluorescein.
The conjugation of either isomer of DTAF to compounds of interest can generally be accomplished by dissolving equal molar amounts of DTAF and a compound having a reactive amine (primary or secondary) or a hydroxyl group in an appropriate solvent such as water, methanol, dimethylformamide or dimethylsulfoxide. If the reactive amine is a salt, a suitable base is added to the reaction mixture to form the free base of the reactive amine. After completion of the reaction, the conjugates can be purified by using either thin-layer or column chromatography.
The biologically interesting compounds employed in the claimed invention must have at least one primary or secondary amino group or hydroxyl group by which it can react with and attach to a carbon atom of the triazine ring. The reactive group may be inherent in the biologically active compounds as it is in the quinidine, procainamide, thyroxine and the aminoglycoside antibiotics, or it may be introduced into the compound by derivatization. Examples of biological compounds that require the formation of amino derivatives before DTAF conjugates can be prepared include theophylline, valproic acid, phenobarbital, phenytoin, primidone, disopyramide, digoxin, chloramphenicol, aspirin, acetaminophen, carbamazepine, desimprmine, and nortriptyline.
Structures illustrating the most preferred compounds of the disclosed invention include the following: 8-AMINOMETHYLTHEOPHYLINE-DTAF
2-ETHYL-5-AMINOPENTANOIC ACID-DTAF
and
2-PROPYL-5-AMINOPENTANOIC ACID-DTAF
The following examples will demonstrate the preparation of specific DTAF conjugates within the scope of the claimed invention. Note that in the following examples, DTAF may be either isomer unless specifically stated.
Example I
Gentamicin-DTAF
Gentamicin sulfate (200mg) was dissolved in 1 ml of distilled water. The pH was adjusted to 9.0
with approximately 0.8ml of 1 .OM sodium hydroxide. DTAF (20mg) was dissolved in 1.5my of
methanol and added dropwise to the gentamicin solution with stirring and allowed to react for one
hour. The reaction mixture was added to a DEAE cellulose medium mesh column and the resulting
gentamicin-DTAF eluted with 0.1 M phosphate buffer at pH 8.0.
Example II Tobramycin-DTAF Tobramycin (250mg) was dissolved in 2ml of carbonate buffer, 0.1 M, pH 9.0. DTAF (20mg) was dissolved in 1 ml of methanol and added to 1 ml of the tobramycin solution. After about five minutes, the reaction mixture was purified by application to a 20ml DEAE cellulose column and equilibrated with 0.1 M phosphate buffer at pH 8.0. The reaction product was eluted with the same buffer.
Example III
Amikacin-DTAF
Amikacin (24mg) was dissolved in 0.2ml of water, and 4.5mg of DTAF were suspended in 0.2ml of methanol. The methanolic suspension of DTAF was added to the amikacin solution with stirring. The small particles of DTAF rapidly dissolved, so the reaction mixture was not stirred. After thirty minutes the reaction mixture was applied to a 17ml column of DEAE cellulose equilibrated with pH 8.1 phosphate buffer, 0.1 M. The reaction product was eluted with the same buffer.
Example IV
Desethyl-N-acetyl-procaina mide-DTAF
Paraacetamidobenzoic acid (1.799) and 1.159 of N-hydroxysuccinimide were dissolved in 15ml of pyridine. Then, 2.3g N,N'-dicyclohexylcarbodiimide was added and dissolved. The reaction mixture was chilled in a refrigerator for two hours and then filtered. Crystals were rinsed with about 2ml of acetone. N-ethylethylenediamine (0.88g) was added to the combined pyridine-acetone filtrate. The mixture was stirred for two hours and then chilled in a refrigerator for about twenty-four hours. The resulting crystals were filtered and rinsed with acetone. The crystals (2.09) were dissolved in 50ml of distilled water, and the solution adjusted to pH 10 with 6N sodium hydroxide solution. A white precipitate was filtered off and dried in a dessicator.The DTAF conjugate was formed by dissolving equal molar amounts of desethyl-N-acetylprocainamide and DTAF in methanol. The reaction was completed in about ten minutes. Purification was performed by silica gel thin layer chromatography, using a developing solvent of chloroform/acetone (1 :1).
Example V
N-p-acetamidobenzoyl Ethylene Diamine-DTAF
The procedure of example IV was repeated using 0.9g of ethylenediamine in place of Nethylethylenediamine. The reaction mixture was stirred for one hour, and chilled for one and one-half hours. The precipitate was filtered and rinsed with acetone. The DTAF conjugate was prepared the same as in Example IV, but methanol was used as the developing solvent.
Example VI
N-p-acetamidobenzoyl-N'-ethyl-N'-amino-acetyl Ethylene Diam ine-DTAF
Desethyl-N-acetyl procainamide (1.259) from example IV and 0.89 of chloroacetyl chloride were dissolved in 25ml of acetone and refluxed for two hours. After refluxing, the mixture was filtered and the filtrate evaporated to dryness. The yellow residue from the filtrate and 0.75g of sodium iodide were dissolved in 20ml of acetone and refluxed for one hour. The solution was filtered and the filtrate evaporated to dryness. The red residue was dissolved in 20ml of methanol. Concentrated ammonium hydroxide (20ml) was added to the solution, which was refluxed for one and one-half hours. After cooling, the mixture was extracted twice with 20ml of chloroform.The combined extracts were dried over sodium sulfate, filtered, and evaporated. The DTAF conjugate was prepared the same as in example IV and purified by thin-layer chromatography (chloroform/acetone (1 :)).
Example VII
Amino-primidone-DTAF
Primidone (1.1 g) was dissolved in 1 Oml of concentrated sulfuric acid. Another solution of 1 ml concentrated nitric acid and 2ml of concentrated sulfuric acid was added to the reaction mixture slowly without cooling. The mixture was then shaken at room temperature for forty-five minutes, and the reaction mixture was poured over 50ml ice and crystals of para-nitro-primidone were filtered and rinsed with water. The crystals (1.1 7g), melting point 225 0--2280C, were dissolved in 200ml of hot ethanol. Iron powder (1.5g) and 100ml of water were added. The mixture was heated to boiling, 2ml of concentrated hydrochloric acid were added, and the mixture was refluxed for two hours. The hot mixture was filtered and the filtrate evaporated to dryness. The residue was dissolved in 100% ethanol and precipitated out by adding diethyl ether. The precipitate was filtered to give 0.8g of brown hydroscopic crystals. The DTAF conjugate was formed by dissolving 5mg of DTAF and 5mg of the brown crystals in 0.5ml of methanol. The reaction was complete in 10 minutes and the conjugate was purified by silica gel thin-layer chromatography using chloroform/methanol (3:1) as the developing solvent. Final purification was performed by thin-layer chromatography using chloroform/methanol (2:1).
Example VIII 2-Ethyl-amino-pentanoic Acid-DTAF
Delta-5-valerolactam (7.5g) was dissolved in 60ml of dry tetrahydrofuran, under a dry nitrogen atmosphere and n-butyllithium (1.6M, 90ml) in hexane were added dropwise to the reaction flask and chilled with a dry iceacetone bath. After all the n-butyllithium was added, the reaction mixture was stirred at room temperature for one hour, refluxed for thirty minutes, and cooled to room temperature (still under dry nitrogen atmosphere). 1-Bromoethane (8.09) was slowly added to the reaction flask while the flask was chilled in an ice bath. The mixture was then stirred for sixteen hours at room temperature and then 100ml of water was added slowly. This mixture was stirred at room temperature for thirty minutes and the organic layer separated.The aqueous layer was extracted with 50ml of diethyl ether and the organic layers combined and dried over sodium sulfate. The solvent was evaporated to give a dark oil, which crystallized on standing, and was recrystallized from petroleum ether to give 3.89 of product. This product (2.8g) was refluxed in 25ml of 6N hydrochloric acid for six hours. The water was evaporated to give a dark, thick oil. The DTAF conjugate was formed by dissolving equimolar amounts of 2-ethyl-5-amino-pentanoic acid and DTAF in methanol. The reaction was completed in about ten minutes. The product was purified by silica gel thin-layer chromatography with chloroform/methanol (3:1) as the developing solvent.
Example X
D-Thyroxine-DTAF
DTAF (5mg) was dissolved in 0.5ml of methanol. Smg of D-thyroxine was added and then dimethylsulfoxide was added dropwise until a clear solution was formed. Two drops of triethylamine were added and a conjugate formed after about sixteen hours. The product was purified by silica gel thin-layer chromatography using chloroform/methanol (3:1) as the developing solvent.
Example XI
L-Thyroxine-DTAF
This conjugate was prepared following the procedure of Example X, but substituting L-thyroxine.
Example XII 3,3',5-TrHiodo-L-thyronine-DTAF This conjugate was prepared following the procedure of Example X, but substituting 3,3',5triiodo-L-thyronine.
Example XIII 5-Amino-dibenzocycloheptane-DTAF The following were dissolved in 50ml of methanol: 8.00gel of ammonium acetate, 630mg of sodium cyanoborohydride, and 2.10g of 10,11-dihydro-5H-dibenzo [a,d] cycloheptent-5-one.The solution was refluxed for twenty-four hours and then evaporated to dryness. A tan residue remained, which was dissolved in 25ml of 2N hydrochloric acid and extracted twice with 25ml dichloromethane.
6N sodium hydroxide was added to the aqueous phase until the pH reached 14. A brown oil began to form and the solution was chilled in a freezer for 1 6 hours. All water was evaporated and the residue was taken up in methanol and filtered. The filtrate was evaporated to give a white residue. The DTAF conjugate was formed by dissolving equimolar amounts of DTAF and the white residue in methanol.
The product was purified by silica gel thin-layer chromatography using chloroform/acetone (1 :1) as developing solvent.
Example XIV
Dibenzosuberone Hydrazone-DTAF
10,1 -Dihydro-5H-dibenzo [a,d] cyclohepten-5-one (10g) and 18g of dimethylhydrazine were refluxed for twenty-four hours in 100% ethanol. 100ml of distilled water were added and the yellow solution was extracted with diethyl ether until extracts were colorless. The combined ether extracts were washed with 25ml of 2N hydrochloric acid. The organic phase was then dried over sodium sulfate and evaporated. The residue was a thick orange oil, dibenzosuberone dimethylhydrazone. This product (2.0g) was refluxed for twelve hours with 39 of hydrazine in 1 Oml of 100% ethanol. The reaction mixture was poured over 1 Oml of ice water then extracted twice with 25ml of diethyl ether.The combined ether extracts were dried over sodium sulfate and evaporated to dryness to give a yellow oil, dibenzosuberone hydrazone. The DTAF conjugate was prepared the same as in Example XIII, but using chloroform/methanol (3:1) as the developing solvent.
Example XV 5-(y-Aminopropylidene)-5H-dibenzo[a,d]-1 0,11 -dihydrocycloheptene-DTAF
5-(y-Bromopropylidene)5H-dibenzo [a,d]-lO,l 1 -dihydrocycloheptene and its precursor 5 cyciopropyl-5-hydroxy-5H-dibenzo [a,d]- 10.11 -dihydrocycloheptene were prepared by the procedure described in Jounal Organic Chemistry, Vol. 27, pages 4134-4137 (1962) by R. D. Hoffsomer, D.
Taub, and N. L. Wendler. Procedure (b) for preparation of end product, 5-(y-bromopropylidene)-5H
dibenzo [a,d]-10,11 -dihydrocycloheptene, was followed substituting the bromopropylidene compound for the chloropropylidene compound. The DTAF conjugate was formed by dissolving equimolar
amounts of DTAF and the amine in methanol. An access amount of triethylamine was added and the
reaction was completed in thirty minutes. The reaction product was purified by silica gel thin-layer
chromatography using chloroform/methanol (2:1) as the developing solvent.
Example XVI N-Aminoacetyli m inostilbene-DTAF Iminodibenzyl (6.0g) was dissolved in 30ml of chloroform. Chloroacetyl chloride (6my) was added
and the mixture was refluxed for forty-five minutes. Water (60ml) was added and the mixture was
stirred for thirty minutes at room temperature. The chloroform layer was separated and dried over
sodium sulfate and evaporated to dryness. The residue was dissolved in 25ml of acetone and a solution
of 4.5g sodium iodide dissolved in 25ml of acetone was added. This mixture was refluxed for thirty
minutes, 100ml of water was added, and the reaction product was extracted twice with 50ml of
chloroform and evaporated to dryness. The residue was dissolved in 40ml of methanol. Concentrated
ammonium hydroxide (60ml) was added and the mixture was refluxed for one hour.The solution was
evaporated to dryness and the residue was taken up in 100ml of chloroform and washed twice with 30ml of 2N hydrochloric acid. The organic phase was dried over sodium sulfate and evaporated to
dryness. The DTAF conjugate was prepared by dissolving 5mg of DTAF and 5mg of the amine in 0.5ml of methanol. Conjugate formed in ten minutes and was purified the same as Example Xlil.
Example XVII N-Aminoacetyidesipramine-DTAF Desipramine hydrochloride (1.33g) and 0.809 of chloracetyl chloride was dissolved in 25ml
chloroform. This was refluxed for two hours, then the chloroform was evaporated. The residue was
dissolved in 25ml of acetone. Sodium iodide (0.759) was added, and the solution was refluxed for
thirty minutes. The solution was filtered and the precipitated salt was rinsed with acetone. The acetone
filtrate was evaporated and the residue was taken up in 20ml of methanol. Concentrated ammonium
hydroxide (20ml) was added and the solution was refluxed for one hour. The reaction mixture was
extracted three times with 25ml of chloroform and combined extracts were dried over sodium sulfate,
filtered and evaporated.The conjugate was prepared by dissolving 5mg each of DTAF and the amine in
0.5ml of methanol. About five drops of dimethylsulfoxide were added to dissolve the precipitate. The
reaction was completed in ten minutes and purified by using the procedure of Example XIII using
chloroform/methanol (3:1) as the developing solvent.
Example XVIII
8-Aminomethyl-theophylline-DTAF
DTAF (1 Omg) and 8-aminomethyl-theophylline (5mg) were dissolved in 0.5ml of
dimethylsulfoxide. After five minutes the reaction was complete and the conjugate was purified by
silica gel thin-layer chromatography, using chloroform/acetone (1 :1) as the developing solvent.
Example IXX 8-Aminoethyl-theophylline-DTAF Procedure same as Example XVII I substituting 8-aminoethyltheophylline for the methyl
derivative.
Example XX
Quinidine-DTAF
DTAF (5mg) and anhydrous quinidine (5mg) were dissolved in 0.5ml of dimethylformamide. After
sixteen hours the reaction was complete and the conjugate was purified by silica gel thin-layer
chromatography, using chloroform/methanol (3:1) as the developing solvent.
As mentioned above, the fluorescently labeled tracers prepared according to this invention can be
used in a variety of immunoassay procedures. The following assays are offered to demonstrate the
suitability of the representative sampling of these tracers in fluoroscence polarization assays.
All Examples followed the same basic protocol:
1) A small voluem of standard or test serum is delivered into a test tube and diluted with buffer;
2) A small volume of concentrated fluorescent tracer containing surfactant is then added to each
tube;
3) Finally, a volume of diluted antiserum is added; and
4) The reaction mixture is incubated at room temperature.
Example XXI
Valproic Acid Assay 2-Ethyl-5-.amino-pentanoic Acid DTAF Conjugate
Materials Required:
1) Buffer: 0.1M phosphate, pH 7.5, containing 0.01% (w/v) sodium azide and 0.01% (w/v) bovine.
gamma globulin (BGG).
2) Tracer: 2-ethyl-5-amino-pentanoic acid DTAF conjugate 50x 1 0-9M in 0.1 M tris hydrochloride buffer, pH 7.8, containing 0.1% (w/v) sodium dodecyl sulfate, 0.01% (w/v) bovine gamma globulin, and 0.01% (w/v) sodium azide.
3) Antibody: Sheep antiserum to valproic acid diluted to 1 to 3.75 in buffer.
4) Standards or unknowns: Human serum (or other biological fluid) containing valproic acid in the concentration range 0 to 150 g ug/ml.
5) Fluorescence polarimeter: Instrument capable of reading the polarization of fluorescense of a 1 xl 0-9M fluorescein solution to + 0.001 polarization unit.
Protocol: 1) 0.75 of standard or unknown sample placed in a 12x75nm disposable culture tube (cuvette). This is accomplished by pipetting 20,u1 of standard or unknown into a predilution container followed by 500y1 of buffer. Next, 20y1 of diluted sample is pipetted into the 1 2x75 culture tube followed by 400y1 of buffer.
2) 40y1 of tracer and 800 l of buffer are added to the cuvette.
3) 40y1 of antiserum and 800,u1 of buffer are added to the cuvette. The contents of the cuvette are mixed and incubated for approximately 15 minutes at room temperature.
4) The fluorescence polarization is read. Typical results are presented in Table
Table I ValproicAcid Conc. (gjml) Polarization
0 0.217
12.5 0.186
25 0.165
50 0.132
100 0.099
150 0.081
The polarization changes in a regular manner as the concentration of valproic acid is varied allowing the construction of a standard curve. Unknown samples are treated in an identical manner; from the polarization of fluorescence of the unknown sample, the concentration of valproic acid in the unknown sample may be determined by reference to the standard curve.
Example XXII
Gentamicin Assay
Materials:
1) Buffer: (See valproic acid assay).
2) Tracer: Gentamicin-DTAF at 1 00nM in a trihydrochloride buffer pH 7.5 containing 0.125% sodium dodecyl sulfate, 0.01% sodium azide, and 0.01% bovine gamma globulin.
3) Antibody: Rabbit or sheep antisera to gentamicin diluted appropriately in buffer.
4) Standards or unknowns: Human serum (or other biological fluid) containing gentamicin.
5) Fluorescence polarimeter: (See valproic acid assay).
Protocol:
1) 1.8ul of standard or unknown sample is placed in a 12x75nM disposable culture tube (cuvette). This is done by pipetting 20 l of sample followed by 200,u1 of buffer. Next 20 l of diluted sample is pipetted into the curvette followed by 200ul of buffer.
2) 40 l of tracer and 1000ul of buffer are added to the cuvette.
3) 40y1 of antibody and 1 OOOyi of buffer are added, the contents of the cuvette are mixed and incubated for approximately fifteen minutes at room temperature.
4) The fluorescence polarization is read following the incubation. Typical results are presented in
Table II.
Table II
Genatmicin Concentration f g/miJ Polarization
0 0.178
0.5 0.158
1.0 0.140
2.0 0.115
4.0 0.090
8.0 0.074
The polarization changes in a regular manner allowing construction of a standard curve. Unknown samples are tested in an identical manner, and the gentamicin content is determined by reference to the standard curve. The utility of the gentamicin-DTAF tracer for determining the concentration of entamicin in biological samples is thereby illustrated.
Example XXIII
N-Acetyl Procainamide Assay
Materials required:
1) Buffer: (See valproic acid assay).
2) Tracer: Desethyl-N-acetyl procainamide-DTAF conjugate at a concentration of 50x 10-9M in a 5.75% (w/v) solution of sodium toluene sulfonate.
3) Antiserum: Rabbit antiserum to N-acetyl-procainamide diluted one to six in buffer.
4) Standards or unknowns: Human serum (or other biological fluid).
5) Fluorescence polarimeter: (See valproic acid assay).
Protocol:
1) 0.4881 of standard or unknown in placed in a cuvette by pipetting 1 oil of sample into a predilution container and mixing with 200,us of buffer. Tenyl of diluted sample is next pipetted into the cuvette followed by 200ul of buffer.
2) 4081 of tracer and 1000yl of buffer are added to the cuvette.
3) 4081 of antiserum and 1000yl of buffer are next added to the cuvette. The contents of the cuvette are mixed and incubated at room temperature for approximately 1 5 minuteqat room temperature.
4) The fluorescence polarization is read following the 15-minute incubation period. Typical results for N-acetyl-procainamide are presented in Table Ill.
Table Ill N-Ace tyl Procainamide (ug/mli Polarization
0 0.239
1 0.218
2 0.209
4 0.190
8 0.173
16 0.158
A standard curve can be constructed from the data in Table III. Unknown samples treated identically to the standards can be quantitated by reference to the standard curve, thereby illustrating the usefuiness of the des-ethyl-N-acetyl procainamide-DTAF conjugate for the determination of Nacetyl procainamide in biological fluids.
Claims (20)
1. A fluorescent compound of the formula:
wherein X is a biologically interesting moiety having a molecular weight of less than 2000 and a reactive substituent selected from the group consisting of a primary or secondary amine or hydroxyl group by which it attaches to a carbon atom of the triazine ring; and Z is selected from the group consisting of 4-aminofluorescein and 5-aminofluorescein attached through the 4-amino or 5-amino, respectively to the number 2 carbon of the triazine ring.
2. The compound according to Claim 1 wherein X is an aminoglycoside antibiotic.
3. The compound according to Claim 2 wherein the aminoglycoside is gentamicin.
4. The compound according to Claim 2 wherein the aminoglycoside is tubramycin.
5. The compound according to Claim 2 wherein the aminoglycoside is amikacin.
6. The compound according to Claim 1 wherein X is amino-phenobarbital.
7. The compound according to Claim 1 wherein X is desethyl-N-acetylprocainamide.
8. The compound according to Claim 1 wherein X is N-para-acetamidobenzoyl ethylenediamine.
9. The compound according to Claim 1 wherein X is N-acetamidobenzoyl-Nt-ethyl-N'-amino acetyl ethylenediamine.
10. The compound according to Claim 1 wherein X is para-aminoprimidone.
11. The compound according to Claim 1 wherein X is 2-ethyl-5-aminopentanoic acid.
1 2. The compound according to Claim 1 wherein X is D-thyroxine.
13. The compound according to Claim 1 wherein X is L-tyroxine.
14. The compound according to Claim 1 wherein X is 2-propyl-5-aminopentanoic acid.
1 5. The compound according to Claim 1 wherein X is 3,3',5-triiodo-L-thyronine.
1 6. The compound according to Claim 1 wherein X is 2-(2-aminoethyl)-5,5-diphenyl hydantoin.
1 7. The compound according to Claim 1 wherein X is 8-aminomethyl theophylline.
18. The compound according to Claim 1 wherein X is 8-(2-aminoethyl) theophylline.
19. The compound according to Claim 1 wherein X is quinidine.
20. A fluorescent compound as claimed in Claim 1 and according to any one of the Examples herein.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17355380A | 1980-07-30 | 1980-07-30 |
Publications (2)
Publication Number | Publication Date |
---|---|
GB2081257A true GB2081257A (en) | 1982-02-17 |
GB2081257B GB2081257B (en) | 1984-11-07 |
Family
ID=22632537
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB8118754A Expired GB2081257B (en) | 1980-07-30 | 1981-06-18 | Biologically interesting compounds labeled with chlorotriazinyl-aminofluorescein |
Country Status (9)
Country | Link |
---|---|
JP (1) | JPS5758695A (en) |
AU (1) | AU554360B2 (en) |
BE (1) | BE889788A (en) |
CA (1) | CA1160626A (en) |
DE (1) | DE3129705A1 (en) |
FR (1) | FR2487835A1 (en) |
GB (1) | GB2081257B (en) |
IT (1) | IT1137630B (en) |
SE (1) | SE8104227L (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0175586A2 (en) * | 1984-09-21 | 1986-03-26 | Ortho Diagnostic Systems Inc. | Fluorescence polarization immunoassay for heavy antigens. |
EP0199042A1 (en) * | 1985-03-26 | 1986-10-29 | Abbott Laboratories | Procainamide assay, tracers, immunogens and antibodies |
EP0199963A1 (en) * | 1985-04-01 | 1986-11-05 | Abbott Laboratories | Ethosuximide assay tracers, immunogens and antibodies |
EP0210410A1 (en) * | 1985-07-22 | 1987-02-04 | Abbott Laboratories | Fluorescence polarization immunoassay and reagents for measurement of C-reactive protein |
EP0218010A2 (en) * | 1985-07-10 | 1987-04-15 | Abbott Laboratories | Ligand detection method and substituted carboxyfluorescein tracers therefor |
EP0254120A2 (en) * | 1986-07-14 | 1988-01-27 | Abbott Laboratories | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies |
US4902630A (en) * | 1985-07-22 | 1990-02-20 | Abbott Laboratories | Fluorescence polarization immunoassy and reagents for measurement of c-reactive protein |
EP0457213A2 (en) * | 1990-05-16 | 1991-11-21 | Abbott Laboratories | Barbiturate assay, tracers, immunogens, antibodies and kit |
EP0542627A1 (en) * | 1991-11-15 | 1993-05-19 | Clonatec | Diagnostic reagent, its applications in a method for determining an analyte and apparatus for carrying out this method |
US5986094A (en) * | 1996-04-24 | 1999-11-16 | Roche Diagnostics Corporation | 4'-methyl substituted fluorescein derivatives |
WO2005036169A2 (en) * | 2003-10-03 | 2005-04-21 | Cumbre Inc. | Fluorescent probes for ribosomes and method of use |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4476228A (en) * | 1982-11-08 | 1984-10-09 | Abbott Laboratories | Determination of unsaturated thyroxine binding protein sites using fluorescence polarization techniques |
US4476229A (en) * | 1982-11-08 | 1984-10-09 | Abbott Laboratories | Substituted carboxyfluoresceins |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3998943A (en) * | 1973-10-02 | 1976-12-21 | Syva Company | Double receptor fluorescent immunoassay |
CA1086306A (en) * | 1976-12-06 | 1980-09-23 | Dennis Blakeslee | Immunofluorescence reagent and process for preparing same |
US4213904A (en) * | 1979-02-28 | 1980-07-22 | Haugland Richard P | Fluorescent labeling reagents containing the fluorescein and eosin chromophores |
-
1981
- 1981-06-15 CA CA000379747A patent/CA1160626A/en not_active Expired
- 1981-06-18 GB GB8118754A patent/GB2081257B/en not_active Expired
- 1981-06-22 AU AU72036/81A patent/AU554360B2/en not_active Ceased
- 1981-07-07 SE SE8104227A patent/SE8104227L/en not_active Application Discontinuation
- 1981-07-28 DE DE19813129705 patent/DE3129705A1/en active Granted
- 1981-07-28 IT IT23207/81A patent/IT1137630B/en active
- 1981-07-29 FR FR8114768A patent/FR2487835A1/en active Granted
- 1981-07-29 BE BE0/205525A patent/BE889788A/en not_active IP Right Cessation
- 1981-07-30 JP JP56118573A patent/JPS5758695A/en active Pending
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0175586A2 (en) * | 1984-09-21 | 1986-03-26 | Ortho Diagnostic Systems Inc. | Fluorescence polarization immunoassay for heavy antigens. |
EP0175586A3 (en) * | 1984-09-21 | 1987-07-29 | Ortho Diagnostic Systems Inc. | Fluorescence polarization immunoassay for heavy antigens. |
EP0199042A1 (en) * | 1985-03-26 | 1986-10-29 | Abbott Laboratories | Procainamide assay, tracers, immunogens and antibodies |
EP0199963A1 (en) * | 1985-04-01 | 1986-11-05 | Abbott Laboratories | Ethosuximide assay tracers, immunogens and antibodies |
EP0218010A3 (en) * | 1985-07-10 | 1988-01-07 | Abbott Laboratories | Ligand detection method and substituted carboxyfluorescein tracers therefor |
EP0218010A2 (en) * | 1985-07-10 | 1987-04-15 | Abbott Laboratories | Ligand detection method and substituted carboxyfluorescein tracers therefor |
US4902630A (en) * | 1985-07-22 | 1990-02-20 | Abbott Laboratories | Fluorescence polarization immunoassy and reagents for measurement of c-reactive protein |
EP0210410A1 (en) * | 1985-07-22 | 1987-02-04 | Abbott Laboratories | Fluorescence polarization immunoassay and reagents for measurement of C-reactive protein |
EP0254120A2 (en) * | 1986-07-14 | 1988-01-27 | Abbott Laboratories | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies |
EP0254120A3 (en) * | 1986-07-14 | 1990-09-12 | Abbott Laboratories | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies |
US5260441A (en) * | 1986-07-14 | 1993-11-09 | Abbott Laboratories | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies |
EP0457213A2 (en) * | 1990-05-16 | 1991-11-21 | Abbott Laboratories | Barbiturate assay, tracers, immunogens, antibodies and kit |
EP0457213A3 (en) * | 1990-05-16 | 1992-09-02 | Abbott Laboratories | Barbiturate assay, tracers, immunogens, antibodies and kit |
EP0542627A1 (en) * | 1991-11-15 | 1993-05-19 | Clonatec | Diagnostic reagent, its applications in a method for determining an analyte and apparatus for carrying out this method |
FR2683911A1 (en) * | 1991-11-15 | 1993-05-21 | Clonatec Sa | DIAGNOSTIC REAGENT, ITS APPLICATIONS IN A METHOD FOR RAPID QUALITATIVE AND QUANTITATIVE DETERMINATION OF AN ANALYTE IN A FLUID TO BE TESTED AND DEVICE FOR IMPLEMENTING SAID METHOD. |
WO1993010452A1 (en) * | 1991-11-15 | 1993-05-27 | Clonatec | Diagnosis reactant, its applications in a method for assaying an analyte and device for implementing such method |
US5986094A (en) * | 1996-04-24 | 1999-11-16 | Roche Diagnostics Corporation | 4'-methyl substituted fluorescein derivatives |
WO2005036169A2 (en) * | 2003-10-03 | 2005-04-21 | Cumbre Inc. | Fluorescent probes for ribosomes and method of use |
WO2005036169A3 (en) * | 2003-10-03 | 2005-09-09 | Cumbre Inc | Fluorescent probes for ribosomes and method of use |
Also Published As
Publication number | Publication date |
---|---|
AU554360B2 (en) | 1986-08-21 |
CA1160626A (en) | 1984-01-17 |
IT8123207A0 (en) | 1981-07-28 |
DE3129705A1 (en) | 1982-05-27 |
IT1137630B (en) | 1986-09-10 |
GB2081257B (en) | 1984-11-07 |
FR2487835B1 (en) | 1984-03-16 |
BE889788A (en) | 1982-01-29 |
FR2487835A1 (en) | 1982-02-05 |
AU7203681A (en) | 1982-02-04 |
DE3129705C2 (en) | 1988-03-10 |
JPS5758695A (en) | 1982-04-08 |
SE8104227L (en) | 1982-01-31 |
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