JP2000319262A - Phenyldiazirine compound and photoaffinity reagent - Google Patents
Phenyldiazirine compound and photoaffinity reagentInfo
- Publication number
- JP2000319262A JP2000319262A JP2000059547A JP2000059547A JP2000319262A JP 2000319262 A JP2000319262 A JP 2000319262A JP 2000059547 A JP2000059547 A JP 2000059547A JP 2000059547 A JP2000059547 A JP 2000059547A JP 2000319262 A JP2000319262 A JP 2000319262A
- Authority
- JP
- Japan
- Prior art keywords
- compound
- group
- general formula
- sugar
- phenyldiazirine
- 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
- -1 Phenyldiazirine compound Chemical class 0.000 title claims abstract description 99
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 44
- 150000001875 compounds Chemical class 0.000 claims abstract description 249
- 125000002947 alkylene group Chemical group 0.000 claims abstract description 17
- 125000000732 arylene group Chemical group 0.000 claims abstract description 13
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 10
- 125000003545 alkoxy group Chemical group 0.000 claims abstract description 8
- 235000000346 sugar Nutrition 0.000 claims description 80
- 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 claims description 46
- 125000003277 amino group Chemical group 0.000 claims description 32
- 238000002372 labelling Methods 0.000 claims description 31
- 238000005222 photoaffinity labeling Methods 0.000 claims description 28
- 229960002685 biotin Drugs 0.000 claims description 23
- 235000020958 biotin Nutrition 0.000 claims description 23
- 239000011616 biotin Substances 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 19
- 150000003839 salts Chemical group 0.000 claims description 18
- 125000003172 aldehyde group Chemical group 0.000 claims description 8
- 125000004432 carbon atom Chemical group C* 0.000 claims description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 6
- 125000006239 protecting group Chemical group 0.000 claims description 6
- 125000004057 biotinyl group Chemical group [H]N1C(=O)N([H])[C@]2([H])[C@@]([H])(SC([H])([H])[C@]12[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(*)=O 0.000 claims description 4
- 230000003993 interaction Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 125000005842 heteroatom Chemical group 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 125000005543 phthalimide group Chemical group 0.000 claims 3
- 239000002262 Schiff base Substances 0.000 claims 1
- 150000004753 Schiff bases Chemical class 0.000 claims 1
- 230000001678 irradiating effect Effects 0.000 claims 1
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 abstract 2
- 102100030878 Cytochrome c oxidase subunit 1 Human genes 0.000 abstract 1
- 101000919849 Homo sapiens Cytochrome c oxidase subunit 1 Proteins 0.000 abstract 1
- 101000605122 Homo sapiens Prostaglandin G/H synthase 1 Proteins 0.000 abstract 1
- 101100133350 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) nhp-1 gene Proteins 0.000 abstract 1
- 150000001299 aldehydes Chemical group 0.000 abstract 1
- 125000002768 hydroxyalkyl group Chemical group 0.000 abstract 1
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 66
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 57
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 39
- 239000002904 solvent Substances 0.000 description 39
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 31
- 238000006243 chemical reaction Methods 0.000 description 31
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 30
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 29
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 21
- 108090000623 proteins and genes Proteins 0.000 description 21
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 18
- 238000000746 purification Methods 0.000 description 18
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 17
- 239000000741 silica gel Substances 0.000 description 17
- 229910002027 silica gel Inorganic materials 0.000 description 17
- 125000006850 spacer group Chemical group 0.000 description 17
- 239000003480 eluent Substances 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 15
- 239000011541 reaction mixture Substances 0.000 description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 13
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 13
- 238000004128 high performance liquid chromatography Methods 0.000 description 13
- 102000005962 receptors Human genes 0.000 description 13
- 108020003175 receptors Proteins 0.000 description 13
- 239000000523 sample Substances 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 10
- CWNPOQFCIIFQDM-UHFFFAOYSA-N 3-nitrobenzyl alcohol Chemical compound OCC1=CC=CC([N+]([O-])=O)=C1 CWNPOQFCIIFQDM-UHFFFAOYSA-N 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 9
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 8
- 102000004856 Lectins Human genes 0.000 description 8
- 108090001090 Lectins Proteins 0.000 description 8
- 239000002523 lectin Substances 0.000 description 8
- 239000003446 ligand Substances 0.000 description 8
- HJUGFYREWKUQJT-UHFFFAOYSA-N tetrabromomethane Chemical compound BrC(Br)(Br)Br HJUGFYREWKUQJT-UHFFFAOYSA-N 0.000 description 8
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 8
- OAKWVPQFGUEGCB-UHFFFAOYSA-N 3-phenyl-1h-diazirine Chemical class N1N=C1C1=CC=CC=C1 OAKWVPQFGUEGCB-UHFFFAOYSA-N 0.000 description 7
- YXHKONLOYHBTNS-UHFFFAOYSA-N Diazomethane Chemical group C=[N+]=[N-] YXHKONLOYHBTNS-UHFFFAOYSA-N 0.000 description 7
- 238000001816 cooling Methods 0.000 description 7
- 238000010586 diagram Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 7
- 239000012046 mixed solvent Substances 0.000 description 7
- 229910000027 potassium carbonate Inorganic materials 0.000 description 7
- 230000002194 synthesizing effect Effects 0.000 description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 6
- 108090001008 Avidin Proteins 0.000 description 6
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- QLTSDROPCWIKKY-PMCTYKHCSA-N beta-D-glucosaminyl-(1->4)-beta-D-glucosamine Chemical group O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O)[C@@H](CO)O1 QLTSDROPCWIKKY-PMCTYKHCSA-N 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- MEKOFIRRDATTAG-UHFFFAOYSA-N 2,2,5,8-tetramethyl-3,4-dihydrochromen-6-ol Chemical compound C1CC(C)(C)OC2=C1C(C)=C(O)C=C2C MEKOFIRRDATTAG-UHFFFAOYSA-N 0.000 description 5
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 5
- 239000008101 lactose Substances 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 5
- KFEUJDWYNGMDBV-UHFFFAOYSA-N (N-Acetyl)-glucosamin-4-beta-galaktosid Natural products OC1C(NC(=O)C)C(O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 KFEUJDWYNGMDBV-UHFFFAOYSA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- HESSGHHCXGBPAJ-UHFFFAOYSA-N N-acetyllactosamine Natural products CC(=O)NC(C=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O HESSGHHCXGBPAJ-UHFFFAOYSA-N 0.000 description 4
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 150000001555 benzenes Chemical class 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 239000013068 control sample Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 description 4
- 239000012312 sodium hydride Substances 0.000 description 4
- 229910000104 sodium hydride Inorganic materials 0.000 description 4
- HKAVADYDPYUPRD-UHFFFAOYSA-N 1h-pyrazine-2-thione Chemical compound SC1=CN=CC=N1 HKAVADYDPYUPRD-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- PNIWLNAGKUGXDO-UHFFFAOYSA-N Lactosamine Natural products OC1C(N)C(O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 PNIWLNAGKUGXDO-UHFFFAOYSA-N 0.000 description 3
- KFEUJDWYNGMDBV-LODBTCKLSA-N N-acetyllactosamine Chemical compound O[C@@H]1[C@@H](NC(=O)C)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KFEUJDWYNGMDBV-LODBTCKLSA-N 0.000 description 3
- KMXMOOQVJSFNNK-UHFFFAOYSA-N [(2-methylpropan-2-yl)oxycarbonylamino] benzoate Chemical compound CC(C)(C)OC(=O)NOC(=O)C1=CC=CC=C1 KMXMOOQVJSFNNK-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229910052786 argon Inorganic materials 0.000 description 3
- 150000001615 biotins Chemical class 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000011261 inert gas Substances 0.000 description 3
- 150000007530 organic bases Chemical class 0.000 description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 3
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 150000004044 tetrasaccharides Chemical group 0.000 description 3
- 150000004043 trisaccharides Chemical group 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- KECMLGZOQMJIBM-UHFFFAOYSA-N 2-[2-(2-chloroethoxy)ethoxy]ethanol Chemical compound OCCOCCOCCCl KECMLGZOQMJIBM-UHFFFAOYSA-N 0.000 description 2
- CFMZSMGAMPBRBE-UHFFFAOYSA-N 2-hydroxyisoindole-1,3-dione Chemical compound C1=CC=C2C(=O)N(O)C(=O)C2=C1 CFMZSMGAMPBRBE-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- ATHHXGZTWNVVOU-UHFFFAOYSA-N N-methylformamide Chemical compound CNC=O ATHHXGZTWNVVOU-UHFFFAOYSA-N 0.000 description 2
- 240000002853 Nelumbo nucifera Species 0.000 description 2
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 2
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 235000019445 benzyl alcohol Nutrition 0.000 description 2
- RDHPKYGYEGBMSE-UHFFFAOYSA-N bromoethane Chemical compound CCBr RDHPKYGYEGBMSE-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000012230 colorless oil Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 150000004845 diazirines Chemical class 0.000 description 2
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- PQJJJMRNHATNKG-UHFFFAOYSA-N ethyl bromoacetate Chemical compound CCOC(=O)CBr PQJJJMRNHATNKG-UHFFFAOYSA-N 0.000 description 2
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 125000000468 ketone group Chemical group 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 2
- 150000007522 mineralic acids Chemical class 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 239000004533 oil dispersion Substances 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000003444 phase transfer catalyst Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 125000005630 sialyl group Chemical group 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 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 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical class N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- 125000004791 2-fluoroethoxy group Chemical group FCCO* 0.000 description 1
- FXQYOKDNWCSKFS-UHFFFAOYSA-N 3-phenyl-1-(trifluoromethyl)diazirine Chemical compound FC(F)(F)N1N=C1C1=CC=CC=C1 FXQYOKDNWCSKFS-UHFFFAOYSA-N 0.000 description 1
- XWSSUYOEOWLFEI-UHFFFAOYSA-N 3-phenylpyridazine Chemical group C1=CC=CC=C1C1=CC=CN=N1 XWSSUYOEOWLFEI-UHFFFAOYSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229920002670 Fructan Polymers 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- DRAJWRKLRBNJRQ-UHFFFAOYSA-N Hydroxycarbamic acid Chemical compound ONC(O)=O DRAJWRKLRBNJRQ-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229920000057 Mannan Polymers 0.000 description 1
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- OPFJDXRVMFKJJO-ZHHKINOHSA-N N-{[3-(2-benzamido-4-methyl-1,3-thiazol-5-yl)-pyrazol-5-yl]carbonyl}-G-dR-G-dD-dD-dD-NH2 Chemical compound S1C(C=2NN=C(C=2)C(=O)NCC(=O)N[C@H](CCCN=C(N)N)C(=O)NCC(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(N)=O)=C(C)N=C1NC(=O)C1=CC=CC=C1 OPFJDXRVMFKJJO-ZHHKINOHSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 235000004443 Ricinus communis Nutrition 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- NIGUVXFURDGQKZ-UQTBNESHSA-N alpha-Neup5Ac-(2->3)-beta-D-Galp-(1->4)-[alpha-L-Fucp-(1->3)]-beta-D-GlcpNAc Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@H]2[C@@H]([C@@H](O[C@]3(O[C@H]([C@H](NC(C)=O)[C@@H](O)C3)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O NIGUVXFURDGQKZ-UQTBNESHSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000002344 aminooxy group Chemical group [H]N([H])O[*] 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000031709 bromination Effects 0.000 description 1
- 238000005893 bromination reaction Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000004106 butoxy group Chemical group [*]OC([H])([H])C([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 229940126086 compound 21 Drugs 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- KNFXXAGQEUUZAZ-UHFFFAOYSA-N ethyl ethaneperoxoate Chemical compound CCOOC(C)=O KNFXXAGQEUUZAZ-UHFFFAOYSA-N 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000010436 fluorite Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 1
- 150000003271 galactooligosaccharides Chemical class 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 238000002000 high resolution fast-atom bombardment mass spectrometry Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- DOVBXGDYENZJBJ-ONMPCKGSSA-N lactosamine Chemical compound O=C[C@H](N)[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O DOVBXGDYENZJBJ-ONMPCKGSSA-N 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 125000004957 naphthylene group Chemical group 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 125000006353 oxyethylene group Chemical group 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 125000004115 pentoxy group Chemical group [*]OC([H])([H])C([H])([H])C([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 1
- 239000007856 photoaffinity label Substances 0.000 description 1
- 125000005544 phthalimido group Chemical group 0.000 description 1
- 125000000612 phthaloyl group Chemical group C(C=1C(C(=O)*)=CC=CC1)(=O)* 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 125000002572 propoxy group Chemical group [*]OC([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- AGDSCTQQXMDDCV-UHFFFAOYSA-M sodium;2-iodoacetate Chemical compound [Na+].[O-]C(=O)CI AGDSCTQQXMDDCV-UHFFFAOYSA-M 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- JOMQIRBLMOXBAZ-UHFFFAOYSA-N sulfamoyl benzoate Chemical compound NS(=O)(=O)OC(=O)C1=CC=CC=C1 JOMQIRBLMOXBAZ-UHFFFAOYSA-N 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- DRDVJQOGFWAVLH-UHFFFAOYSA-N tert-butyl n-hydroxycarbamate Chemical compound CC(C)(C)OC(=O)NO DRDVJQOGFWAVLH-UHFFFAOYSA-N 0.000 description 1
- 125000000383 tetramethylene group Chemical group [H]C([H])([*:1])C([H])([H])C([H])([H])C([H])([H])[*:2] 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 238000012929 ultra trace analysis Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Saccharide Compounds (AREA)
- Plural Heterocyclic Compounds (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、新規な光反応性フ
ェニルジアジリン化合物、光反応性ビオチン化フェニル
ジアジリン化合物、及び光反応性糖結合ビオチン化フェ
ニルジアジリン化合物に係わるものであり、更に、本発
明は生体物質構造解析のプローブへの用途が期待される
該光反応性ビオチン化フェニルジアジリン化合物からな
る光反応性標識試薬、及び光反応性糖結合ビオチン化フ
ェニルジアジリン化合物からなる光親和性標識試薬に係
わるものである。TECHNICAL FIELD The present invention relates to a novel photoreactive phenyldiazirine compound, a photoreactive biotinylated phenyldiazirine compound, and a photoreactive sugar-bonded biotinylated phenyldiazirine compound. The present invention provides a photoreactive labeling reagent comprising the photoreactive biotinylated phenyldiazirine compound, which is expected to be used as a probe for structural analysis of a biological material, and a photoreactive sugar-binding biotinylated phenyldiazirine compound. It relates to the affinity labeling reagent.
【０００２】[0002]
【従来技術】光親和性標識試薬は、薬物あるいはリガン
ドと蛋白質との相互作用部位を解析できる有用なプロー
ブとして知られている。蛋白質を光反応により標識する
光反応基としてはアジド基が古くから知られていたが、
より優れた光反応基としてジアジリン基が注目されてき
ている。薬物や生体物質由来のリガンドへのジアジリン
基の導入は、一般にアミド結合やエステル結合により行
われ、さらに微量検出のために放射性の誘導体に変換さ
れている。2. Description of the Related Art A photoaffinity labeling reagent is known as a useful probe capable of analyzing an interaction site between a drug or a ligand and a protein. Azide groups have long been known as photoreactive groups for labeling proteins by photoreaction,
Diazirine groups have been attracting attention as better photoreactive groups. Introduction of a diazirine group into a ligand derived from a drug or a biological substance is generally performed by an amide bond or an ester bond, and further converted to a radioactive derivative for detection of a trace amount.
【０００３】ごく最近になり、蛋白質の微量検出とアフ
ィニティー精製を同時に実現するものとして、分子内
に、スペーサー化合物を介してビオチン残基を有するフ
ェニルジアジリン誘導体が開発され、例えば、生体物質
由来のリガンドとしての糖化合物を該ジアジリン誘導体
のカルボキシル基に結合させることにより非放射性の光
親和性標識試薬の合成が可能であることが報告されてい
る（有機合成化学協会誌、第56巻第7号、第581〜590頁
(1998)；ファルマシア、Vol.34、No.8、第772〜776頁(1
998)；Biochem.J.(1998)330、1209-1215）。しかし、こ
の方法では、該ジアジリン誘導体に糖化合物を結合させ
る場合、該糖化合物に予めアミノ基を導入し、該ジアジ
リン誘導体が有するカルボキシル基と、ジシクロヘキシ
ルカルボジイミド（ＤＣＣ）等の縮合剤の存在下で反応
させることが必要であり、操作が煩雑であった。一方、
生体物質を特異的に修飾する方法として、オキシアミノ
基の利用が注目されているが、該オキシアミノ基を光反
応性化合物に導入し、糖化合物と結合させようとする試
みは未だ成されていない。[0003] More recently, a phenyldiazirine derivative having a biotin residue in the molecule via a spacer compound has been developed as a means for simultaneously realizing the detection of a trace amount of protein and affinity purification. It has been reported that a nonradioactive photoaffinity labeling reagent can be synthesized by binding a sugar compound as a ligand to the carboxyl group of the diazirine derivative (Journal of the Society of Synthetic Organic Chemistry, Vol. 56, No. 7) Pp. 581-590
(1998); Pharmacia, Vol. 34, No. 8, 772-776 (1
998); Biochem. J. (1998) 330, 1209-1215). However, in this method, when a sugar compound is bonded to the diazirine derivative, an amino group is introduced into the sugar compound in advance, and the carboxyl group of the diazirine derivative is combined with a condensing agent such as dicyclohexylcarbodiimide (DCC). The reaction was required, and the operation was complicated. on the other hand,
The use of oxyamino groups has attracted attention as a method for specifically modifying biological substances, but attempts to introduce the oxyamino groups into photoreactive compounds and bind them to sugar compounds have not yet been made. Absent.
【０００４】[0004]
【発明が解決しようとする課題】本発明は、放射性標識
を施す必要がない光反応性プローブへ導くことが可能な
新規な光反応性ビオチン化フェニルジアジリン化合物を
含む光反応性標識試薬、及び、糖と相互作用しうる蛋白
質（糖受容体）などの生体高分子の機能や構造を研究す
るうえでの有用性が期待される非放射性の光反応性糖結
合ビオチン化フェニルジアジリン化合物を含む光親和性
標識試薬を提供することを目的とする。本発明の更なる
目的は、これらの標識試薬を構成するために有用な新規
なビオチン化フェニルジアジリン化合物、及び糖結合ビ
オチン化フェニルジアジリン化合物、所望によりビオチ
ン残基が切断性のスペーサーを介してフェニルジアジリ
ン化合物と連結している該化合物、加えてこれらの化合
物を取得するための合成中間体である新規なフェニルジ
アジリン化合物を提供すること、並びにこの新規糖結合
ビオチン化フェニルジアジリン化合物の製法及び該化合
物による糖受容体の標識方法を提供することにある。SUMMARY OF THE INVENTION The present invention provides a photoreactive labeling reagent comprising a novel photoreactive biotinylated phenyldiazirine compound that can lead to a photoreactive probe that does not need to be radiolabeled. Including non-radioactive photoreactive sugar-linked biotinylated phenyldiazirine compounds expected to be useful in studying the function and structure of biological macromolecules such as proteins (sugar receptors) that can interact with sugar It is an object to provide a photoaffinity labeling reagent. It is a further object of the present invention to provide novel biotinylated phenyldiazirine compounds useful for constructing these labeling reagents, and sugar-linked biotinylated phenyldiazirine compounds, optionally with a biotin residue via a cleavable spacer. To provide a novel phenyldiazirine compound which is a synthetic intermediate for obtaining these compounds, and a novel sugar-linked biotinylated phenyldiazirine compound And a method for labeling a sugar receptor with the compound.
【０００５】[0005]
【課題を解決するための手段】本発明の要旨は、前記一
般式（II）〜(IV)の少なくとも一つで表されるビオチン
化フェニルジアジリン化合物、及びその合成中間体であ
る前記一般式（I）で表されるフェニルジアジリン化合
物、さらに、該一般式（II）〜(IV)の少なくとも一つか
ら誘導され、各式に対応して前記一般式（V）〜(VII)の
少なくとも一つで表される糖結合ビオチン化フェニルジ
アジリン化合物に存し、他の要旨は、該一般式（II）〜
(IV)の少なくとも一つで表される化合物を含む光反応性
標識試薬、及び該一般式（V）〜(VII)の少なくとも一つ
で表される糖結合ビオチン化フェニルジアジリン化合物
からなる光親和性標識試薬に存する。本発明の更なる要
旨は、一般式（II）〜(IV)の少なくとも一つで表される
ビオチン化フェニルジアジリン化合物に還元末端を有す
る糖化合物を反応させることよりなる、一般式（V）〜
(VII)の少なくとも一つで表される糖結合ビオチン化フ
ェニルジアジリン化合物の製造方法及びこれらの該糖結
合ビオチン化フェニルジアジリン化合物を用いる糖受容
体の標識方法に存する。The gist of the present invention is to provide a biotinylated phenyldiazirine compound represented by at least one of the above-mentioned general formulas (II) to (IV) and the above-mentioned general formula which is a synthetic intermediate thereof. A phenyldiazirine compound represented by (I), further derived from at least one of the general formulas (II) to (IV), and corresponding to each of the formulas (V) to (VII) Another aspect of the present invention resides in a sugar-linked biotinylated phenyldiazirine compound represented by the formula (II):
A photoreactive labeling reagent containing a compound represented by at least one of (IV), and a light comprising a sugar-linked biotinylated phenyldiazirine compound represented by at least one of the general formulas (V) to (VII). Affinity labeling reagent. A further gist of the present invention is that a biotinylated phenyldiazirine compound represented by at least one of the general formulas (II) to (IV) is reacted with a saccharide compound having a reducing end, and the general formula (V) ~
The present invention relates to a method for producing a sugar-linked biotinylated phenyldiazirine compound represented by at least one of (VII) and a method for labeling a sugar receptor using the sugar-linked biotinylated phenyldiazirine compound.
【０００６】[0006]
【発明の実施の形態】本発明における光親和性標識試薬
を構成する化合物を生成するための中間体化合物として
は、本質的には糖受容体蛋白質を光反応により標識する
光反応基としてのジアジリン基を有するトリフルオロメ
チルフェニルジアジリンを基本骨格とし、そのベンゼン
核に糖などのリガンドが結合し得るアミノ基を有する基
と標識化合物を有する基がそれぞれ結合したフェニルジ
アジリン誘導体であれば良い。詳しくは、該フェニルジ
アジリン誘導体において、アミノ基、アミノ基の塩、保
護アミノ基等から選ばれる基はアルキレン基等のスペー
サーを介してベンゼン核に結合し、且つリガンドとの結
合能に及ぼすジアジリン型の光反応基による影響を少な
くするために光反応基の結合位に対しP-位に結合してい
る。BEST MODE FOR CARRYING OUT THE INVENTION As an intermediate compound for producing a compound constituting a photoaffinity labeling reagent in the present invention, diazirine as a photoreactive group for labeling a sugar receptor protein by photoreaction is essentially used. Any phenyldiazirine derivative may be used as long as it has trifluoromethylphenyldiazirine as a basic skeleton and a benzene nucleus in which a group having an amino group to which a ligand such as a sugar can bind and a group having a labeling compound are respectively bonded. Specifically, in the phenyldiazirine derivative, a group selected from an amino group, a salt of an amino group, a protected amino group, and the like binds to a benzene nucleus via a spacer such as an alkylene group and affects the binding ability with a ligand. In order to reduce the influence of the type photoreactive group, it is bonded to the P-position relative to the bonding position of the photoreactive group.
【０００７】又、標識化合物を有する基は、ポリオキシ
エチレン基等を含む親水性スペーサーを介して標識化合
物、例えばビオチンがベンゼン核に結合されている。そ
の際、標識化合物、即ちビオチンはスペーサーの末端ア
ミノ基を介してベンゼン核に結合することができる。一
方、ビオチン化された糖受容体（ラベル化蛋白質）をア
ビジン固定化担体（固定化アビジン）を用いてアフィニ
ティー精製し、再遊離・回収を所望する場合、分離・精
製に通常使用される固定化アビジンとビオチンとの結合
力が強く、ラベル化蛋白質の回収率が低下することがあ
るので、ラベル化蛋白質とビオチンを緩和な条件で切り
離す事が出来るよう切断性を有するスペーサーを介して
結合させるのが好ましく、例えば、アシルスルホンアミ
ド等の切断性スペーサーを、ポリオキシエチレン基端に
導入したスペーサーを介してビオチンをベンゼン核に連
結させることが出来る。In the group having a labeling compound, a labeling compound, for example, biotin is bound to a benzene nucleus via a hydrophilic spacer containing a polyoxyethylene group or the like. At that time, the labeling compound, ie, biotin, can bind to the benzene nucleus via the terminal amino group of the spacer. On the other hand, a biotinylated sugar receptor (labeled protein) is affinity-purified using an avidin-immobilized carrier (immobilized avidin), and when re-release / recovery is desired, immobilization usually used for separation / purification is performed. Since the binding force between avidin and biotin is strong and the recovery rate of labeled protein may decrease, it is necessary to bind the labeled protein and biotin via a cleavable spacer so that they can be separated under mild conditions. Preferably, for example, biotin can be linked to a benzene nucleus via a cleavable spacer such as acylsulfonamide via a spacer introduced at the polyoxyethylene group end.
【０００８】本発明の上記一般式（II）で表される光反
応性のビオチン化フェニルジアジリン化合物は、この誘
導体を代表するものである。本発明の下記一般式（I）
で表される化合物は、該一般式（II）で表されるビオチ
ン化フェニルジアジリン化合物を合成するための中間体
であり、新規化合物である。The photoreactive biotinylated phenyldiazirine compound represented by the general formula (II) of the present invention is representative of this derivative. The following general formula (I) of the present invention
Is an intermediate for synthesizing the biotinylated phenyldiazirine compound represented by the general formula (II), and is a novel compound.
【０００９】[0009]
【化８】 Embedded image
【００１０】一般式（I）中、Ｒ1は−ＮＨＰ1又は−Ｃ
ＯＸ1を表し、Ｒ2は、アルデヒド基、ヒドロキシ低級ア
ルキル基、ハロゲン化低級アルキル基、フタルイミドオ
キシ低級アルキル基、又は−(ＣＨ2)x−Ｏ−ＮＨＰ1を
表し、Ｐ1はアミノ保護基を表し、Ｘ1はアルコキシ基，
ヒドロキシ基，又は−ＮＨＳＯ2−Ｘ2−−ＣＯ−Ｘ3を
表し、Ｘ2はアリーレン基又はアルキレン基を表し、Ｘ3
はアルコキシ基又はヒドロキシ基を表し、ｍは１〜４、
好ましくは１〜２の整数を表し、ｎは１〜６、好ましく
は１〜４の整数を表し、ｘは１〜４、好ましくは１〜２
の整数を表す。In the general formula (I), R 1 is -NHP 1 or -C
OX 1 , R 2 represents an aldehyde group, a hydroxy lower alkyl group, a halogenated lower alkyl group, a phthalimidooxy lower alkyl group, or — (CH 2 ) x —O-NHP 1 , and P 1 represents an amino protecting group X 1 represents an alkoxy group,
Hydroxy group, or an -NHSO 2 -X 2 --CO-X 3 , X 2 represents an arylene group or an alkylene group, X 3
Represents an alkoxy group or a hydroxy group, m represents 1 to 4,
Preferably, n represents an integer of 1 to 2, n represents an integer of 1 to 6, preferably 1 to 4, and x represents 1 to 4, preferably 1 to 2.
Represents an integer.
【００１１】本発明において、低級アルキル基とは、メ
チル、エチル、プロピル、ブチル等の炭素数１〜４のア
ルキル基を表し、直鎖状が好ましい。また、アルコキシ
基としては、炭素数１〜６のアルコキシ基であり、例え
ばメトキシ基、エトキシ基、プロポキシ基、ブトキシ
基、ペントキシ基等が挙げられる。アリーレン基として
は、メチル基、エチル基等の置換基を有していてもよい
フェニレン基、ナフチレン基等の炭素数５〜１４のアリ
ーレン基が挙げられるが、フェニレン基が好ましい。ア
ルキレン基としては、メチレン基、エチレン基、プロピ
レン基、ブチレン基等の炭素数１〜６のアルキレン基が
挙げられ、直鎖状の基が好ましい。アミノ保護基として
は、ｔ−ブトキシカルボニル基、トリフルオロアセチル
基、ベンジルオキシカルボニル基、ｐ−メトキシベンジ
ルオキシカルボニル、ｐ−トルエンスルホニル基、９−
フルオレニルメチルオキシカルボニル基、トリチル基、
フタロイル基、ｏ−ニトロフェニルスルフェニル基、３
−ニトロ−２−ピリジンスルフェニル基等が挙げられ
る。これらの内ｔ−ブトキシカルボニル基が簡便であ
る。In the present invention, the term "lower alkyl group" means an alkyl group having 1 to 4 carbon atoms such as methyl, ethyl, propyl and butyl, and is preferably linear. The alkoxy group is an alkoxy group having 1 to 6 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, a butoxy group, and a pentoxy group. Examples of the arylene group include a phenylene group which may have a substituent such as a methyl group and an ethyl group, an arylene group having 5 to 14 carbon atoms such as a naphthylene group, and a phenylene group is preferable. Examples of the alkylene group include an alkylene group having 1 to 6 carbon atoms such as a methylene group, an ethylene group, a propylene group, and a butylene group, and a linear group is preferable. Examples of the amino protecting group include t-butoxycarbonyl group, trifluoroacetyl group, benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl, p-toluenesulfonyl group, 9-
Fluorenylmethyloxycarbonyl group, trityl group,
Phthaloyl group, o-nitrophenylsulfenyl group, 3
-Nitro-2-pyridinesulfenyl group. Of these, the t-butoxycarbonyl group is convenient.
【００１２】本発明のビオチン化フェニルジアジリン化
合物は、下記一般式（II）で表される化合物であり、上
記一般式（I）の化合物をビオチン化し、所望によりＲ2
をアミノオキシ低級アルキル基に変換することにより得
られる。The biotinylated phenyldiazirine compound of the present invention is a compound represented by the following general formula (II). The compound of the above general formula (I) is biotinylated, and if desired, R 2
To an aminooxy lower alkyl group.
【００１３】[0013]
【化９】 Embedded image
【００１４】一般式（II）中、Ｒ3は、フタルイミド
基、アミノ基、−ＮＨＰ1又はアミノ基の塩を表し、Ｐ1
はアミノ保護基を表し、ｍは１〜４、好ましくは１〜２
の整数を表し、ｎは１〜６、好ましくは１〜４の整数を
表し、ｘは１〜４、好ましくは１〜２の整数を表す。
又、アミノ保護基は、上記一般式（I）におけるものと
同義を表す。アミノ基の塩としては、塩酸、硫酸、硝
酸、リン酸等の無機酸の塩、或いは酢酸、トリフルオロ
酢酸、プロピオン酸、メタンスルホン酸等の有機酸の塩
が挙げられる。これらの内、トリフルオロ酢酸は、一般
式（V）の化合物を生成するのに有利である。[0014] In the general formula (II), R 3 represents phthalimido group, an amino group, a salt of -NHP 1 or amino groups, P 1
Represents an amino-protecting group, and m is 1-4, preferably 1-2.
And n represents an integer of 1 to 6, preferably 1 to 4, and x represents an integer of 1 to 4, preferably 1 to 2.
The amino-protecting group has the same meaning as that in the above formula (I). Examples of the salt of an amino group include salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid, and salts of organic acids such as acetic acid, trifluoroacetic acid, propionic acid, and methanesulfonic acid. Of these, trifluoroacetic acid is advantageous for producing the compound of the general formula (V).
【００１５】Ｙ1は−ＮＨ−又は−ＣＯＮＨＳＯ2−Ｘ2
−ＣＯ−を表し、Ｘ2はアリーレン基又はアルキレン基
を表し、Ｙ2はビオチン又はビオチン誘導体残基を表
す。又、アリーレン基及びアルキレン基は上記一般式
(I)におけるものと同義を表す。本発明でビオチン誘導
体とは、ビオチンの末端基（−ＯＨ）がアミノ基又はカ
ルボキシル基と反応し得る基に置き換わったビオチンを
意味し、例えば、カルボキシル基と反応し得る基を有す
るものとして、(ビオチン)−ＮＨ−Ｚｑ−ＮＨ2で表さ
れる末端にアミノ基を有する化合物、好ましくはビオチ
ンヒドラジドが挙げられる。本発明の一般式(II)で示さ
れる化合物のより具体的な化合物は、下記一般式(III)
及び(IV)で示される化合物である。Y 1 is —NH— or —CONHSO 2 —X 2
It represents -CO-, X 2 represents an arylene group or an alkylene group, Y 2 is represents a biotin or a biotin derivative residue. Further, an arylene group and an alkylene group have the general formula
Represents the same meaning as in (I). In the present invention, the biotin derivative means biotin in which the terminal group (-OH) of biotin is replaced by a group capable of reacting with an amino group or a carboxyl group. For example, as a biotin derivative having a group capable of reacting with a carboxyl group, compounds having an amino group at the terminal represented by biotin) -NH-Zq-NH 2, with preference given to biotin hydrazide. More specific compound of the compound represented by the general formula (II) of the present invention, the following general formula (III)
And (IV).
【００１６】[0016]
【化１０】 一般式(III)中、Ｒ3、Ｐ1、ｍ、ｎ及びｘは、一般式(I
I)におけるものと同義を表す。Embedded image In the general formula (III), R 3 , P 1 , m, n and x are represented by the general formula (I
Represents the same meaning as in I).
【００１７】[0017]
【化１１】 一般式(IV)中、Ｒ3、Ｐ1、ｍ、ｎ及びｘは、上記一般式
(II)におけるものと同義を表し、Ｘ2はアリーレン基又
はアルキレン基を表し、上記一般式(I)におけるものと
同義を表す。Ｚは、酸素、硫黄、窒素等のヘテロ原子で
中断されていてもよい炭素数１〜２０のアルキレン基を
表し、例えば、メチレン基、エチレン基、プロピレン
基、（ポリ）オキシエチレン基等が挙げられる。ｑは０
又は１を表す。Embedded image In the general formula (IV), R 3 , P 1 , m, n and x represent the above general formula
X 2 has the same meaning as in (II), and X 2 represents an arylene group or an alkylene group, and has the same meaning as in formula (I). Z represents an alkylene group having 1 to 20 carbon atoms which may be interrupted by a hetero atom such as oxygen, sulfur or nitrogen, and examples thereof include a methylene group, an ethylene group, a propylene group, and a (poly) oxyethylene group. Can be q is 0
Or represents 1.
【００１８】本発明の下記一般式(V)〜(VII)で表され
る、糖結合ビオチン化フェニルジアジリン化合物は、一
般式（II）〜(IV)において、末端アミノ基或いはアミノ
基の塩を有するビオチン化フェニルジアジリン化合物
に、還元末端を有する糖化合物の還元末端のアルデヒド
基を反応させることにより得られる化合物である。The sugar-bonded biotinylated phenyldiazirine compound represented by the following general formulas (V) to (VII) according to the present invention is a compound represented by the general formula (II) to (IV), which has a terminal amino group or a salt of an amino group. Is a compound obtained by reacting a biotinylated phenyldiazirine compound having the following formula with an aldehyde group at the reducing end of a sugar compound having a reducing end.
【００１９】[0019]
【化１２】 Embedded image
【００２０】一般式(V)中、Ｒ4は、還元末端をもつ糖化
合物の残基を表し、ｍは１〜４、好ましくは１〜２の整
数を表し、ｎは１〜６、好ましくは１〜４の整数を表
し、ｘは１〜４、好ましくは１〜２の整数を表す。Ｙ1
は−ＮＨ−又は−ＣＯＮＨＳＯ2−Ｘ2−ＣＯ−を表し、
Ｘ2はアリーレン基又はアルキレン基を表し、Ｙ2はビオ
チン又はビオチン誘導体残基を表し、上記一般式(II)に
おけるものと同義を表す。本発明の下記一般式(VI)及び
(VII)で示される化合物は、一般式(V)で示される化合物
のより具体的な化合物である。In the general formula (V), R 4 represents a residue of a sugar compound having a reducing end, m represents an integer of 1-4, preferably 1-2, and n represents 1-6, preferably X represents an integer of 1 to 4, and x represents an integer of 1 to 4, preferably 1 or 2. Y 1
It is -NH- or -CONHSO 2 -X 2 -CO- represent,
X 2 represents an arylene group or an alkylene group, Y 2 represents biotin or a biotin derivative residue, and has the same meaning as in the above formula (II). The following general formula (VI) of the present invention and
The compound represented by (VII) is a more specific compound of the compound represented by formula (V).
【００２１】[0021]
【化１３】 一般式(VI)中、Ｒ4、ｍ、ｎ、ｘは上記一般式(V)におけ
るものと同義を表す。Embedded image In the general formula (VI), R 4 , m, n, and x have the same meanings as those in the general formula (V).
【００２２】[0022]
【化１４】 一般式（VII）中、Ｒ4、ｍ、ｎ、ｘ及びＸ2は、上記一
般式(V)におけるものと同義を表し、Ｚ及びｑは、上記
一般式(IV)におけるものと同義を表す。一般式(VII)で
示される化合物は、切断性スペーサーを有する糖結合ビ
オチン化フェニルジアジリン化合物であり、特にＸ2が
フェニレン基、ｑ＝０の化合物は、ラベル蛋白質の分離
・回収に有用な光親和性標識試薬を構成する化合物とし
て好ましい。Embedded image In the general formula (VII), R 4 , m, n, x and X 2 have the same meaning as in the above general formula (V), and Z and q have the same meaning as in the above general formula (IV) . The compound represented by the general formula (VII) is a sugar-bonded biotinylated phenyldiazirine compound having a cleavable spacer, and in particular, a compound in which X 2 is a phenylene group and q = 0 is useful for separating and recovering a labeled protein. Preferred as a compound constituting the photoaffinity labeling reagent.
【００２３】一般式（II）〜(IV)で示される化合物と反
応させる糖化合物としては、還元末端を持つ多糖類、オ
リゴ糖類、単糖類、例えば、ムチン型糖類、Asn型糖
類、シアリル糖類、グリコサミノグリカン、ラクトサミ
ン、N-アセチルラクトサミン、ラクトサミンオリゴ糖、
シアリルラクトサミン、グルカン、マンナン、フルクタ
ン、ガラクタン、ポリウロン酸、オリゴアミノ酸、ポリ
アミノ糖、ガラクトオリゴ糖などが挙げられる。Ｒ4の
より具体的な例としては、N-アセチルラクトシル基、シ
アリル−α2-3-ラクトシル基、シアリル−α2-3-N-アセ
チルラクトサミニル基、ルイスX型三糖残基、シアリル
ルイスX型四糖残基、又はキトビオース残基が挙げられ
るが、下記模式図に示す結合が可能な糖化合物であれば
これらに限定されない。即ち、本発明の一般式（II）で
示される化合物において、Ｒ3がアミノ基又はアミノ基
の塩である化合物と還元末端を有する糖化合物との結合
は選択的で、その結合は以下のように示される。Examples of the saccharide compound to be reacted with the compounds represented by the general formulas (II) to (IV) include polysaccharides, oligosaccharides, and monosaccharides having a reducing end, for example, mucin-type saccharide, Asn-type saccharide, sialyl saccharide, Glycosaminoglycan, lactosamine, N-acetyllactosamine, lactosamine oligosaccharide,
Sialyl lactosamine, glucan, mannan, fructan, galactan, polyuronic acid, oligoamino acids, polyamino sugars, galactooligosaccharides and the like can be mentioned. More specific examples of R 4 include N-acetyllactosyl group, sialyl-α2-3-lactosyl group, sialyl-α2-3-N-acetyllactosaminyl group, Lewis X-type trisaccharide residue, sialyl Lewis Examples thereof include an X-type tetrasaccharide residue and a chitobiose residue, but are not limited thereto as long as they are sugar compounds capable of bonding as shown in the following schematic diagrams. That is, in the compound represented by the general formula (II) of the present invention, the bond between the compound in which R 3 is an amino group or a salt of an amino group and the sugar compound having a reducing end is selective, and the bond is as follows. Is shown in
【００２４】[0024]
【化１５】 Embedded image
【００２５】本発明の一般式(II)〜(IV)で示される化合
物において、Ｒ3がアミノ基又はアミノ基の塩である化
合物と、還元末端を有する糖化合物との反応はｐＨ依存
性であり、該反応は弱酸性下有利に進行する。通常該反
応はｐＨ１〜７で進行するが、好ましくは緩衝液を用い
ｐＨ４〜５に調整されて行われる。本発明の一般式（I
I)〜(IV)で示される光反応性ビチオン化フェニルジアジ
リン化合物の一種は、そのフェニル骨格にスペーサーを
介してオキシアミノ基が導入されている特徴を有し、こ
のオキシアミノ基は、還元末端を有する糖化合物の還元
末端のアルデヒド基と、糖鎖水酸基の保護などを特に必
要とせず、また従来必要とされているＤＣＣ等の縮合剤
も存在せしめることなく、単に糖化合物と混合するだけ
でシッフ塩基形成反応により結合し、一般式(V)〜(VII)
で示される糖結合ビオチン化フェニルジアジリン化合物
を生成する。そして、この化合物によって本発明の光親
和性標識試薬を構成することができる。該試薬は、この
化合物の機能を害さない限り、水、緩衝剤、安定化剤，
塩等の添加物を含んでいてもよい。また、一般式(II)〜
(IV)の化合物のオキシアミノ基は、アルデヒド基以外に
ケトン基、カルボキシル基とも結合し得るので、糖化合
物がこれらの反応基を有する場合はその官能基とオキシ
アミノ基を反応させてもよい。なお、光親和性標識試薬
とは、広義には、光反応性基を有する化合物にリガンド
を結合した標識試薬を指称するが、本明細書において
「光親和性標識試薬」とは、光反応性糖受容体親和性標
識試薬を意味するものとし、糖受容体とは糖と親和性を
有する、すなわち糖との特異的な相互作用により糖に結
合性を示す蛋白質を意味し、具体的には糖に特異的なレ
クチン、レセプター、酵素、抗体などを包含する。In the compounds represented by the general formulas (II) to (IV) of the present invention, the reaction of a compound wherein R 3 is an amino group or a salt of an amino group with a sugar compound having a reducing end is pH-dependent. Yes, the reaction advantageously proceeds under weak acidity. Usually, the reaction proceeds at pH 1 to 7, but is preferably performed by adjusting the pH to 4 to 5 using a buffer solution. The general formula (I) of the present invention
One of the photoreactive bitionized phenyldiazirine compounds represented by I) to (IV) has a feature that an oxyamino group is introduced into the phenyl skeleton via a spacer, and this oxyamino group is reduced. The aldehyde group at the reducing end of the sugar compound having a terminal and the protection of the hydroxyl group of the sugar chain are not particularly required, and the saccharide compound is simply mixed with the sugar compound without the need for a condensing agent such as DCC conventionally required. In general formulas (V) to (VII)
To produce a sugar-linked biotinylated phenyldiazirine compound represented by the formula: Then, this compound can constitute the photoaffinity labeling reagent of the present invention. The reagent may be any of water, buffers, stabilizers, as long as the function of the compound is not impaired.
An additive such as a salt may be included. In addition, the general formula (II)
Since the oxyamino group of the compound (IV) can bind to a ketone group and a carboxyl group in addition to the aldehyde group, when the sugar compound has these reactive groups, the functional group may be reacted with the oxyamino group. . Note that, in a broad sense, the photoaffinity labeling reagent refers to a labeling reagent in which a ligand is bound to a compound having a photoreactive group. In this specification, the term “photoaffinity labeling reagent” refers to a photoreactive labeling reagent. The term "sugar receptor affinity labeling reagent" refers to a sugar receptor, and a sugar receptor refers to a protein that has affinity for sugar, that is, a protein that binds to sugar by a specific interaction with sugar. Includes lectins, receptors, enzymes, antibodies, etc. specific to sugars.
【００２６】本発明の光親和性標識試薬と種々の蛋白質
を含む混合系において、該標識試薬の糖化合物と糖受容
体との相互作用をおこなわせると同時又はその後に、光
を照射すると、光親和性標識試薬の糖化合物に特異的に
相互作用する蛋白質（糖受容体）の、該糖化合物との相
互作用部位（アミノ酸）に該標識試薬のジアジリン基が
光反応によりクロスリンクする。更に、このビオチンを
含む該光親和性標識試薬でラベル化された蛋白質（ラベ
ル化蛋白質）は、ビオチンを検出する自体公知の検出系
によって高感度での検出が可能であり、アビジン、スト
レプトアビジン等を担体に結合したアビジンカラムを利
用することにより、アフィニティークロマトグラフィー
によって目的蛋白質を容易に精製することが可能にな
る。更に、本発明においては、光親和性標識試薬を構成
する糖化合物としてビオチンが切断性スペーサーを介し
て結合された化合物を適用することにより、アビジンカ
ラムによるアフィニティークロマトグラフィーを行う
際、緩和な条件でビオチン部分を容易に切り離すことが
でき、目的蛋白質を高い回収率で取得することが可能で
ある。それ故、糖化合物を結合した本発明の光親和性標
識試薬は、糖鎖関連蛋白質の構造及び機能解析に極めて
有用なプローブ又は精製手段となることが期待されるの
である。In a mixed system containing the photoaffinity labeling reagent of the present invention and various proteins, irradiation with light is performed simultaneously with or after the interaction between the sugar compound of the labeling reagent and the sugar receptor. The diazirine group of the labeling reagent cross-links to the site (amino acid) of the protein (sugar receptor) that specifically interacts with the sugar compound of the affinity labeling reagent by a photoreaction. Furthermore, a protein (labeled protein) labeled with the photoaffinity labeling reagent containing biotin can be detected with high sensitivity by a detection system known per se for detecting biotin, such as avidin and streptavidin. By using an avidin column in which is bound to a carrier, the target protein can be easily purified by affinity chromatography. Furthermore, in the present invention, by applying a compound in which biotin is bound via a cleavable spacer as a sugar compound constituting the photoaffinity labeling reagent, the affinity chromatography using an avidin column can be performed under mild conditions. The biotin moiety can be easily separated, and the target protein can be obtained at a high recovery rate. Therefore, the photoaffinity labeling reagent of the present invention to which a sugar compound is bound is expected to be a very useful probe or purification means for analyzing the structure and function of a sugar chain-related protein.
【００２７】本発明の一般式（II）で示され、オキシア
ミノ基をフェニル骨格に有する光反応性ビチオン化フェ
ニルジアジリン化合物に、そのオキシアミノ基と選択的
に反応する基、例えばアルデヒド基、ケトン基、カルボ
キシル基などを有するか、或いは必要に応じこれらの基
が導入された薬物或いは糖化合物以外の生体由来成分を
リガンドとして結合させることにより、広義な意味での
光親和性標識試薬に誘導することもできる。そして、こ
の試薬は、薬物あるいは糖化合物以外の生体由来物質に
親和性を持つ蛋白質の構造及び機能解析のプローブとな
り又単離精製も可能とする。この様にして、多種多様な
リガンドを連接する手法で得ることができプローブライ
ブラリーと、質量分析装置による超微量解析とを組み合
わせる応用は、蛋白質機能の高速度・高精度解析への展
望を開くものとして期待される。従って、一般式（II）
で示される本発明化合物は極めて広範な有用性を有する
化合物である。The photoreactive bitionized phenyldiazirine compound represented by the general formula (II) having an oxyamino group on the phenyl skeleton according to the present invention can be selectively reacted with the oxyamino group, for example, an aldehyde group. Derivation of photoaffinity labeling reagent in a broad sense by binding as a ligand a drug or a sugar compound having a ketone group, a carboxyl group, or the like, if necessary, into which these groups have been introduced. You can also. This reagent serves as a probe for analyzing the structure and function of a protein having an affinity for a biological substance other than a drug or a sugar compound, and also enables isolation and purification. In this way, the application of a probe library that can be obtained by a method that links a wide variety of ligands with ultra-trace analysis using a mass spectrometer opens the prospects for high-speed, high-precision analysis of protein functions. Expected as something. Therefore, the general formula (II)
The compounds of the present invention represented by are compounds having extremely wide utility.
【００２８】本発明化合物の合成の一例を図１〜６に示
す反応スキームに基づいて以下に説明する。まず、図１
に示す反応工程に従い、一般式（I）で示される本発明
化合物、即ち、図中化合物(4)〜(7)の製造工程を説明す
る。出発原料である化合物(1)、［2-メトキシ-4-(1-アシ゛-2,
2,2-トリフルオロエチル）ヘ゛ンス゛アルテ゛ヒト゛］は、橋本らの方法（M.H
ashimoto,Y.Kanaoka,Y.Hatanaka,Heterocycles.46,119-
122(1997))により調製することができる。化合物(1)
は、例えばジクロロメタンなどの溶媒中、不活性ガス雰
囲気下、−２０℃付近でBBr3を作用させることにより化
合物(2)を生成する。生成した化合物(2)に、化合物(3)
を無水炭酸カリウムなどの塩基及びBu4NIなどの相関移
動触媒の存在下反応させることにより、化合物(4)を生
成することができる。この反応は、シ゛メチルホルムアミト゛(DMF)
中、不活性ガス雰囲気下、６０℃付近で行うことができ
る。なお、化合物(3)は、式Ｐ1ＮＨ(ＣＨ2)m（ＯＣ
2Ｈ4）n−Ｂｒにおいて、m=n=2、Ｐ1がt-フ゛トキシカルホ゛ニル基
である化合物であり、畑中らの方法（Y.Hatanaka,M.Has
himoto,Y.Kanaoka,Bioorg.Med.Chem.2,1367-1373(199
4)）により調製することができる。An example of the synthesis of the compound of the present invention is described below based on the reaction scheme shown in FIGS. First, FIG.
The production process of the compound of the present invention represented by the general formula (I), that is, the compounds (4) to (7) in the figure, will be described according to the reaction process shown in (1). Starting compound (1), [2-methoxy-4- (1-acid-2,
2,2-trifluoroethyl) benzene [art human] was prepared by the method of Hashimoto et al. (MH
ashimoto, Y.Kanaoka, Y.Hatanaka, Heterocycles.46,119-
122 (1997)). Compound (1)
Produces compound (2) by allowing BBr 3 to act in a solvent such as dichloromethane under an inert gas atmosphere at around −20 ° C. Compound (3) is added to generated compound (2).
Is reacted in the presence of a base such as anhydrous potassium carbonate and a phase transfer catalyst such as Bu 4 NI to produce compound (4). This reaction is carried out using methylformamide (DMF).
It can be carried out at around 60 ° C. in an inert gas atmosphere. Compound (3) has the formula P 1 NH (CH 2 ) m (OC
2 H 4 ) n -Br, a compound in which m = n = 2 and P 1 is a t-ethoxycarbonyl group, which is described by Hatanaka et al. (Y. Hatanaka, M. Has
himoto, Y.Kanaoka, Bioorg.Med.Chem. 2,1367-1373 (199
4)).
【００２９】化合物(4)は、例えばエタノールなどの溶
媒中、NaBH4により室温付近で還元して化合物(5)とする
ことができる。還元反応生成物は、精製処理することな
く、次の反応工程に供することができる。生成した化合
物(5)は、四臭化炭素とトリフェニルホスフィンによ
り、例えばジクロルメタンなどの溶媒中でブロム化する
ことにより、化合物(6)へと導く。この反応は、０℃付
近に冷却して行うことが好ましい。化合物(6)は、N-ヒ
ドロキシフタルイミドと無水炭酸カリウムなどの塩基の
存在下反応させることにより、化合物(7)を得ることが
できる。この反応は、シ゛メチルスルホキシト゛（DMSO）などの溶媒
中、室温付近で行うことができる。Compound (4) can be reduced to near-room temperature with NaBH 4 in a solvent such as ethanol to give compound (5). The reduction reaction product can be subjected to the next reaction step without purification treatment. The resulting compound (5) is brominated with carbon tetrabromide and triphenylphosphine in a solvent such as dichloromethane to lead to compound (6). This reaction is preferably performed by cooling to around 0 ° C. Compound (7) can be obtained by reacting compound (6) with N-hydroxyphthalimide in the presence of a base such as anhydrous potassium carbonate. This reaction can be performed in a solvent such as dimethylsulfoxide (DMSO) at around room temperature.
【００３０】次いで、図２に則り、化合物(7)をビオチ
ン化し、一般式（II）中、特に一般式(III)で示される
本発明化合物、即ち、図中化合物(8)〜(10)の製造工程
を説明する。化合物(7)に、トリフルオロ酢酸を例えば
ジクロロメタンなどの有機溶媒中、０℃付近で作用させ
た後、トリエチルアミン（Et3N)などの有機塩基の存在
下、例えばDMFなどの溶媒中、ビオチンのN-ヒドロキシ
スクシンイミドエステル（biotin-OSu）を室温付近で反
応させることによりビオチン化した化合物(8)を得るこ
とができる。Next, the compound (7) is biotinylated according to FIG. 2, and the compound of the present invention represented by the general formula (II), particularly the compound of the general formula (III), ie, the compounds (8) to (10) Will be described. Compound (7) is treated with trifluoroacetic acid in an organic solvent such as dichloromethane at about 0 ° C., and then biotin is added in the presence of an organic base such as triethylamine (Et 3 N) in a solvent such as DMF. The biotinylated compound (8) can be obtained by reacting N-hydroxysuccinimide ester (biotin-OSu) at around room temperature.
【００３１】化合物(8)に、例えばメタノール溶媒中、
無水ヒドラジンを室温付近で作用させることにより化合
物(9)を生成することができるが、この化合物(9)はカラ
ムクロマトグラフィーによる精製中に分解し易いので、
精製処理することなく次の反応に用いることが好まし
い。化合物(8)とヒドラジンとの反応生成物である化合
物(9)をそのまま、例えばクロロホルムとアセトニトリ
ルとの混合溶媒中で、トリエチルアミンなどの有機塩基
の存在下、ジ−ｔ−ブチルジカルボナート｛[(CH3)3 CO
CO]2O｝と室温付近で反応させることにより化合物(10)
を得ることができる。Compound (8) is added, for example, in a methanol solvent,
Compound (9) can be produced by allowing anhydrous hydrazine to act at around room temperature, but since compound (9) is easily decomposed during purification by column chromatography,
It is preferable to use for the next reaction without purification treatment. Compound (9), which is a reaction product of compound (8) and hydrazine, is used as it is, for example, in a mixed solvent of chloroform and acetonitrile in the presence of an organic base such as triethylamine to prepare di-t-butyl dicarbonate {[ (CH 3 ) 3 CO
Compound (10) by reacting with CO] 2 O｝ near room temperature
Can be obtained.
【００３２】更に、図３及び４に示すように、上記工程
で得たビオチン化フェニルジアジリン化合物に還元末端
を有する糖化合物を導入し、一般式(V)中、特に一般式
（VI）で示される本発明化合物、即ち化合物(12)〜(16)
を生成することができる。化合物(10)に、トリフルオロ
酢酸を、例えばジクロロメタンなどの有機溶媒中、０℃
付近で作用させることにより化合物(11)を得ることがで
きる。生成した化合物(11)は、直ちに還元末端を有する
各種の糖化合物と反応させて、それぞれの糖化合物の結
合したビオチン化フェニルジアジリン化合物を得ること
ができる。例えば、化合物(11)を含水アセトニトリル中
でN-アセチルラクトサミン（Galβ1-4ＧlcNAc）と３７
℃付近で反応させることにより化合物(12)を取得するこ
とができる。同様に、シアリル-α2-3-ラクトース（Neu
NAcα2-3Galβ1-4Glc）、シアリル-α2-3-N-アセチルラ
クトサミン（NeuNAcα2-3Galβ1-4GlcNAc）、ルイスX型
三糖（Galβ1-4（Fucα1-3）GlcNAc）、またはシアリル
ルイスX型四糖（NeuNAcα2-3Galβ1-4（Fucα1-3）GlcN
Ac）と各々反応させることにより化合物(13)〜(16)をそ
れぞれ得ることができる。又、化合物(10)に作用させる
トリフルオロ酢酸に代えて、塩酸／酢酸、塩酸／ジオキ
サン混合物等を使用し、無機酸の塩を生成することもで
きる。Further, as shown in FIGS. 3 and 4, a saccharide compound having a reducing end is introduced into the biotinylated phenyldiazirine compound obtained in the above step, and the compound represented by the general formula (V), particularly the general formula (VI) Compounds of the present invention shown, that is, compounds (12) to (16)
Can be generated. Compound (10) is treated with trifluoroacetic acid in an organic solvent such as dichloromethane at 0 ° C.
By acting in the vicinity, compound (11) can be obtained. The produced compound (11) can be immediately reacted with various sugar compounds having a reducing end to obtain a biotinylated phenyldiazirine compound to which each sugar compound is bound. For example, compound (11) is mixed with N-acetyllactosamine (Galβ1-4GlcNAc) in hydrous acetonitrile for 37 hours.
Compound (12) can be obtained by reacting at around ° C. Similarly, sialyl-α2-3-lactose (Neu
NAcα2-3Galβ1-4Glc), sialyl-α2-3-N-acetyllactosamine (NeuNAcα2-3Galβ1-4GlcNAc), Lewis X type trisaccharide (Galβ1-4 (Fucα1-3) GlcNAc), or sialyl Lewis X type tetrasaccharide ( NeuNAcα2-3Galβ1-4 (Fucα1-3) GlcN
Compounds (13) to (16) can be obtained by reacting each with Ac). Further, instead of trifluoroacetic acid acting on the compound (10), a salt of an inorganic acid can be produced by using a mixture of hydrochloric acid / acetic acid, hydrochloric acid / dioxane, or the like.
【００３３】次に、図５及び図６に示す反応工程に従
い、一般式(I)で示される化合物、即ち、図中化合物(1
8)〜(20),(22)、(23)、(25)、(26)、一般式（IV）及び
(VII)で示される切断性スペーサーを有する化合物、即
ち、図中化合物(28)及び(30)、加えて一般式(VI)の化合
物(29)の製造工程を説明する。化合物(2)は、図１に示
す工程により化合物(1)から合成する。化合物(2)に化合
物(17)を無水炭酸カリウムなどの塩基及びBu4NIなどの
相関移動触媒の存在下反応させることにより、化合物(1
8)を生成することができ、この反応は、シ゛メチルスルホキシト゛(D
MSO)中、不活性ガス雰囲気下、６０℃付近で行うことが
できる。なお、化合物(18)は、一般式(I)において、Ｒ2
がアルデヒド基、Ｒ1がＣＯＸ1で、Ｘ1がエトキシ基、m
=1、n=３である化合物である。Next, according to the reaction steps shown in FIGS. 5 and 6, the compound represented by the general formula (I), that is, the compound (1
8) to (20), (22), (23), (25), (26), general formula (IV) and
The production process of the compound having a cleavable spacer represented by (VII), that is, the compounds (28) and (30) in the figure, and the compound (29) of the general formula (VI) will be described. Compound (2) is synthesized from compound (1) by the steps shown in FIG. Compound (2) was reacted with compound (17) in the presence of a base such as anhydrous potassium carbonate and a phase transfer catalyst such as Bu 4 NI to give compound (1).
8), and the reaction is carried out with methylsulfoxide (D
MSO) in an inert gas atmosphere at around 60 ° C. The compound (18), in the general formula (I), R 2
Is an aldehyde group, R 1 is COX 1 , X 1 is an ethoxy group, m
= 1, n = 3.
【００３４】一方、化合物(17)は以下のようにして合成
出来る。即ち、2-[2-(2-クロロエトキシ)エトキシ]エタノールを水素化ナ
トリウムを含むトルエン溶媒中、−７０℃でブロム酢酸
エチルと反応させ、得られた反応混合物から溶媒を留去
して得た残渣をカラムクロマトグラフィーにより精製す
る。精製した残渣をＮ−メチルピロリドン等の溶媒に溶
解した後、臭化エチル及び臭化ナトリウムを加えて65℃
で反応させ、反応物から溶媒を留去後、更にカラムクロ
マトグラフィーにより精製する。On the other hand, compound (17) can be synthesized as follows. That is, 2- [2- (2-chloroethoxy) ethoxy] ethanol is reacted with ethyl bromoacetate in a toluene solvent containing sodium hydride at -70 ° C., and the solvent is distilled off from the obtained reaction mixture. The residue obtained is purified by column chromatography. After the purified residue was dissolved in a solvent such as N-methylpyrrolidone, ethyl bromide and sodium bromide were added and
After removing the solvent from the reaction product, the reaction product is further purified by column chromatography.
【００３５】化合物(18)は、例えばエタノールなどの溶
媒中、NaBH4により室温付近で還元して化合物(19)とす
ることができる。生成した化合物(19)は、四臭化炭素と
トリフェニルホスフィンにより、例えばジクロルメタン
などの溶媒中でブロム化することにより、化合物(20)へ
と導かれる。この反応は、０℃付近に冷却して行うこと
が好ましい。化合物(20)は、N-ヒドロキシカルバミン酸
t-ブチルエステル等のN-ヒドロキシカルバミン酸アル
キルエステル（化合物21）と水素化ナトリウムなどの塩
基の存在下反応させることにより、化合物(22)を得るこ
とができる。この反応は、テトラヒト゛ロフラン（THF）などの溶
媒中、０℃付近で行うことができる。Compound (18) can be reduced to a compound (19) with NaBH 4 at around room temperature in a solvent such as ethanol. The produced compound (19) is led to compound (20) by bromination with carbon tetrabromide and triphenylphosphine in a solvent such as dichloromethane. This reaction is preferably performed by cooling to around 0 ° C. Compound (20) is N-hydroxycarbamic acid
Compound (22) can be obtained by reacting with an N-hydroxycarbamic acid alkyl ester such as t-butyl ester (compound 21) in the presence of a base such as sodium hydride. This reaction can be carried out at around 0 ° C. in a solvent such as tetrahydrofuran (THF).
【００３６】化合物(22)は、例えばエタノールとアルカ
リ水溶液との混合溶媒中で加水分解することにより化合
物(23)を生成する。化合物(23)とカルボニルジイミダゾ
ールをTHF等の溶媒に溶解し、これにスペーサーに切断
性を付与するための化合物であるスルファモイル安息香
酸エステルなどの化合物(24)をジイソプロピルエチルア
ミンなどの有機塩基の存在下反応させ、化合物(25)に導
く。切断性を付与するために使用される化合物(24)は、
本発明の標識試薬に結合している糖化合物の種類、並び
に該糖化合物に特異的に相互作用する糖の受容体である
蛋白質の種類等を考慮して適宜選択されるが、スルファ
モイル安息香酸エステルは、緩和な条件で切断され、且
つ安定性も良いので好ましい。The compound (22) is hydrolyzed, for example, in a mixed solvent of ethanol and an aqueous alkali solution to produce a compound (23). Compound (23) and carbonyldiimidazole are dissolved in a solvent such as THF, and compound (24) such as sulfamoylbenzoate, which is a compound for imparting cleavability to the spacer, is added to an organic base such as diisopropylethylamine. The reaction is carried out below to lead to compound (25). Compound (24) used to impart cleavability,
It is appropriately selected in consideration of the type of the sugar compound bound to the labeling reagent of the present invention and the type of the protein which is a sugar receptor that specifically interacts with the sugar compound. Is preferred because it is cut under mild conditions and has good stability.
【００３７】化合物(25)は、例えばエタノールとアルカ
リ水溶液との混合溶媒中で加水分解することにより化合
物(26)を生成し、化合物(26)をＮ−ヒドロキシスクシン
イミドとカルボジイミド類（ＤＣＣ等）を反応させ活性
エステルとした後、ビオチンヒドラジドである化合物(2
7)などビオチン誘導体を反応させてビオチン化し、切断
性スペーサーを有するビオチン化フェニルジアジリン化
合物(28)とする。この化合物(28)に対し、前記図３及び
図４に示す工程と同様にして糖化合物（キトビオース：
GlcNAcβ1-4GlcNAc）を導入して切断性スペーサーを有
する糖結合ビオチン化フェニルジアジリン化合物(30)を
形成し、他方、前記化合物(11)に還元末端を有する糖化
合物としてキトビオースを導入して、切断性スペーサー
を有しない化合物(29)を生成する。The compound (25) is hydrolyzed, for example, in a mixed solvent of ethanol and an aqueous alkali solution to produce a compound (26), and the compound (26) is converted from N-hydroxysuccinimide and carbodiimides (DCC, etc.). After the reaction to form an active ester, the compound (2
A biotin derivative such as 7) is reacted and biotinylated to obtain a biotinylated phenyldiazirine compound (28) having a cleavable spacer. This compound (28) was reacted with a saccharide compound (chitobiose:
GlcNAcβ1-4GlcNAc) to form a sugar-linked biotinylated phenyldiazirine compound (30) having a cleavable spacer, while introducing chitobiose as a sugar compound having a reducing end into the compound (11) to cleave it. Compound (29) having no spacer is produced.
【００３８】[0038]
【実施例】以下、本発明を実施例によりさらに詳細に説
明するが、本発明はその要旨を超えない限り、これらの
実施例に限定されるものではない。なお、実施例中、各
化合物番号は、図１〜図６に記載の化合物番号に対応す
る。EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples unless it exceeds the gist. In addition, each compound number in an Example respond | corresponds to the compound number described in FIGS.
【００３９】実施例１ （化合物(1) → (2)） 化合物(1)3.66ｇ(0.015mol)を30mlのジクロロメタンに
溶解し、アルゴンガス雰囲気下、-20℃でこの溶液にBBr
33.76ｇ(0.015mol)をゆっくり滴下した。滴下後、溶液
を０℃で１時間かくはんした後、水を０℃で加えた。反
応混合物をジクロロメタンで抽出し、ジクロロメタン層
を無水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、
残渣をシリカゲルカラム（溶離剤；ジクロロメタン：ヘ
キサン＝１：２）で精製して3.18ｇの白色固体として化
合物(2)｛2-ヒト゛ロキシ-4-[3-(トリフルオロメチル)-3H-シ゛アシ゛リン-3-イ
ル]ヘ゛ンス゛アルテ゛ヒト゛｝を得た（収率：92%）。 IR(nujol):v=3170,1665cm-1;1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=11.05(s,1H),9.92
(s,1H),7.60(d,J=8.0Hz,1H),6.78(d,J=8.0Hz,1H),6.77
(br s,1H); ＭＳ（ＥＩ+）：ｍ／ｚ（％）:230(38)[Ｍ+]; ＨＲＭＳ：Ｃ9Ｈ5Ｆ3Ｎ2Ｏ2 ［Ｍ+］＝230.0303(計算
値),230.0291(実測値)Example 1 (Compound (1) → (2)) 3.66 g (0.015 mol) of compound (1) was dissolved in 30 ml of dichloromethane, and BBr was added to the solution at -20 ° C under an argon gas atmosphere.
3. 3.76 g (0.015 mol) was slowly added dropwise. After the dropwise addition, the solution was stirred at 0 ° C for 1 hour, and water was added at 0 ° C. The reaction mixture was extracted with dichloromethane, and the dichloromethane layer was dried over anhydrous magnesium sulfate.
The residue was purified by a silica gel column (eluent; dichloromethane: hexane = 1: 2) to give 3.18 g of a white solid of compound (2) {2-human-peroxy-4- [3- (trifluoromethyl) -3H-diacetic)- [3-Ill] benzene (human) was obtained (yield: 92%). IR (nujol): v = 3170,1665cm -1; 1 HNMR (400MHz, CDCl 3, 25 ℃): δ = 11.05 (s, 1H), 9.92
(s, 1H), 7.60 (d, J = 8.0Hz, 1H), 6.78 (d, J = 8.0Hz, 1H), 6.77
(br s, 1H); MS (EI + ): m / z (%): 230 (38) [M + ]; HRMS: C 9 H 5 F 3 N 2 O 2 [M + ] = 230.0303 (calculated value) ), 230.0291 (actual value)
【００４０】実施例２ （化合物(2)+(3) → (4)） 化合物(２）２．７６ｇ（０．０１２ｍｏｌ）、化合物
（３)｛2-[2-(2-tert-フ゛トキシカルホ゛ニルアミノエトキシ)エトキシ]エチル フ゛
ロミト゛｝４．５０ｇ（０．０１４４ｍｏｌ）、無水炭酸カ
リウム１．６６ｇ（０．０１２ｍｏｌ）、Ｂｕ4ＮＩ５
５４ｍｇ（１．５ｍｍｏｌ）、及びＤＭＦ１５ｍｌの混
合物をアルゴンガス雰囲気下、６０℃に１４時間かくは
んした。その後、反応混合物の溶媒を減圧留去し、得ら
れた残渣に水を加え、酢酸エチルで抽出した。酢酸エチ
ル層を水、０．１Ｎ ＨＣ１、及び水で順次洗浄して無
水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣
をシリカゲルカラム（溶離剤；酢酸エチル：ヘキサン＝
１：１）で精製して５．３３ｇの淡黄色油状物質として
化合物（４)｛2-[2-[2-(2-tert-フ゛トキシカルホ゛ニルアミノエトキシ)エト
キシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-シ゛アシ゛リン-3-イル]ヘ゛ンス゛アルテ
゛ヒト゛｝を得た（収率：９６％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=10.5(s,1H),7.85(d,
J=8.1Hz,1H),6.86(d,J=8.1Hz,1H),6.75(br s,1H),5.00
(br,1H),4.27(t,J=4.8Hz,2H),3.92(t,J=4.8Hz,2H),3.7
(m,2H),3.6(m,2H),3.54(t,J=5.1Hz,2H),3.3(m,2H),1.43
(s,9H); ＭＳ（ＦＡＢ+）：ｍ／ｚ（％）：484（84）［Ｍ＋Ｎ
ａ］+，462（15）［Ｍ＋Ｈ］+； ＨＲＭＳ：Ｃ20Ｈ27Ｆ3Ｎ3Ｏ6 ［Ｍ＋Ｈ］+＝462.1852
(計算値),462.1874(実測値)Example 2 (Compound (2) + (3) → (4)) 2.76 g (0.012 mol) of compound (2), compound (3) {2- [2- (2-tert-ethoxycarboxynil) Aminoethoxy) ethoxy] ethyl fluorite 4.50 g (0.0144 mol), anhydrous potassium carbonate 1.66 g (0.012 mol), Bu 4 NI5
A mixture of 54 mg (1.5 mmol) and 15 ml of DMF was stirred at 60 ° C. for 14 hours under an argon gas atmosphere. Thereafter, the solvent of the reaction mixture was distilled off under reduced pressure, water was added to the obtained residue, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with water, 0.1N HCl and water, dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was subjected to a silica gel column (eluent; ethyl acetate: hexane =
Purified by 1: 1) to give 5.33 g of compound (4) {2- [2- [2- (2-tert-butoxycarbonylaminoethoxy) ethoxy] ethoxy] -4- [3-] as a pale yellow oily substance. (Trifluoromethyl) -3H-siadolin-3-yl] benzene (human) was obtained (yield: 96%). 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 10.5 (s, 1 H), 7.85 (d,
J = 8.1Hz, 1H), 6.86 (d, J = 8.1Hz, 1H), 6.75 (br s, 1H), 5.00
(br, 1H), 4.27 (t, J = 4.8Hz, 2H), 3.92 (t, J = 4.8Hz, 2H), 3.7
(m, 2H), 3.6 (m, 2H), 3.54 (t, J = 5.1Hz, 2H), 3.3 (m, 2H), 1.43
(s, 9H); MS (FAB + ): m / z (%): 484 (84) [M + N
a] + , 462 (15) [M + H] + ; HRMS: C 20 H 27 F 3 N 3 O 6 [M + H] + = 462.1852.
(Calculated), 462.1874 (actual)
【００４１】実施例３（化合物（４）→（５）） 化合物（４）３．６９ｇ（８ｍｍｏ１）をエタノール４
０ｍ１に溶解し、この溶液にＮａＢＨ4３０３ｍｇ（８
ｍｍｏ１）をゆっくり加えた．反応混合物を室温で３０
分かくはんした後、溶媒を減圧留去して得られた残渣に
水を加え、０℃で１Ｎ ＨＣｌを加えることによりｐＨ
３付近に調節した後、酢酸エチルて抽出した。酢酸エチ
ル層を水、飽和重曹水、及び飽和食塩水で順次洗浄して
無水硫酸マグネシウムて乾燥後、溶媒を減圧留去し、淡
黄色油状物質として粗化合物(５)｛2-[2-[2-(2-tert-フ゛
トキシカルホ゛ニルアミノエトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-シ゛
アシ゛リン-3-イル]ヘ゛ンシ゛ルアルコール｝を得た。粗化合物（５）は更
に精製することなく直ちに次の反応に用いた。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=7.26(d,J=8.0Hz,1
H),6.79(d,J=8.0Hz,1H),6.66(br s,1H),6.01(br,1H),5.
02(br,1H),4.66(s,2H),4.22(t,J=4.8Hz,2H),3.84(t,J=
4.8Hz,2H),3.7(m,2H),3.6(m,2H),3.51(t,J=5.1Hz,2H),
3.3(m,2H),1.45(s,9H); ＭＳ（ＦＡＢ+）:ｍ／ｚ（％）:486（100）［Ｍ＋Ｎ
ａ］+，464（16）［Ｍ＋Ｈ］+; ＨＲＭＳ：Ｃ20Ｈ29Ｆ3Ｎ3Ｏ6 ［Ｍ＋Ｈ］+＝464.2008
(計算値),464.1997(実測値)Example 3 (Compound (4) → (5)) 3.69 g (8 mmol) of compound (4) was added to ethanol 4
0 ml, and 303 mg of NaBH 4 (8 mg) was added to this solution.
mmo1) was added slowly. The reaction mixture is kept at room temperature for 30 minutes.
After stirring, water was added to the residue obtained by evaporating the solvent under reduced pressure, and 1N HCl was added at 0 ° C. to adjust the pH.
After adjusting to around 3, the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with water, saturated aqueous sodium bicarbonate, and saturated saline, dried over anhydrous magnesium sulfate, and dried under reduced pressure to remove the solvent under reduced pressure to give crude compound (5) {2- [2- [ 2- (2-tert-ethoxycarbonylaminoethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-siadicrin-3-yl] benzyl alcohol was obtained. The crude compound (5) was used for the next reaction immediately without further purification. 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 7.26 (d, J = 8.0 Hz, 1
H), 6.79 (d, J = 8.0Hz, 1H), 6.66 (br s, 1H), 6.01 (br, 1H), 5.
02 (br, 1H), 4.66 (s, 2H), 4.22 (t, J = 4.8Hz, 2H), 3.84 (t, J =
4.8Hz, 2H), 3.7 (m, 2H), 3.6 (m, 2H), 3.51 (t, J = 5.1Hz, 2H),
3.3 (m, 2H), 1.45 (s, 9H); MS (FAB + ): m / z (%): 486 (100) [M + N
a] + , 464 (16) [M + H] + ; HRMS: C 20 H 29 F 3 N 3 O 6 [M + H] + = 464.2008
(Calculated), 464.1997 (actual)
【００４２】実施例４（化合物（５）→（６）） 上記の方法で化合物（４）３．６９ｇ（８ｍｍｏ１）か
ら得られた粗化合物（５）と四臭化炭素３．３２ｇ
（０．０１ｍｏｌ）とを１６ｍｌのジクロロメタンに溶
解し、０℃に冷却しながらトリフェニルホスフィン３．
１５ｇ（０．０１２ｍｏｌ）をゆっくりと加えた。１０
分後、無水炭酸カリウム１．６６ｇ（０．０１２ｍｏ
ｌ）を反応混合物に加え、０℃でさらに３０分かくはん
した。その後、反応混合物に水を加え、ジクロロメタン
で抽出し、ジクロロメタン層を無水硫酸マグネシウムで
乾燥後、溶媒を減圧留去し、残渣をシリカゲルカラム
（溶離剤；酢酸エチル：ヘキサン＝１：１）で精製して
３．６２ｇの淡黄色油状物質として化合物(６)｛2-[2-
[2-(2-tert-フ゛トキシカルホ゛ニルアミノエトキシ)エトキシ]エトキシ]-4-[3-(トリフ
ルオロメチル)-3H-シ゛アシ゛リン-3-イル]ヘ゛ンシ゛ルフ゛ロミト゛｝を得た（収
率：８６％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=7.35(d,J=8.1Hz,1
H),6.77(d,J=8.1Hz,1H),6.65(br s,1H),5.00(br,1H),4.
51(s,2H),4.21(t,J=4.8Hz,2H),3.91(t,J=4.8Hz,2H),3.7
5(m,2H),3.65(m,2H),3.55(t,J=5.1Hz,2H),3.3(m,2H),1.
43(s,9H); ＭＳ(ＦＡＢ+）:ｍ／ｚ(％):550（41）［Ｍ＋Ｎａ］+,5
48(40),528(12)［Ｍ＋Ｈ］+,526(11); ＨＲＭＳ：(79Ｂｒ)Ｃ20Ｈ28ＢｒＦ3Ｎ3Ｏ5 ［Ｍ＋Ｈ］
+＝526.1164(計算値),526.1193(実測値)Example 4 (Compound (5) → (6)) Crude compound (5) obtained from 3.69 g (8 mmol) of compound (4) by the above method and 3.32 g of carbon tetrabromide
(0.01 mol) was dissolved in 16 ml of dichloromethane, and triphenylphosphine was cooled to 0 ° C.
15 g (0.012 mol) were added slowly. 10
Minutes later, 1.66 g of anhydrous potassium carbonate (0.012 mol
l) was added to the reaction mixture and stirred at 0 ° C. for a further 30 minutes. Thereafter, water was added to the reaction mixture, extracted with dichloromethane, the dichloromethane layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified with a silica gel column (eluent; ethyl acetate: hexane = 1: 1). Compound (6) {2- [2-
[2- (2-tert-Pethoxycarbonylaminoethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-siacindin-3-yl] benzoylperomitone was obtained (yield: 86%). . 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 7.35 (d, J = 8.1 Hz, 1
H), 6.77 (d, J = 8.1Hz, 1H), 6.65 (br s, 1H), 5.00 (br, 1H), 4.
51 (s, 2H), 4.21 (t, J = 4.8Hz, 2H), 3.91 (t, J = 4.8Hz, 2H), 3.7
5 (m, 2H), 3.65 (m, 2H), 3.55 (t, J = 5.1Hz, 2H), 3.3 (m, 2H), 1.
43 (s, 9H); MS (FAB + ): m / z (%): 550 (41) [M + Na] + , 5.
48 (40), 528 (12 ) [M + H] +, 526 (11); HRMS :( 79 Br) C 20 H 28 BrF 3 N 3 O 5 [M + H]
+ = 526.1164 (calculated value), 526.1193 (actual value)
【００４３】実施例５（化合物（６）→（７）） 化合物（６）２．６３ｇ（５ｍｍｏｌ）、N-ヒドロキシ
フタルイミド９７９ｍｇ（６ｍｍｏｌ）、無水炭酸カリ
ウム６９１ｍｇ（５ｍｍｏｌ）、及びDMSO１０ｍｌの混
合物を室温で１２時間かくはんした。その後、反応混合
物の溶媒をオイルポンプを用いて減圧留去し、得られた
残渣に水を加え、酢酸エチルで抽出した。酢酸エチル層
を水、ｌＮ ＮａＯＨ及び飽和食塩水で順次洗浄して無
水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣
をシリカゲルカラム（溶離剤；ヘキサン：アセトン＝
３：１）で精製して２．５９ｇの淡黄色油状物質として
化合物(７)｛2-[2-[2-(2-tert-フ゛トキシカルホ゛ニルアミノエトキシ)エトキ
シ]エトキシ]-4-[3-(トリフルオロメチル)-3H-シ゛アシ゛リン-3-イル]ヘ゛ンシ゛ルオキシ
フタルイミト゛｝を得た（収率：８５％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=7.82-7.73(AB,4H),
7.54(d,J=7.9Hz,1H),6.81(d,J=7.9Hz,1H),6.66(br s,1
H),5.27(s,2H),5.05(br,1H),4.15(t,J=4.8Hz,2H),3.86
(t,J=4.8Hz,2H),3.7(m,2H),3.6(m,2H),3.54(t,J=5.2Hz,
2H),3.3(m,2H),1.42(s,9H); ＭＳ（ＦＡＢ+）:ｍ／ｚ（％）:631（100）［Ｍ＋Ｎ
ａ］+,609(9)［Ｍ＋Ｈ］+;ＨＲＭＳ：Ｃ28Ｈ32Ｆ3Ｎ4Ｏ
8 ［Ｍ＋Ｈ］+＝609.2172(計算値),609.2164(実測値)Example 5 (Compound (6) → (7)) A mixture of 2.63 g (5 mmol) of compound (6), 979 mg (6 mmol) of N-hydroxyphthalimide, 691 mg (5 mmol) of anhydrous potassium carbonate and 10 ml of DMSO was added at room temperature. And stirred for 12 hours. Thereafter, the solvent of the reaction mixture was distilled off under reduced pressure using an oil pump, water was added to the obtained residue, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with water, 1N NaOH and saturated saline, dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was subjected to a silica gel column (eluent; hexane: acetone =
3: 1) to give 2.59 g of compound (7) {2- [2- [2- (2-tert-butoxycarbonylaminoethoxy) ethoxy] ethoxy] -4- [3- as a pale yellow oily substance. (Trifluoromethyl) -3H-diacidolin-3-yl] benzoyloxyphthalimito was obtained (yield: 85%). 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 7.82-7.73 (AB, 4H),
7.54 (d, J = 7.9Hz, 1H), 6.81 (d, J = 7.9Hz, 1H), 6.66 (br s, 1H
H), 5.27 (s, 2H), 5.05 (br, 1H), 4.15 (t, J = 4.8Hz, 2H), 3.86
(t, J = 4.8Hz, 2H), 3.7 (m, 2H), 3.6 (m, 2H), 3.54 (t, J = 5.2Hz,
2H), 3.3 (m, 2H), 1.42 (s, 9H); MS (FAB + ): m / z (%): 631 (100) [M + N
a] + , 609 (9) [M + H] + ; HRMS: C 28 H 32 F 3 N 4 O
8 [M + H] + = 609.2172 (calculated value), 609.2164 (actual value)
【００４４】実施例６（化合物（７）→（８）） 化合物（７）３０４ｍｇ（０．５ｍｍｏｌ）をジクロロ
メタン０．５ｍｌに溶解し、０℃に冷却しながらトリフ
ルオロ酢酸０．５ｍｌをゆっくりと加え、反応混合物を
０℃で１時間かくはんした。溶媒を減圧留去して得られ
た残渣にトルエン１ｍｌを加え、減圧留去した。この操
作を計３回繰り返し、残存する過剰のトリフルオロ酢酸
を除いて得られる黄色油状残渣をＤＭＦ０．５ｍｌに溶
解し、ビオチンＮ−ヒドロキシスクシンイミドエステル
１７１ｍｇ（０．５ｍｍｏｌ）を２ｍｌのＤＭＦに溶解
した溶液を加え、触媒量のトリエチルアミンの存在下反
応混合物を室温で１４時間かくはんした。その後、溶媒
を減庄留去し、得られた残渣をジクロロメタンとメタノ
ール１：１の混合溶媒に溶解し、１Ｎ ＮａＯＨ、飽和
食塩水、０．１Ｎ ＨＣｌ、及び飽和食塩水で順次洗浄
して無水硫酸マグネシウムで乾燥後、溶媒を減圧留去
し、残渣をシリカゲルカラム（溶離剤：クロロホルム：
エタノール＝１０：１）で精製して３２０ｍｇの無色固
体として化合物(８)｛2-[2-[2-(2-ヒ゛オチニルアミノエトキシ)エトキシ]
エトキシ]-4-[3-(トリフルオロメチル)-3H-シ゛アシ゛リン-3-イル]ヘ゛ンシ゛ルオキシフタ
ルイミト゛｝を得た（収率：８７％）。1 HNMR(400MHz,CDCl3,25℃):δ=7.83-7.73(AB,4H),7.53
(d,J=7.9Hz,1H),6.84(s,1H),6.81(d,J=7.9Hz,1H),6.64
(s,1H),6.61(s,1H),5.64(s,1H),5.26(s,2H),4.5(m,1H),
4.3(m,1H),4.15(t,J=4.8Hz,2H),3.90(t,J=4.8Hz,2H),3.
7(m,2H),3.65(m,2H),3.55(m,2H),3.4(br m,2H),3.1(m,1
H),2.9(m,1H),2.72(d,J=12.5Hz,1H),2.19(t,J=7.5Hz,2
H),1.8-1.6(m,4H),1.4(m,2H); MS(FAB+):m/z(%):757(16),[M+Na]+,735(62)[M+H]+; HRMS:C33H38F3N6O8S [M+H]+=735.2424(計算値),735.245
8(実測値)Example 6 (Compound (7) → (8)) 304 mg (0.5 mmol) of compound (7) was dissolved in 0.5 ml of dichloromethane, and 0.5 ml of trifluoroacetic acid was slowly added thereto while cooling to 0 ° C. In addition, the reaction mixture was stirred at 0 ° C. for 1 hour. The solvent was distilled off under reduced pressure, 1 ml of toluene was added to the residue obtained, and the solvent was distilled off under reduced pressure. This operation was repeated three times in total, and the yellow oily residue obtained by removing the remaining excess trifluoroacetic acid was dissolved in 0.5 ml of DMF, and 171 mg (0.5 mmol) of biotin N-hydroxysuccinimide ester was dissolved in 2 ml of DMF. The solution was added and the reaction mixture was stirred at room temperature for 14 hours in the presence of a catalytic amount of triethylamine. Thereafter, the solvent was distilled off, and the obtained residue was dissolved in a mixed solvent of dichloromethane and methanol 1: 1. The mixture was washed with 1N NaOH, saturated saline, 0.1N HCl, and saturated saline in that order, and dried. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was subjected to a silica gel column (eluent: chloroform:
Purification with ethanol = 10: 1) to give 320 mg of compound (8) {2- [2- [2- (2-diotinylaminoethoxy) ethoxy] as a colorless solid.
Ethoxy] -4- [3- (trifluoromethyl) -3H-diacidin-3-yl] benzoyloxyphthalimito was obtained (yield: 87%). 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 7.83-7.73 (AB, 4H), 7.53
(d, J = 7.9Hz, 1H), 6.84 (s, 1H), 6.81 (d, J = 7.9Hz, 1H), 6.64
(s, 1H), 6.61 (s, 1H), 5.64 (s, 1H), 5.26 (s, 2H), 4.5 (m, 1H),
4.3 (m, 1H), 4.15 (t, J = 4.8Hz, 2H), 3.90 (t, J = 4.8Hz, 2H), 3.
7 (m, 2H), 3.65 (m, 2H), 3.55 (m, 2H), 3.4 (br m, 2H), 3.1 (m, 1
H), 2.9 (m, 1H), 2.72 (d, J = 12.5Hz, 1H), 2.19 (t, J = 7.5Hz, 2
H), 1.8-1.6 (m, 4H), 1.4 (m, 2H); MS (FAB + ): m / z (%): 757 (16), [M + Na] + , 735 (62) [M + H] + ; HRMS: C 33 H 38 F 3 N 6 O 8 S [M + H] + = 735.2424 (calculated), 735.245
8 (actual value)
【００４５】実施例７（化合物（８）→（９）） 化合物（８）７３ｍｇ（０．１ｍｍｏｌ）を無水ヒドラ
ジンの１Ｍメタノール溶液１ｍｌに溶解し、室温で３０
分間反応させた。その後、溶媒を減圧留去して得られた
残渣にトルエン１ｍｌを加え、減圧留去した。この操作
を計３回繰り返し、残存する過剰のヒドラジンを除いて
無色固体として粗化合物（９）を得た。粗化合物（９）
は精製することなく直ちに次の反応に用いた。Example 7 (Compound (8) → (9)) 73 mg (0.1 mmol) of compound (8) was dissolved in 1 ml of a 1M solution of anhydrous hydrazine in methanol, and the solution was added at room temperature.
Allowed to react for minutes. Thereafter, 1 ml of toluene was added to the residue obtained by evaporating the solvent under reduced pressure, and the mixture was evaporated under reduced pressure. This operation was repeated three times in total, and a crude compound (9) was obtained as a colorless solid by removing the remaining excess hydrazine. Crude compound (9)
Was used for the next reaction immediately without purification.
【００４６】実施例８（化合物：（９）→（１０）） 上記の方法で化合物（８）７３ｍｇ（０．１ｍｍｏｌ）
から得られた粗化合物（９）をクロロホルムとアセトニ
トリル１：１の混合溶媒１ｍｌに溶解し、［（ＣＨ3）3
ＣＯＣＯ］2Ｏ １０９ｍｇ（０．５ｍｍｏｌ）とトリ
エチルアミン５１ｍｇ（０．５ｍｍｏｌ）を加えて室温
で１４時間反応させた。その後、溶媒を減圧留去し、残
渣をシリカゲルカラム（溶離剤；クロロホルム：エタノ
ール＝１０：１）で精製して６４ｍｇの無色固体として
化含物(１０)｛2-[2-[2-(2-ヒ゛オチニルアミノエトキシ)エトキシ]エトキシ]-
4-[3-(トリフルオロメチル)-3H-シ゛アシ゛リン-3-イル]ヘ゛ンシ゛ルオキシカルハ゛ミン酸
tert-フ゛チルエステル｝を得た（収率：９５％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=8.31(s,1H),7.39(d,
J=7.9Hz,1H),6.97(br,1H),6.81(d,J=7.9Hz,1H),6.64(s,
1H),6.58(s,1H),5.66(s,1H),4.91(s,2H),4.5(m,1H),4.3
(m,1H),4.16(t,J=4.5Hz,2H),3.87(t,J=4.5Hz,2H),3.7
(m,2H),3.65(m,2H),3.55(m,2H),3.4(br m,2H),3.1(m,1
H),2.9(m,1H),2.70(d,J=12.8Hz,1H),2.17(t,J=7.3Hz,2
H),1.8-1.6(m,4H),1.45(s,9H),1.4(m,2H); ＵＶ／ＶＩＳ（ＭｅＯＨ）:λmax（ε）＝360(400),283
(2600); ＭＳ（ＦＡＢ+）:ｍ／ｚ（％）:727(100),［Ｍ＋Ｎａ］
+,705(12)［Ｍ＋Ｈ］+;ＨＲＭＳ：Ｃ30Ｈ44Ｆ3Ｎ6Ｏ8Ｓ
［Ｍ＋Ｈ］+＝705.2893(計算値),705.2853(実測値)Example 8 (Compound: (9) → (10)) 73 mg (0.1 mmol) of compound (8) by the above method.
The crude compound (9) obtained from was dissolved in 1 ml of a mixed solvent of chloroform and acetonitrile 1: 1 and [(CH 3 ) 3
[COCO] 2 O 109 mg (0.5 mmol) and triethylamine 51 mg (0.5 mmol) were added and reacted at room temperature for 14 hours. Thereafter, the solvent was distilled off under reduced pressure, and the residue was purified by a silica gel column (eluent; chloroform: ethanol = 10: 1) to give 64 mg of a colorless solid (10) ｛2- [2- [2- ( 2-diotinylaminoethoxy) ethoxy] ethoxy]-
4- [3- (trifluoromethyl) -3H-siadicrin-3-yl] benzoyloxycarbamic acid
Tert-butyl ester was obtained (yield: 95%). 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 8.31 (s, 1 H), 7.39 (d,
J = 7.9Hz, 1H), 6.97 (br, 1H), 6.81 (d, J = 7.9Hz, 1H), 6.64 (s,
1H), 6.58 (s, 1H), 5.66 (s, 1H), 4.91 (s, 2H), 4.5 (m, 1H), 4.3
(m, 1H), 4.16 (t, J = 4.5Hz, 2H), 3.87 (t, J = 4.5Hz, 2H), 3.7
(m, 2H), 3.65 (m, 2H), 3.55 (m, 2H), 3.4 (br m, 2H), 3.1 (m, 1
H), 2.9 (m, 1H), 2.70 (d, J = 12.8Hz, 1H), 2.17 (t, J = 7.3Hz, 2
H), 1.8-1.6 (m, 4H), 1.45 (s, 9H), 1.4 (m, 2H); UV / VIS (MeOH): λ max (ε) = 360 (400), 283
(2600); MS (FAB + ): m / z (%): 727 (100), [M + Na].
+ , 705 (12) [M + H] + ; HRMS: C 30 H 44 F 3 N 6 O 8 S
[M + H] + = 705.2893 (calculated value), 705.22853 (actual value)
【００４７】実施例９（化合物（１０）→（１１）） 化合物（１０）７ｍｇ（０．０１ｍｍｏｌ）をジクロロ
メタン０．１ｍｌに溶解し、０℃に冷却しながらトリフ
ルオロ酢酸０．１ｍｌをゆっくりと加え、０℃で１時間
反応させた。その後、溶媒を減圧留去して得られた残渣
にトルエン０．１ｍｌを加え、減圧留去した。この操作
を計３回繰り返し、残存する過剰のトリフルオロ酢酸を
除き、淡黄色固体として化合物（１１）を得た。化合物
（１１）は精製することなく直ちに糖化合物類との縮合
反応に用いた。Example 9 (Compound (10) → (11)) 7 mg (0.01 mmol) of compound (10) was dissolved in 0.1 ml of dichloromethane, and 0.1 ml of trifluoroacetic acid was slowly added thereto while cooling to 0 ° C. The reaction was performed at 0 ° C. for 1 hour. Thereafter, 0.1 ml of toluene was added to the residue obtained by evaporating the solvent under reduced pressure, and the mixture was evaporated under reduced pressure. This operation was repeated three times in total, and the remaining excess trifluoroacetic acid was removed to obtain Compound (11) as a pale yellow solid. Compound (11) was used immediately for condensation reaction with sugar compounds without purification.
【００４８】実施例１０（化合物（１１）→（１２）） 上記の方法で化合物（１０）７ｍｇ（０．０１ｍｍｏ
ｌ）から調製した粗化合物（１１）をアセトニトリル
０．１ｍｌに溶解し、Ｎ−アセチルラクトサミン１．９
ｍｇ（０．００５ｍｍｏｌ）の水溶液を加え、３７℃で
４０時間反応させた。反応混合物をシリカゲルカラム
（センシュー科学AQUASILＳＳ−１２５１−１２０、
４．６ｆ ｘ ２５０ｍｍ）を用いてＨＰＬＣにより精
製した。収量は、メタノール溶液のＵＶスペクトルを測
定し、化合物（１１）のＵＶスペクトル（３６１ｎｍ
（ｅ＝３８６））を基準として求め０．００３１ｍｍｏ
ｌの化合物（１２）を得た。〈収率６３％）。 ＨＰＬＣ条件:90％aq.ＣＨ3ＣＮ→75％aq.ＣＨ3ＣＮ、
２０min、１ｍｌ／minＭＳ（ＦＡＢ-）:m/z(%):968(64)
［Ｍ−Ｈ］-; ＨＲＭＳ;Ｃ39Ｈ57Ｆ3Ｎ7０16Ｓ（[Ｍ−Ｈ]-）:968.353
5(計算値)、968.3586(実測値)Example 10 (Compound (11) → (12)) 7 mg (0.01 mmol) of compound (10) was obtained by the method described above.
The crude compound (11) prepared from 1) was dissolved in 0.1 ml of acetonitrile, and N-acetyllactosamine 1.9 was dissolved.
mg (0.005 mmol) of an aqueous solution was added and reacted at 37 ° C. for 40 hours. The reaction mixture was applied to a silica gel column (Sensyu Kagaku AQUASILSS-1251-120,
(4.6f x 250mm) using HPLC. The yield was determined by measuring the UV spectrum of a methanol solution and examining the UV spectrum (361 nm) of compound (11).
(E = 386)) and 0.0031 mmo
1 of compound (12) was obtained. <63% yield). HPLC conditions: 90% aq. CH 3 CN → 75% aq. CH 3 CN,
20min, 1ml / minMS (FAB - ): m / z (%): 968 (64)
[M-H] -; HRMS ; C 39 H 57 F 3 N 7 0 16 S ([M-H] -): 968.353
5 (calculated), 968.3586 (actual)
【００４９】実施例１１（化合物（１１）→（１３）） 実施例９の方法で化合物（１０）３．５ｍｇ（０．００
５ｍｍｏｌ）から調製した化合物（１１）とシアリル−
α２−３−ラクトース１．６ｍｇ（０．００２５ｍｍｏ
ｌ）を実施例１０と同様の条件で反応させた。同様にＨ
ＰＬＣ精製後０．００１２ｍｍｏｌの化合物（１３）を
得た。（収率４８％）。 ＨＰＬＣ条件:95％aq.ＣＨ3ＣＮ→50％aq.ＣＨ3ＣＮ、
１０min、１ｍｌ／minNegative ion FAB-MS（３-nitr
obenzylalcohol）ｍ／ｚ：１２１８[Ｍ−Ｈ]-ＨＲ-ＦＡ
Ｂ-ＭＳ （３-nitrobenzylalcohol） Ｃ48Ｈ71Ｆ3Ｎ7Ｏ24Ｓ（[Ｍ‐Ｈ]-）：1218.4223(計算
値)、1218.4257(実測値)Example 11 (Compound (11) → (13)) By the method of Example 9, 3.5 mg (0.00%) of compound (10)
5 mmol) and sialyl-
1.6 mg of α2-3-lactose (0.0025 mmol
l) was reacted under the same conditions as in Example 10. Similarly H
After PLC purification, 0.0012 mmol of compound (13) was obtained. (Yield 48%). HPLC conditions: 95% aq. CH 3 CN → 50% aq. CH 3 CN,
10 min, 1 ml / min Negative ion FAB-MS (3-nitr
obenzylalcohol) m / z: 1218 [ M-H] - HR-FA
B-MS (3-nitrobenzylalcohol) C 48 H 71 F 3 N 7 O 24 S ([M-H] -): 1218.4223 ( calculated), 1218.4257 (found)
【００５０】実施例１２（化合物（１１）→（１４）） 実施例９の方法で化合物（１０）４．３ｍｇ（０．００
６ｍｍｏｌ）から調製した化合物（１１）とシアリル−
α２−３−Ｎ−アセチルラクトサミン（純度９５％）
１．０ｍｇ（０．００１４ｍｍｏｌ）を実施例１０と同
様の条件で反応させた。同様にＨＰＬＣ精製後０．００
０８５ｍｍｏｌの化合物（１４）を得た。（収率６１
％）。 ＨＰＬＣ条件:85%aq.ＣＨ3ＣＮ→70%aq.ＣＨ3ＣＮ、15ｍ
ｉｎ、1ｍｌ/ｍｉｎ Negative ion FAB-MS（３-nitrobenzylalcohol）ｍ／
ｚ：１２５９[Ｍ−Ｈ]-ＨＲ-ＦＡＢ-ＭＳ （３-nitrobe
nzylalcohol） Ｃ50Ｈ74Ｆ3Ｎ8Ｏ24Ｓ（[Ｍ−Ｈ]-）：1259.4489(計算
値)、1259.4507(実測値)Example 12 (Compound (11) → (14)) According to the method of Example 9, 4.3 mg (0.00%) of compound (10)
6 mmol) and the compound (11) and sialyl-
α2-3-N-acetyllactosamine (95% purity)
1.0 mg (0.0014 mmol) was reacted under the same conditions as in Example 10. Similarly after HPLC purification
085 mmol of compound (14) was obtained. (Yield 61
%). HPLC conditions: 85% aq. CH 3 CN → 70% aq. CH 3 CN, 15 m
in, 1ml / min Negative ion FAB-MS (3-nitrobenzylalcohol) m /
z: 1259 [M-H] - HR-FAB-MS (3-nitrobe
nzylalcohol) C 50 H 74 F 3 N 8 O 24 S ([M-H] -): 1259.4489 ( calculated), 1259.4507 (found)
【００５１】実施例１３（化合物（１１）→（１５）） 実施例９の方法で化合物（１０）ｌ．４ｍｇ（０．００
２ｍｍｏｌ）から調製した化合物（ｌｌ）とルイスＸ型
三糖０．５ｍｇ（０．０００９４ｍｍｏｌ）を実施例１
０と同様の条件で反応させた。同様にＨＰＬＣ精製後
０．０００７３ｍｍｏｌの化合物（１５）を得た。（収
率７８％）。 HPLC条件:９０％ａｑ．CH3CN→７０％ａｑ．CH3CN、１
５min、１ｍｌ／min ＭＳ（ＦＡＢ-）:m/z(%):1114(47)［Ｍ−Ｈ］-; ＨＲＭＳ; Ｃ45Ｈ66Ｆ3Ｎ7Ｏ20Ｓ（[Ｍ−Ｈ]-）:1114.4
114(計算値)、1114.4163(実測値)Example 13 (Compound (11) → (15)) Compound (10) was prepared in the same manner as in Example 9. 4 mg (0.00
Example 1 was prepared by mixing the compound (11) prepared from 2 mmol) with 0.5 mg (0.00094 mmol) of the Lewis X-type trisaccharide.
The reaction was carried out under the same conditions as in Example 1. Similarly after HPLC purification
0.00073 mmol of compound (15) was obtained. (Yield 78%). HPLC conditions: 90% aq. CH 3 CN → 70% aq. CH 3 CN, 1
5min, 1ml / min MS (FAB -): m / z (%): 1114 (47) [M-H] -; HRMS; C 45 H 66 F 3 N 7 O 20 S ([M-H] -) : 1114.4
114 (calculated), 1114.4163 (actual)
【００５２】実施例１４（化合物（１１）→（１６）） 実施例９の方法で化合物（１０）１．７ｍｇ（０．００
２４ｍｍｏｌ）から調製した化合物（１１）とシアリル
ルイスＸ型四糖（純度９５％）１ｍｇ（０．００１２ｍ
ｍｏｌ）を実施例１０と同様の条件で反応させた。同様
にＨＰＬＣ精製後０．０００８５ｍｍｏｌの化含物（１
６）を得た。（収率７１％）。 ＨＰＬＣ条件:85%aq.ＣＨ3ＣＮ→65%aq.ＣＨ3ＣＮ、１
５min、１ｍｌ／min ＭＳ（ＦＡＢ-）:m/z(%):1405(29)［Ｍ−Ｈ］-; ＨＲＭＳ; Ｃ56Ｈ84Ｆ3Ｎ8Ｏ28Ｓ（[Ｍ−Ｈ]-）：1405.
5068(計算値)、1405.5143(実測値)Example 14 (Compound (11) → (16)) According to the method of Example 9, 1.7 mg (0.00%) of compound (10)
24 mmol) and 1 mg (0.0012 m) of sialyl Lewis type X tetrasaccharide (purity 95%)
mol) was reacted under the same conditions as in Example 10. Similarly, after HPLC purification, 0.00085 mmol of the compound (1
6) was obtained. (Yield 71%). HPLC conditions: 85% aq. CH 3 CN → 65% aq. CH 3 CN, 1
5min, 1ml / min MS (FAB -): m / z (%): 1405 (29) [M-H] -; HRMS; C 56 H 84 F 3 N 8 O 28 S ([M-H] -) : 1405.
5068 (calculated value), 1405.5143 (actual value)
【００５３】参考例１ （レクチンの光親和性標識実
験） 化合物（１２）〜化合物（１６）による光親和性標識実
験は畑中ら（Y.Hatanaka、M.Hashimoto、H.Nishihara、
H.Narimatsu、Y.Kanaoka、Carbohydr.Res.,294、95-108
(1996)）の方法に準じて以下の通り行った。０．１Ｍリ
ン酸緩衝液、ｐＨ７.６中に、化合物（１２）〜化合物
（１６）のいずれかの光親和性標識試薬（０．１ｍＭ）
及びレクチン（サブユニット濃度０．１ｍＭ）、を含む
光親和性標識試科を各０．０５ｍｌ調製した。別に対照
試料として、化合物（１２）〜化合物（１６）のいずれ
かの光親和性標識試薬（０．１ｍＭ）、レクチン（サブ
ユニットあたり０．１ｍＭ）、及び阻害剤０．１Ｍを含
む０．１Ｍリン酸緩衝液、ｐＨ７．６を各0.05ml調製し
た。光親和性標識、レクチン、及び対照実験における阻
害剤の組み合わせは、この順に以下の通りである。Reference Example 1 (Lectin Photoaffinity Labeling Experiment) Photoaffinity labeling experiments with Compounds (12) to (16) were performed by Hatanaka et al. (Y. Hatanaka, M. Hashimoto, H. Nishihara,
H. Narimatsu, Y. Kanaoka, Carbohydr. Res., 294, 95-108
(1996)). A photoaffinity labeling reagent (0.1 mM) of any of compounds (12) to (16) in 0.1 M phosphate buffer, pH 7.6
And lectin (subunit concentration: 0.1 mM), 0.05 ml of each photoaffinity labeling sample was prepared. Separately, as a control sample, 0.1 M containing the photoaffinity labeling reagent (0.1 mM), lectin (0.1 mM per subunit), and 0.1 M of the inhibitor of any of compounds (12) to (16) Phosphate buffer, pH 7.6, 0.05 ml each was prepared. The combinations of photoaffinity labels, lectins, and inhibitors in control experiments are, in this order, as follows.
【００５４】 化合物(12) ヒママメレクチン(RCA) N-アセチルラクトサミン 化合物(13) 小麦胚芽レクチン(WGA) シアリル-α2-3-ラクトース 化合物(14) イヌエンジュレクチン(MAL) シアリル-α2-3-ラクトース 化合物(15) ロータスレクチン(Lotus) メチル-α-フコピラノシド 化合物(16) イヌエンジュレクチン(MAL) シアリル-α2-3-ラクトース 以上のようにして調製した、光親和性標識試薬試料及び
対照試料を遮光下、２５℃で３０分間インキュベートし
た後、３０Ｗ長波長ＵＶランプ（フナコシ、XX-15）を
用いて、上方５ｃｍの距離から、氷上０℃で１時間照射
した。照射後の各試料は、常法に従い12% SDS-ポリア
クリルアミド電気泳動で分離後、PVDF膜に転写して化学
発光解析により光標識バンドを検出した。対照実験によ
り、光標識が阻害されることから、標識は各レクチンに
特異的であることを確認した。結果を表−１に示す。Compound (12) Castor lectin (RCA) N-acetyllactosamine Compound (13) Wheat germ lectin (WGA) Sialyl-α2-3-lactose Compound (14) Canine endectin (MAL) Sialyl-α2-3- Lactose Compound (15) Lotus Lectin (Lotus) Methyl-α-fucopyranoside Compound (16) Inuendurectin (MAL) Sialyl-α2-3-lactose Photoaffinity labeling reagent sample and control sample prepared as described above are shielded from light After incubating at 25 ° C. for 30 minutes under the above conditions, irradiation was performed at 0 ° C. for 1 hour on ice from a distance of 5 cm above using a 30 W long wavelength UV lamp (Funakoshi, XX-15). Each sample after irradiation was separated by 12% SDS-polyacrylamide electrophoresis according to a conventional method, transferred to a PVDF membrane, and a photolabeled band was detected by chemiluminescence analysis. Control experiments confirmed that the label was specific for each lectin, as the light label was inhibited. The results are shown in Table 1.
【００５５】[0055]
【表１】 [Table 1]
【００５６】実施例１５（化合物（１７）） 水素化ナトリウム８.０ｇ(６０%油状分散液 ０.２ｍｏ
ｌ)をトルエン２００ｍｌに懸濁し、-７０℃で2-[2-(2-クロロエト
キシ)エトキシ]エタノール３３.７ｇ(０.２ｍｏｌ)をゆっくり加え
た。この混合物にフ゛ロム酢酸エチル３３.４ｇ(０.２ｍｏｌ)を
-７０℃でゆっくり滴下し、３０分後反応混合物を室温
に放置した。２時間後、反応混合物に０℃で酢酸を加え
て中和後、溶媒を減圧留去し、残渣をシリカゲルカラム
（溶離剤；酢酸エチル：ヘキサン＝３：２）で精製して
３８.８ｇの無色油状物質を得た。これをＮ−メチル−
２−ピロリドン２００ｍｌに溶解し、臭化エチル１６３
ｇ(１.５ｍｏｌ)と臭化ナトリウム３.１ｇ(０.０３ｍｏ
ｌ)を加えて６５℃に４８時間加熱した。溶媒を減圧留
去して残渣をシリカゲルカラム（溶離剤；酢酸エチル：
ヘキサン＝３：２）で精製して３２.３ｇの無色油状物
質として化合物（１７)｛2-[2-(2-フ゛ロムエトキシ)エトキシ]エトキシ
酢酸エチル｝を得た（収率：７６％）。 1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=1.28(3H,t,J=7.3Hz,
-CH2-CH3 ),3.47(2H,t,J=6.3Hz,BrCH2),3.68-3.75(8H,m,
O-CH2-),3.81(2H,t,J=6.3Hz,BrCH2 CH2 O),4.15(2H,s,O-C
H2-CO-),4.23(2H,q,J=7.3Hz,-CH2 -CH3)Example 15 (Compound (17)) 8.0 g of sodium hydride (0.2 mol of a 60% oil dispersion)
l) was suspended in 200 ml of toluene, and 33.7 g (0.2 mol) of 2- [2- (2-chloroethoxy) ethoxy] ethanol was slowly added at -70 ° C. To this mixture was added 33.4 g (0.2 mol) of ethyl bromoacetate.
The mixture was slowly added dropwise at -70 ° C, and after 30 minutes, the reaction mixture was left at room temperature. After 2 hours, the reaction mixture was neutralized by adding acetic acid at 0 ° C., the solvent was distilled off under reduced pressure, and the residue was purified by a silica gel column (eluent; ethyl acetate: hexane = 3: 2) to give 38.8 g of the residue. A colorless oil was obtained. This is N-methyl-
Dissolve in 200 ml of 2-pyrrolidone and add 163 ethyl bromide.
g (1.5 mol) and 3.1 g of sodium bromide (0.03 mol)
1) was added and heated to 65 ° C. for 48 hours. The solvent was distilled off under reduced pressure, and the residue was subjected to silica gel column (eluent; ethyl acetate:
Purification with hexane = 3: 2) gave 32.3 g of compound (17) {ethyl 2- [2- (2-fluoroethoxy) ethoxy] ethoxyacetate} as a colorless oil (yield: 76%). . 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 1.28 (3H, t, J = 7.3 Hz,
-CH 2 -CH 3 ), 3.47 (2H, t, J = 6.3Hz, BrCH 2 ), 3.68-3.75 (8H, m,
O-CH 2- ), 3.81 (2H, t, J = 6.3Hz, BrCH 2 CH 2 O), 4.15 (2H, s, OC
H 2 -CO-), 4.23 (2H, q, J = 7.3Hz,-CH 2 -CH 3 )
【００５７】実施例１６（化合物(２)+(１７)→(１
８)） 化合物(２)４.６２ｇ(２０ｍｍｏｌ)、化合物(１７)７.
１８ｇ(２４ｍｍｏｌ)、無水炭酸カリウム２.７６ｇ(２
０ｍｍｏｌ)、Ｂｕ4ＮＩ ７.３９ｇ(２０ｍｍｏｌ)、及
びＤＭＳＯ ２０ｍｌの混合物をアルゴンガス雰囲気
下、６０℃に１４時間撹拌した。その後、反応液に水を
加え、酢酸エチルで抽出した。酢酸エチル層を水、０．
１Ｎ ＨＣｌ、及び水で順次洗浄して無水硫酸マグネシ
ウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲルカ
ラム（溶離剤；ジエチルエーテル：ヘキサン＝３：１）
で精製して６.５８ｇの淡黄色油状物質として化合物
（１８）｛2-[2-[2-(2-(エトキシカルホ゛ニルメトキシ)エトキシ)エトキシ]エトキ
シ]-4-[3-(トリフルオロメチル)-3H-シ゛アシ゛リン-3-イル]ヘ゛ンス゛アルテ゛ヒト゛｝
を得た（収率：７６％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=1.28(3H,t,J=7.3Hz,
-CH2-CH3 ),3.67-3.93(12H,m,O-CH2-),4.14(2H,s,O-CH2-
CO-),4.20(2H,q,J=7.3Hz,-CH2 -CH3),4.52(2H,s,Ph-CH
2-),6.74(1H,s,Ph-H),6.85(1H,d,J=8.1Hz,Ph-H),7.84(1
H,d,J=8.1Hz,Ph-H); ＵＶ(ＥｔＯＨ)ｎｍ(ε):210(16,368),289.5(1,272)Example 16 (Compound (2) + (17) → (1
8)) 4.62 g (20 mmol) of compound (2), 7.2 of compound (17)
18 g (24 mmol), 2.76 g of anhydrous potassium carbonate (2
0 mmol), 7.39 g (20 mmol) of Bu 4 NI, and 20 ml of DMSO were stirred at 60 ° C. for 14 hours under an argon gas atmosphere. Thereafter, water was added to the reaction solution, and extracted with ethyl acetate. The ethyl acetate layer was washed with water, 0.1 mL.
After sequentially washing with 1N HCl and water and drying over anhydrous magnesium sulfate, the solvent is distilled off under reduced pressure, and the residue is subjected to a silica gel column (eluent; diethyl ether: hexane = 3: 1).
Compound (18) {2- [2- [2- (2- (ethoxycarbonylmethoxy) ethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) as a pale yellow oily substance ) -3H-Siacylin-3-yl] benzartehuman
Was obtained (yield: 76%). 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 1.28 (3H, t, J = 7.3 Hz,
-CH 2 - CH 3 ), 3.67-3.93 (12H, m, O-CH 2- ), 4.14 (2H, s, O-CH 2-
CO -), 4.20 (2H, q, J = 7.3Hz, - CH 2 -CH 3), 4.52 (2H, s, Ph-CH
2- ), 6.74 (1H, s, Ph-H), 6.85 (1H, d, J = 8.1 Hz, Ph-H), 7.84 (1
H, d, J = 8.1Hz, Ph-H); UV (EtOH) nm (ε): 210 (16,368), 289.5 (1,272)
【００５８】実施例１７（化合物(１８)→(１９)） 化合物(１８)２.１７ｇ（５ｍｍｏｌ）を、エタノール
１０ｍｌに溶解し、この溶液にＮａＢＨ4 １８９ｍｇ
（５ｍｍｏｌ）をゆっくり加えた。反応混合物を室温で
３０分撹拌した後、反応液に０℃で酢酸を加えることに
よりｐＨ３付近に調節した後溶媒を減圧留去し、残渣を
酢酸エチルで抽出した。酢酸エチル層を０.１Ｎ ＨＣ
ｌ、水、及び飽和食塩水で順次洗浄して無水硫酸マグネ
シウムで乾燥後、溶媒を減圧留去し、残渣をシリカゲル
カラム（溶離剤；酢酸エチル：ヘキサン＝１：１）で精
製して１.７２ｇの淡黄色油状物質として化合物（１９)
｛2-[2-[2-(2-(エトキシカルホ゛ニルメトキシ)エトキシ)エトキシ]エトキシ]-4-[3-
(トリフルオロメチル)-3H-シ゛アシ゛リン-3-イル]ヘ゛ンシ゛ルアルコール｝を得た
（収率：７６％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=1.28(3H,t,J=7.3Hz,
-CH2-CH3 ),3.43-3.87(12H,m,O-CH2-),4.12(2H,s,O-CH2-
CO-),4.20(2H,q,J=7.3Hz,-CH2 -CH3),4.52(2H,s,Ph-CH
2-),6.62(1H,s,Ph-H),6.79(1H,d,J=7.8Hz,Ph-H),7.30(1
H,d,J=7.8Hz,Ph-H); ＵＶ(ＥｔＯＨ)ｎｍ(ε):206.5(24,660),227(10,244),2
80(2,311)Example 17 (Compound (18) → (19)) 2.17 g (5 mmol) of compound (18) was dissolved in 10 ml of ethanol, and 189 mg of NaBH 4 was added to this solution.
(5 mmol) was added slowly. After stirring the reaction mixture at room temperature for 30 minutes, acetic acid was added to the reaction solution at 0 ° C. to adjust the pH to around 3, then the solvent was distilled off under reduced pressure, and the residue was extracted with ethyl acetate. The ethyl acetate layer was washed with 0.1N HC
After washing with water, saturated brine and drying over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified on a silica gel column (eluent; ethyl acetate: hexane = 1: 1) to give 1. Compound (19) as a pale yellow oily substance (72 g)
｛2- [2- [2- (2- (ethoxycarbonylmethoxy) ethoxy) ethoxy] ethoxy] -4- [3-
(Trifluoromethyl) -3H-siadolin-3-yl] benzyl alcohol was obtained (yield: 76%). 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 1.28 (3H, t, J = 7.3 Hz,
-CH 2 - CH 3 ), 3.43-3.87 (12H, m, O-CH 2- ), 4.12 (2H, s, O-CH 2-
CO -), 4.20 (2H, q, J = 7.3Hz, - CH 2 -CH 3), 4.52 (2H, s, Ph-CH
2- ), 6.62 (1H, s, Ph-H), 6.79 (1H, d, J = 7.8Hz, Ph-H), 7.30 (1
H, d, J = 7.8Hz, Ph-H); UV (EtOH) nm (ε): 206.5 (24,660), 227 (10,244), 2
80 (2,311)
【００５９】実施例１８（化合物(１９)→(２０)） 化合物(１９)３.６０ｇ（８ｍｍｏｌ）と四臭化炭素３.
３２ｇ（１０ｍｍｏｌ）とを、１６ｍｌのジクロロメタ
ンに溶解し、０℃に冷却しながらトリフェニルホスフィ
ン３.１５ｇ（１２ ｍｍｏｌ）をゆっくりと加えた。３
０分後、反応混合物に水を加え、ジクロロメタンで抽出
し、ジクロロメタン層を無水硫酸マグネシウムで乾燥
後、溶媒を減圧留去し、残渣をシリカゲルカラム（溶離
剤；酢酸エチル：ヘキサン＝１：１）で精製して３.３
７ｇの淡黄色油状物質として化合物（２０）｛2-[2-[2-
(2-(エトキシカルホ゛ニルメトキシ)エトキシ)エトキシ]エトキシ]-4-[3-(トリフルオロメチ
ル)-3H-シ゛アシ゛リン-3-イル]ヘ゛ンシ゛ルフ゛ロミト゛｝を得た（収率：82
％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=1.28(3H,t,J=7.3Hz,
-CH2-CH3 ),3.67-3.91(12H,m,O-CH2-),4.14(2H,s,O-CH2-
CO-),4.20(2H,q,J=7.3Hz,-CH2 -CH3),4.52(2H,s,Ph-CH
2-),6.65(1H,s,Ph-H),6.77(1H,d,J=8.0Hz,Ph-H),7.35(1
H,d,J=8.0Hz,Ph-H); Positive ion FABMS (3-nitrobenzylalcohol)ｍ／ｚ：
５３５（Ｍ＋Ｈ）+ ＵＶ(ＥｔＯＨ)ｎｍ(ε):209(23,783),238.5(8,873),29
0(2,927)Example 18 (Compound (19) → (20)) 3.60 g (8 mmol) of compound (19) and 3.60 g of carbon tetrabromide.
32 g (10 mmol) was dissolved in 16 ml of dichloromethane, and 3.15 g (12 mmol) of triphenylphosphine was slowly added while cooling to 0 ° C. 3
After 0 minute, water was added to the reaction mixture, extracted with dichloromethane, the dichloromethane layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was subjected to a silica gel column (eluent; ethyl acetate: hexane = 1: 1). Purification by 3.3
Compound (20) {2- [2- [2-
(2- (Ethoxycarbonylmethoxy) ethoxy) ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-siacinidin-3-yl] benzenesulfuromito was obtained (yield: 82
%). 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 1.28 (3H, t, J = 7.3 Hz,
-CH 2 - CH 3 ), 3.67-3.91 (12H, m, O-CH 2- ), 4.14 (2H, s, O-CH 2-
CO -), 4.20 (2H, q, J = 7.3Hz, - CH 2 -CH 3), 4.52 (2H, s, Ph-CH
2- ), 6.65 (1H, s, Ph-H), 6.77 (1H, d, J = 8.0Hz, Ph-H), 7.35 (1
H, d, J = 8.0Hz, Ph-H); Positive ion FABMS (3-nitrobenzylalcohol) m / z:
535 (M + H) + UV (EtOH) nm (ε): 209 (23,783), 238.5 (8,873), 29
0 (2,927)
【００６０】実施例１９（化合物（２０）＋（２１）→
（２２）） 水素化ナトリウム２１６ｍｇ（６０%油状分散液 ５.４
ｍｍｏｌ）をＴＨＦ１０ｍｌに懸濁し、-２０℃に冷却
しながらＮ-ヒドロキシカルバミン酸 tert-ブチル(２
１)７１８ｍｇ（６ｍｍｏｌ）を加えた。１５分後、化
合物(２０)２.３１ｇ（４．５ｍｍｏｌ）を−１５℃に
冷却しながら加え、反応混合物を０℃で撹拌した。２時
間後、反応液に０℃で１Ｎ ＨＣｌを加えることにより
ｐＨ３付近に調節した後、酢酸エチルで抽出した。酢酸
エチル層を無水硫酸マグネシウムで乾燥後、溶媒を減圧
留去し、残渣をシリカゲルカラム（溶離剤；ヘキサン：
ジエチルエーテル＝１：１０）で精製して２.１１ｇの
淡黄色油状物質として化合物（２２）｛2-[2-[2-[2-(エト
キシカルホ゛ニルメトキシ)エトキシ]エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-シ
゛アシ゛リン-3-イル]ヘ゛ンシ゛ルオキシカルハ゛ミン酸 tert-フ゛チルエステル｝を得
た（収率：83％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=1.28(3H,t,J=7.1Hz,
-CH2-CH3 ),1.46(9H,s,C(CH3)3),3.67-3.88(12H,m,O-CH2
-),4.12(2H,s,O-CH2-CO-),4.20(2H,q,J=7.1Hz,- CH2 -C
H3),4.91(2H,s,Ph-CH2-),6.65(1H,s,Ph-H),6.79(1H,d,J
=8.0Hz,Ph-H),7.38(1H,d,J=8.0Hz,Ph-H),7.67(1H,s,N
H); Positive ion FABMS (3-nitrobenzylalcohol)ｍ／ｚ：
５８８（Ｍ＋Ｈ）+ ＵＶ:２０７ｎｍ(ε３４０２９),２２８.５ｎｍ(ε１３
２２８),２８０.５ｎｍ(ε２９５９)Example 19 (Compound (20) + (21) →
(22)) 216 mg of sodium hydride (5.4% of 60% oil dispersion)
mmol) in 10 ml of THF and cooled to -20 ° C
Tert-butyl N-hydroxycarbamate (2
1) 718 mg (6 mmol) was added. 15 minutes later
2.31 g (4.5 mmol) of compound (20) was brought to −15 ° C.
Added with cooling, the reaction mixture was stirred at 0 ° C. 2 o'clock
After a while, add 1N HCl to the reaction at 0 ° C.
After adjusting the pH to around 3, the mixture was extracted with ethyl acetate. Acetic acid
After the ethyl layer was dried over anhydrous magnesium sulfate, the solvent was removed under reduced pressure.
The residue was distilled off, and the residue was purified with a silica gel column (eluent; hexane:
(Diethyl ether = 1: 10) to give 2.11 g of
Compound (22) as a pale yellow oily substance 2- [2- [2- [2- (ethoxy
Xycarboxylmethoxy) ethoxy] ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-cy
[Acylin-3-yl] benzoyloxycarbamic acid tert-butyl ester was obtained.
(Yield: 83%).1 HNMR (400MHz, CDClThree, 25 ° C): δ = 1.28 (3H, t, J = 7.1Hz,
-CHTwo-CH 3 ), 1.46 (9H, s, C (CHThree)Three), 3.67-3.88 (12H, m, O-CHTwo
-), 4.12 (2H, s, O-CHTwo-CO-), 4.20 (2H, q, J = 7.1Hz,- CH 2 -C
HThree), 4.91 (2H, s, Ph-CHTwo-), 6.65 (1H, s, Ph-H), 6.79 (1H, d, J
= 8.0Hz, Ph-H), 7.38 (1H, d, J = 8.0Hz, Ph-H), 7.67 (1H, s, N
H); Positive ion FABMS (3-nitrobenzylalcohol) m / z:
588 (M + H)+ UV: 207 nm (ε34029), 228.5 nm (ε13
228), 280.5 nm (ε2959)
【００６１】実施例２０ （化合物（２２）→（２
３）） 化合物（２２）２．２６ｇ（４ｍｍｏｌ）を１Ｎ Ｎａ
ＯＨ ２０ｍｌとエタノール１８ｍｌの混合溶媒に溶か
し、０℃で３０分間撹拌した。反応液に０℃で１Ｎ Ｈ
Ｃｌを加えることによりｐＨ３付近に調節した後、溶媒
を減圧留去し、残渣をシリカゲルカラム（溶離剤；クロ
ロホルム：メタノール＝５：１）で精製して２．１ｇの
淡黄色油状物質として化合物（２３）｛2-[2-[2-[2-(カル
ホ゛キシメトキシ)エトキシ]エトキシ]エトキシ]-4-[3-(トリフルオロメチル)-3H-シ゛アシ゛
リン-3-イル]ヘ゛ンシ゛ルオキシカルハ゛ミン酸 tert-フ゛チルエステル｝を得た
（収率：98％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=1.46(9H,s,C(C
H3)3),3.69-3.90(12H,m,O-CH 2-),4.13(2H,bs,O-CH2-CO
-),4.91(2H,s,Ph-CH2-),6.65(1H,s,Ph-H),6.81(1H,d,J=
7.8Hz,Ph-H),7.37(1H,d,J=7.8Hz,Ph-H),8.06(1H,bs,NH)Example 20 (Compound (22) → (2
3)) 2.26 g (4 mmol) of compound (22) was added with 1N Na
Dissolved in a mixed solvent of 20 ml of OH and 18 ml of ethanol
And stirred at 0 ° C. for 30 minutes. Add 1N H at 0 ° C
After adjusting the pH to around 3 by adding Cl, the solvent
Was distilled off under reduced pressure, and the residue was subjected to a silica gel column (eluent;
Purified with chloroform: methanol = 5: 1) to give 2.1 g of
Compound (23) as a pale yellow oily substance 2- [2- [2- [2- (Cal
(Methoxymethoxy) ethoxy] ethoxy] ethoxy] -4- [3- (trifluoromethyl) -3H-thiocyanate
[Rin-3-yl] benzoyloxycarbamic acid tert-butyl ester was obtained.
(Yield: 98%).1 HNMR (400MHz, CDClThree, 25 ° C): δ = 1.46 (9H, s, C (C
HThree)Three), 3.69-3.90 (12H, m, O-CH Two-), 4.13 (2H, bs, O-CHTwo-CO
-), 4.91 (2H, s, Ph-CHTwo-), 6.65 (1H, s, Ph-H), 6.81 (1H, d, J =
7.8Hz, Ph-H), 7.37 (1H, d, J = 7.8Hz, Ph-H), 8.06 (1H, bs, NH)
【００６２】実施例２１ （化合物（２３）＋ （２４）
→（２５）） 化合物（２３）５３７ｍｇ（１ｍｍｏｌ）とカルボニル
ジイミダゾール １９４ｍｇ（１．２ｍｍｏｌ）を無水
ＴＨＦ １ｍｌに溶解し、室温で撹拌しながらメチル ス
ルファモイルベンゾエート（２４） ２５８ｍｇ（１．
２ｍｍｏｌ）とジイソプロピルエチルアミン １５８ｍ
ｇ（１．２ｍｍｏｌ）を加え、室温で１８時間反応させ
た。その後、溶媒を減圧留去して得られた残渣に水を加
え、酢酸エチルで抽出した。酢酸エチル層を０．５Ｎ
ＨＣｌ、飽和重曹水、及び飽和食塩水で順次洗浄して無
水硫酸マグネシウムで乾燥後、溶媒を減圧留去し、残渣
をシリカゲルカラム（溶離剤；酢酸エチル：エタノール
＝１０：１）で精製して７３４ｍｇの淡黄色油状物質と
して化合物（２５）を得た（収率：74％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=1.40(9H,s,-C(C
H3)3),3.69-3.90(12H,m,O-CH2-),3.94(3H,s,CH3),4.2(2
H,bs,O-CH2-CO-),5.02(2H,s,Ph-CH2-),6.67(1H,s,Ph-
H),6.80(1H,d,J=7.8Hz,Ph-H),7.34(1H,d,J=7.8Hz,Ph-
H),7.69(1H,s,NH),8.14(4H,s,Ph-H)Example 21 (Compound (23) + (24)
→ (25)) 537 mg (1 mmol) of the compound (23) and 194 mg (1.2 mmol) of carbonyldiimidazole are dissolved in 1 ml of anhydrous THF, and 258 mg (1.
2mmol) and diisopropylethylamine 158m
g (1.2 mmol) was added and reacted at room temperature for 18 hours. Thereafter, water was added to the residue obtained by evaporating the solvent under reduced pressure, and the mixture was extracted with ethyl acetate. 0.5N ethyl acetate layer
After successively washing with HCl, saturated aqueous sodium bicarbonate and saturated saline and drying over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by a silica gel column (eluent; ethyl acetate: ethanol = 10: 1). Compound (25) was obtained as 734 mg of a pale yellow oil (yield: 74%). 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 1.40 (9H, s, -C (C
H 3) 3), 3.69-3.90 ( 12H, m, O-CH 2 -), 3.94 (3H, s, CH 3), 4.2 (2
H, bs, O-CH 2 -CO-), 5.02 (2H, s, Ph-CH 2- ), 6.67 (1H, s, Ph-
H), 6.80 (1H, d, J = 7.8Hz, Ph-H), 7.34 (1H, d, J = 7.8Hz, Ph-H)
H), 7.69 (1H, s, NH), 8.14 (4H, s, Ph-H)
【００６３】実施例２２ （化合物（２５）→（２
６）） 化合物（２５）１２０ｍｇ（０．１５ｍｍｏｌ）を１Ｎ
ＮａＯＨ ０．７５ｍｌとエタノール３．５ｍｌの混合
溶媒に溶かし、０℃で３０分間撹拌した。０℃に冷却し
ながら５Ｎ ＨＣｌ ０．１５ｍｌを加えてｐＨ３付近に
調節した後、酢酸エチルで抽出した。酢酸エチル層を飽
和食塩水で洗浄して無水硫酸マグネシウムで乾燥後、溶
媒を減圧留去し、残渣をシリカゲルカラム（溶離剤；ク
ロロホルム：メタノール＝５：１）で精製して９９ｍｇ
の淡黄色油状物質として化合物（２６）を得た（収率：
８９％）。1 ＨＮＭＲ(400MHz,CDCl3,25℃):δ=1.43(9H,s,-C(C
H3)3),3.68-4.10(12H,m,O-CH2-),4.23(2H,s,O-CH2-CO
-),4.91(2H,s,Ph-CH2-),6.65(1H,s,Ph-H),6.77(1H,d,J=
8.1Hz,Ph-H),7.35(1H,d,J=8.1Hz,Ph-H),8.17-8.23(4H,
m,Ph-H),8.03(1H,bs,-NH)Example 22 (Compound (25) → (2
6)) 120 mg (0.15 mmol) of compound (25) was added to 1N
It was dissolved in a mixed solvent of 0.75 ml of NaOH and 3.5 ml of ethanol and stirred at 0 ° C. for 30 minutes. While cooling to 0 ° C., 0.15 ml of 5N HCl was added to adjust the pH to around 3, followed by extraction with ethyl acetate. The ethyl acetate layer was washed with saturated saline and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by a silica gel column (eluent; chloroform: methanol = 5: 1) to give 99 mg.
Compound (26) was obtained as a pale yellow oily substance (yield:
89%). 1 H NMR (400 MHz, CDCl 3 , 25 ° C.): δ = 1.43 (9H, s, -C (C
H 3) 3), 3.68-4.10 ( 12H, m, O-CH 2 -), 4.23 (2H, s, O-CH 2 -CO
-), 4.91 (2H, s, Ph-CH 2- ), 6.65 (1H, s, Ph-H), 6.77 (1H, d, J =
8.1Hz, Ph-H), 7.35 (1H, d, J = 8.1Hz, Ph-H), 8.17-8.23 (4H,
m, Ph-H), 8.03 (1H, bs, -NH)
【００６４】実施例２３ （化合物（２６）＋（２７）
→（２８）） 化合物（２６）７２ｍｇ（０．１ｍｍｏｌ）をジクロロ
ロメタン１ｍｌに溶解し、Ｎ−ヒドロキシスクシンイミ
ド１３ｍｇ（０．１１ｍｍｏｌ）とＤＣＣ ２３ｍｇ
（０．１１ｍｍｏｌ）を加え、室温で１時間撹拌した。
溶媒を減圧留去し、残渣をＤＭＳＯ １ｍｌに溶解し、
ビオチンヒドラジド（２７）２６ｍｇ（０．１ｍｍｏ
ｌ）を加えて室温で一夜攪拌した。反応液を直接シリカ
ゲルカラム（溶離剤；クロロホルム：メタノール＝５：
１）で精製して１０ｍｇの淡黄色固体として化合物（２
８）を得た（収率：１０％） Positive ion FABMS (3-nitrobenzylalcohol) ｍ／
ｚ：961（Ｍ＋Ｈ）+ Example 23 (Compound (26) + (27)
→ (28)) 72 mg (0.1 mmol) of compound (26) is dissolved in 1 ml of dichloromethane, and 13 mg (0.11 mmol) of N-hydroxysuccinimide and 23 mg of DCC are dissolved.
(0.11 mmol) was added and stirred at room temperature for 1 hour.
The solvent was distilled off under reduced pressure, the residue was dissolved in 1 ml of DMSO,
26 mg of biotin hydrazide (27) (0.1 mmo
l) was added and the mixture was stirred at room temperature overnight. The reaction solution was directly applied to a silica gel column (eluent; chloroform: methanol = 5:
Purified in 1) to give 10 mg of compound (2) as a pale yellow solid.
8) (Yield: 10%) Positive ion FABMS (3-nitrobenzylalcohol) m /
z: 961 (M + H) +
【００６５】実施例２４ （化合物（１１）→（２
９）） 実施例９の方法で化合物（１０）３．５ｍｇ（５μｍｏ
ｌ）から調製した化合物（１１）とキトビオース １．
１ｍｇ（２．５μｍｏｌ）を実施例１０と同様な条件で
反応させた。同様にＨＰＬＣ精製後、１．２μｍｏｌの
化合物（２９)を得た（収率：４８％）。 ＨＰＬＣ条件：８５％ aq.ＣＨ3ＣＮ、１ml/min Positive ion FABMS (3-nitrobenzylalcohol) ｍ／ｚ：
1011（Ｍ＋Ｈ）+，1033（Ｍ＋Ｎａ）+ Example 24 (Compound (11) → (2
9)) 3.5 mg (5 μmo) of compound (10) by the method of Example 9
Compound (11) prepared from l) and chitobiose
1 mg (2.5 μmol) was reacted under the same conditions as in Example 10. Similarly, after HPLC purification, 1.2 μmol of compound (29) was obtained (yield: 48%). HPLC conditions: 85% aq. CH 3 CN, 1 ml / min Positive ion FABMS (3-nitrobenzylalcohol) m / z:
1011 (M + H) + , 1033 (M + Na) +
【００６６】実施例２５ （化合物（２８）→（３
０）） 実施例９の方法で化合物（２８）９．６ｍｇ（１０μｍ
ｏｌ）を脱保護し、キトビオース ２．１ｍｇ（５μｍ
ｏｌ）を実施例１０と同様な条件で反応させた。同様に
ＨＰＬＣ精製後、１．０μｍｏｌの化合物（３０)を得
た（収率：２％）。 ＨＰＬＣ条件：90％ aq.ＣＨ3ＣＮ→80％ ａｑ.ＣＨ3
ＣＮ 20min,１ml/minPositive ion FABMS (3-nitroben
zylalcohol)ｍ/ｚ:1266(Ｍ+Ｈ)+,1288(Ｍ+Ｎａ)+ Example 25 (Compound (28) → (3
0)) 9.6 mg (10 μm) of compound (28) according to the method of Example 9
ol) was deprotected, and chitobiose 2.1 mg (5 μm
ol) was reacted under the same conditions as in Example 10. Similarly, after HPLC purification, 1.0 μmol of compound (30) was obtained (yield: 2%). HPLC conditions: 90% aq.CH 3 CN → 80% aq.CH 3
CN 20min, 1ml / minPositive ion FABMS (3-nitroben
zylalcohol) m / z: 1266 (M + H) + , 1288 (M + Na) +
【００６７】参考例２ （ＷＧＡの光親和性標識実験） 化合物(２９)、化合物(３０)の光親和性標識実験を参考
例１と同様な方法で行った。 ０.１Ｍリン酸緩衝液、ｐ
Ｈ７.６中に、化合物(２９)及び化合物(３０)のいずれ
かの光親和性標識試薬（０.１ｍＭ）及びＷＧＡ（小麦
胚芽レクチン(wheat germ agglutinin);サブユニット濃
度０.１ｍＭ）、を含む光親和性標識試料を各０.０５ｍ
ｌ調製した。別に対照試料として、化合物(２９)及び化
合物(３０)のいずれかの光親和性標識試薬（０.１ｍ
Ｍ）、ＷＧＡ（サブユニット濃度０.１ｍＭ）、及びキ
トビオース０.１Ｍを含む０.１Ｍリン酸緩衝液、ｐＨ
７．６を各０.０５ｍｌ調製した。以上のようにして調
製した、光親和性標識試薬試料及び対照試料を遮光下、
２５℃で３０分間インキュベートした後、３０Ｗ 長波
長ＵＶランプ（フナコシ、XX-15）を用いて、上方５ｃ
ｍの距離から、氷上０℃で１時間照射した。照射後の各
試料は、常法に従い12% SDS-ポリアクリルアミド電気
泳動で分離後、PVDF膜に転写して化学発光解析により光
標識バンドを検出した。対照実験により、分子量２２Ｋ
のＷＧＡバンドの光標識が阻害されることから、標識は
ＷＧＡに特異的であることを確認した。Reference Example 2 (Photoaffinity Labeling Experiment of WGA) The photoaffinity labeling experiment of compound (29) and compound (30) was performed in the same manner as in Reference Example 1. 0.1 M phosphate buffer, p
In H7.6, a photoaffinity labeling reagent (0.1 mM) of either compound (29) or compound (30) and WGA (wheat germ agglutinin; subunit concentration: 0.1 mM) were added. The photoaffinity-labeled sample containing
1 was prepared. Separately, as a control sample, a photoaffinity labeling reagent (0.1 m) of either compound (29) or compound (30) was used.
M), WGA (subunit concentration 0.1 mM), and 0.1 M phosphate buffer containing 0.1 M chitobiose, pH
7.6 was prepared in 0.05 ml each. The light-affinity labeling reagent sample and the control sample prepared as described above were shielded from light,
After incubating for 30 minutes at 25 ° C., use a 30 W long wavelength UV lamp (Funakoshi, XX-15) to extract
Irradiated at 0 ° C. for 1 hour on ice from a distance of m. Each sample after irradiation was separated by 12% SDS-polyacrylamide electrophoresis according to a conventional method, transferred to a PVDF membrane, and a photolabeled band was detected by chemiluminescence analysis. According to a control experiment, the molecular weight was 22K.
Inhibition of the optical labeling of the WGA band indicated that the label was specific to WGA.
【００６８】スペーサーの切断は、照射後の各試料 １
０μｌをとり、７Ｍグアニジン,ＥＤＴＡ ２Ｎａ、１０
ｍＭを含む０.５Ｍトリス塩酸緩衝液、ｐＨ８.５、１０
μｌに溶かし、０.７Ｍ ＤＴＴ １０μｌを加えて室温
２時間反応させた。さらに、１Ｍ ヨード酢酸ナトリウ
ム １０μｌを加えて室温４時間、さらに４Ｍ メルカプ
トエタノールを加えて５時間反応させた後、０.１Ｍリ
ン酸緩衝液、ｐＨ７.６に対して透析した後、同様に電
気泳動により解析した。切断反応後は、化合物(３０)に
よるＷＧＡラベルバンドの分子量２２Ｋが消失すること
により、切断を確認した。The cutting of the spacer was performed for each sample 1 after irradiation.
Take 0 μl and add 7M guanidine, EDTA 2Na, 10
0.5M Tris-HCl buffer containing mM, pH 8.5, 10
The mixture was dissolved in μl, 10 μl of 0.7M DTT was added, and the mixture was reacted at room temperature for 2 hours. Further, 10 μl of 1M sodium iodoacetate was added thereto, and the mixture was reacted at room temperature for 4 hours, and further added with 4M mercaptoethanol. The mixture was reacted for 5 hours, dialyzed against 0.1M phosphate buffer, pH 7.6, and electrophoresed in the same manner. Analyzed by After the cleavage reaction, the disappearance of the molecular weight 22K of the WGA label band by the compound (30) confirmed the cleavage.
【００６９】[0069]
【発明の効果】本発明の一般式(II)で示される化合物、
特にオキシアミノ基およびその塩を有する上記化合物(1
1)は、還元末端を有する糖化合物に、特別の保護処理を
施すことなく、一段階で反応して光反応基（ジアジリ
ン）とビオチンを有する一般式(V)の糖結合フェニルジ
アジリン化合物を導くことができ、この一般式(V)の化
合物は、光親和性標識試薬として、糖と相互作用する蛋
白質（糖受容体）のビオチン標識に極めて有用である。
しかも、該一般式(V)の化合物において、ビオチンが切
断性スペーサーを介してフェニルジアリジン骨格に結合
している化合物は、固定化アビジンからのラベル化した
蛋白質の遊離・回収が容易である点で優れている。従っ
て、一般式(II)の化合物は、光親和性標識試薬を簡便に
製造するための安定した有用な化合物であり、この一般
式(II)の化合物の合成中間体である、一般式(I)の化合
物も有用性の高い物質である。The compound of the present invention represented by the general formula (II),
In particular, the above compound having an oxyamino group and a salt thereof (1
1) reacts the saccharide compound having a reducing end with a photoreactive group (diazirine) and a biotin-containing saccharide-linked phenyldiazirine compound having a photoreactive group (diazirine) and biotin without applying a special protective treatment. The compound of the general formula (V) can be derived and is extremely useful as a photoaffinity labeling reagent for biotin labeling of a protein (sugar receptor) that interacts with sugar.
Moreover, in the compound of the general formula (V), the compound in which biotin is bonded to the phenyldiazine skeleton via a cleavable spacer is easy to release and recover the labeled protein from the immobilized avidin. Is excellent. Therefore, the compound of the general formula (II) is a stable and useful compound for easily producing a photoaffinity labeling reagent, and is a synthetic intermediate of the compound of the general formula (II), ) Is also a highly useful substance.
【図１】 本発明の一般式(I)化合物を合成する一例を
示す反応スキームの概略図である。FIG. 1 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of the general formula (I) of the present invention.
【図２】 本発明の一般式(II)化合物を合成する一例を
示す反応スキームの概略図である。FIG. 2 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of the general formula (II) of the present invention.
【図３】 本発明の一般式(Ｖ)化合物を合成する一例を
示す反応スキームの概略図である。FIG. 3 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of the general formula (V) of the present invention.
【図４】 本発明の一般式(Ｖ)化合物を合成する一例を
示す反応スキームの概略図である。FIG. 4 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of the general formula (V) of the present invention.
【図５】 本発明の一般式(I)化合物及び(IV)化合物を
合成する一例を示す反応スキームの概略図である。FIG. 5 is a schematic diagram of a reaction scheme showing an example of synthesizing the compounds of the general formulas (I) and (IV) of the present invention.
【図６】 本発明の一般式(Ｖ)化合物を合成する一例を
示す反応スキームの概略図である。FIG. 6 is a schematic diagram of a reaction scheme showing an example of synthesizing a compound of the general formula (V) of the present invention.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｇ０１Ｎ 33/532 Ｇ０１Ｎ 33/532 Ａ // Ｇ０１Ｎ 33/53 33/53 Ｕ 33/566 33/566 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) G01N 33/532 G01N 33/532 A // G01N 33/53 33/53 U 33/566 33/566
Claims (19)
アジリン化合物。 【化１】 （式中、Ｒ1は−ＮＨＰ1又は−ＣＯＸ1を表し、Ｒ2は、
アルデヒド基、ヒドロキシ低級アルキル基、ハロゲン化
低級アルキル基、フタルイミドオキシ低級アルキル基、
又は−(ＣＨ2)x−Ｏ−ＮＨＰ1を表し、Ｐ1はアミノ保護
基を表し、Ｘ1はアルコキシ基，ヒドロキシ基，又は−
ＮＨＳＯ2−Ｘ2−ＣＯ−Ｘ3を表し、Ｘ2はアリーレン基
又はアルキレン基を表し、Ｘ3はアルコキシ基又はヒド
ロキシ基を表し、ｍは１〜４の整数を表し、ｎは１〜６
の整数を表し、ｘは１〜４の整数を表す。）1. A phenyldiazirine compound represented by the following general formula (I). Embedded image (Wherein, R 1 represents —NHP 1 or —COX 1 , and R 2 represents
Aldehyde group, hydroxy lower alkyl group, halogenated lower alkyl group, phthalimidooxy lower alkyl group,
Or — (CH 2 ) x —O—NHP 1 , P 1 represents an amino protecting group, and X 1 represents an alkoxy group, a hydroxy group, or —
Represents NHSO 2 -X 2 -CO-X 3 , X 2 represents an arylene group or an alkylene group, X 3 represents an alkoxy group or a hydroxy group, m represents an integer of 1 to 4, n is 1 to 6
And x represents an integer of 1 to 4. )
化フェニルジアジリン化合物。 【化２】 （式中、Ｒ3は、フタルイミド基、アミノ基、−ＮＨＰ1
又はアミノ基の塩を表し、Ｐ1はアミノ保護基を表し、
Ｙ1は−ＮＨ−又は−ＣＯＮＨＳＯ2−Ｘ2−ＣＯ−を表
し、Ｘ2はアリーレン基又はアルキレン基を表し、Ｙ2は
ビオチン又はビオチン誘導体残基を表し、ｍは１〜４の
整数を表し、ｎは１〜６の整数を表し、ｘは１〜４の整
数を表す。）2. A biotinylated phenyldiazirine compound represented by the following general formula (II). Embedded image (Wherein, R 3 is a phthalimide group, an amino group, —NHP 1
Or a salt of an amino group, P 1 represents an amino protecting group,
Y 1 represents -NH- or -CONHSO 2 -X 2 -CO-, X 2 represents an arylene group or an alkylene group, Y 2 represents biotin or a biotin derivative residue, and m is an integer of 1 to 4. In the formula, n represents an integer of 1 to 6, and x represents an integer of 1 to 4. )
記載のビオチン化フェニルジアジリン化合物。 【化３】 （式中、Ｒ3は、フタルイミド基、アミノ基、−ＮＨＰ1
又はアミノ基の塩を表し、Ｐ1はアミノ保護基を表し、
ｍは１〜４の整数を表し、ｎは１〜６の整数を表し、ｘ
は１〜４の整数を表す。）3. A compound represented by the following general formula (III):
The biotinylated phenyldiazirine compound according to the above. Embedded image (Wherein, R 3 is a phthalimide group, an amino group, —NHP 1
Or a salt of an amino group, P 1 represents an amino protecting group,
m represents an integer of 1 to 4, n represents an integer of 1 to 6, x
Represents an integer of 1 to 4. )
記載のビオチン化フェニルジアジリン化合物。 【化４】 （式中、Ｒ3は、フタルイミド基、アミノ基、−ＮＨＰ1
又はアミノ基の塩を表し、Ｐ1はアミノ保護基を表し、
ｍは１〜４の整数を表し、ｎは１〜６の整数を表し、Ｘ
2はアリーレン基又はアルキレン基を表し、Ｚはヘテロ
原子で中断されていてもよい炭素数１〜２０のアルキレ
ン基を表し、ｘは１〜４の整数を表し、ｑは０又は１を
表す。）4. The method according to claim 2, which is represented by the following general formula (IV).
The biotinylated phenyldiazirine compound according to the above. Embedded image (Wherein, R 3 is a phthalimide group, an amino group, —NHP 1
Or a salt of an amino group, P 1 represents an amino protecting group,
m represents an integer of 1 to 4; n represents an integer of 1 to 6;
2 represents an arylene group or an alkylene group, Z represents an alkylene group having 1 to 20 carbon atoms which may be interrupted by a hetero atom, x represents an integer of 1 to 4, and q represents 0 or 1. )
チン化フェニルジアジリン化合物。 【化５】 （式中、Ｒ4は、還元末端をもつ糖化合物の残基を表
し、Ｙ1は−ＮＨ−又は−ＣＯＮＨＳＯ2−Ｘ2−ＣＯ−
を表し、Ｘ2はアリーレン基又はアルキレン基を表し、
Ｙ2はビオチン又はビオチン誘導体残基を表し、ｍは１
〜４の整数を表し、ｎは１〜６の整数を表し、ｘは１〜
４の整数を表す。）5. A sugar-linked biotinylated phenyldiazirine compound represented by the following general formula (V). Embedded image (Wherein, R 4 represents a residue of a sugar compound having a reducing end, and Y 1 represents —NH— or —CONHSO 2 —X 2 —CO—
X 2 represents an arylene group or an alkylene group;
Y 2 represents biotin or a biotin derivative residue;
Represents an integer of 4 to 4, n represents an integer of 1 to 6, x represents 1 to
Represents an integer of 4. )
チン化フェニルジアジリン化合物。 【化６】 （式中、Ｒ4は、還元末端をもつ糖化合物の残基を表
し、ｍは１〜４の整数を表し、ｎは１〜６の整数を表
し、ｘは１〜４の整数を表す。）6. A sugar-linked biotinylated phenyldiazirine compound represented by the following general formula (VI). Embedded image (Wherein, R 4 represents a residue of a sugar compound having a reducing end, m represents an integer of 1 to 4, n represents an integer of 1 to 6, and x represents an integer of 1 to 4. )
オチン化フェニルジアジリン化合物。 【化７】 （式中、Ｒ4は、還元末端をもつ糖化合物の残基を表
し、Ｘ2はアリーレン基又はアルキレン基を表し、Ｚは
ヘテロ原子で中断されていてもよい炭素数１〜２０のア
ルキレン基を表し、ｍは１〜４の整数を表し、ｎは１〜
６の整数を表し、ｘは１〜４の整数を表し、ｑは０又は
１を表す。）7. A sugar-linked biotinylated phenyldiazirine compound represented by the following general formula (VII). Embedded image (Wherein, R 4 represents a residue of a sugar compound having a reducing end, X 2 represents an arylene group or an alkylene group, and Z represents an alkylene group having 1 to 20 carbon atoms which may be interrupted by a hetero atom. And m represents an integer of 1 to 4, and n represents 1 to
Represents an integer of 6, x represents an integer of 1 to 4, and q represents 0 or 1. )
ビオチン化フェニルジアジリン化合物を含む光反応性標
識試薬。但し、一般式(II)中、Ｒ3はアミノ基、又はア
ミノ基の塩を表す。8. A photoreactive labeling reagent comprising the biotinylated phenyldiazirine compound represented by the general formula (II) according to claim 2. However, in the general formula (II), R 3 represents an amino group or a salt of an amino group.
るビオチン化フェニルジアジリン化合物を含む光反応性
標識試薬。但し、一般式（III）中、Ｒ3はアミノ基、又
はアミノ基の塩を表す。9. A photoreactive labeling reagent comprising the biotinylated phenyldiazirine compound represented by the general formula (III) according to claim 3. However, in the general formula (III), R 3 represents an amino group or a salt of the amino group.
るビオチン化フェニルジアジリン化合物を含む光反応性
標識試薬。但し、一般式（IV）中、Ｒ3はアミノ基、又
はアミノ基の塩を表す。10. A photoreactive labeling reagent comprising the biotinylated phenyldiazirine compound represented by the general formula (IV) according to claim 4. However, in the general formula (IV), R 3 represents an amino group or a salt of the amino group.
る糖結合ビオチン化フェニルジアジリン化合物を含む光
親和性標識試薬。11. A photoaffinity labeling reagent comprising the sugar-linked biotinylated phenyldiazirine compound represented by the general formula (V) according to claim 5.
る糖結合ビオチン化フェニルジアジリン化合物を含む光
親和性標識試薬。12. A photoaffinity labeling reagent comprising the sugar-bound biotinylated phenyldiazirine compound represented by the general formula (VI) according to claim 6.
れる糖結合ビオチン化フェニルジアジリン化合物を含む
光親和性標識試薬。13. A photoaffinity labeling reagent comprising the sugar-bound biotinylated phenyldiazirine compound represented by the general formula (VII) according to claim 7.
化フェニルジアジリン化合物と還元末端を有する糖化合
物を反応させ、該還元末端を有する糖化合物の還元末端
のアルデヒド基と該ビオチン化フェニルジアジリン化合
物のアミノ基とのシッフ塩基を形成させることを含む上
記一般式（V）で表される糖結合ビオチン化フェニルジ
アジリン化合物の製造方法。但し、一般式（II）中、Ｒ
3は、アミノ基、又はアミノ基の塩を表す。14. A biotinylated phenyldiazirine compound represented by the above general formula (II) is reacted with a saccharide compound having a reducing terminal, and the aldehyde group at the reducing terminal of the saccharide compound having the reducing terminal is reacted with the biotinylated phenyl compound. A method for producing a sugar-bound biotinylated phenyldiazirine compound represented by the above general formula (V), comprising forming a Schiff base with an amino group of a diazirine compound. However, in the general formula (II), R
3 represents an amino group or a salt of an amino group.
が上記一般式(III)で表され、糖結合ビオチン化フェニ
ルジアジリン化合物が上記一般式(VI)で表される請求項
１４記載の製造方法。15. The method according to claim 14, wherein the biotinylated phenyldiazirine compound is represented by the general formula (III), and the sugar-bound biotinylated phenyldiazirine compound is represented by the general formula (VI).
が上記一般式(IV)で表され、糖結合ビオチン化フェニル
ジアジリン化合物が上記一般式(VII)で表される請求項
１４記載の製造方法。16. The method according to claim 14, wherein the biotinylated phenyldiazirine compound is represented by the general formula (IV), and the sugar-bound biotinylated phenyldiazirine compound is represented by the general formula (VII).
オチン化フェニルジアジリン化合物と糖受容体とを混合
して該フェニルジアジリン化合物の糖化合物残基と糖受
容体との特異的相互作用によって結合させるとともに、
該混合物に光を照射し、該糖受容体の、一般式（V）の
化合物に結合している糖化合物と相互作用をする部位
に、一般式（V）の化合物を光反応により結合させ、該
糖受容体を標識することからなる糖受容体の標識方法。17. A method of mixing a sugar-bound biotinylated phenyldiazirine compound represented by the above general formula (V) with a sugar acceptor to specifically react the sugar compound residue of the phenyldiazirine compound with the sugar acceptor. While being connected by interaction,
Irradiating the mixture with light to bind the compound of the general formula (V) to the site of the sugar receptor that interacts with the sugar compound bound to the compound of the general formula (V) by a photoreaction; A method for labeling a sugar receptor, comprising labeling the sugar receptor.
化合物が上記一般式(VI)で表されることからなる請求項
１７記載の糖受容体の標識方法。18. The method for labeling a sugar receptor according to claim 17, wherein the sugar-bound biotinylated phenyldiazirine compound is represented by the general formula (VI).
化合物が上記一般式(VII)で表されることからなる請求
項１７記載の糖受容体の標識方法。19. The method for labeling a sugar receptor according to claim 17, wherein the sugar-bound biotinylated phenyldiazirine compound is represented by the general formula (VII).
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005291956A (en) * | 2004-03-31 | 2005-10-20 | Hitachi High-Technologies Corp | How to remove sugar |
| JP2006176537A (en) * | 2003-10-16 | 2006-07-06 | Nard Inst Ltd | Biotin compound |
| JPWO2006019116A1 (en) * | 2004-08-17 | 2008-05-08 | 国立大学法人富山大学 | Photoreactive compound, photoreactive polyamine and method for producing polyamine sheet |
| US7804592B2 (en) | 2003-10-16 | 2010-09-28 | Nard Institute, Ltd. | Method for measuring a surface plasmon resonance and noble metal compound used for the same |
-
2000
- 2000-03-03 JP JP2000059547A patent/JP3746181B2/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006176537A (en) * | 2003-10-16 | 2006-07-06 | Nard Inst Ltd | Biotin compound |
| US7804592B2 (en) | 2003-10-16 | 2010-09-28 | Nard Institute, Ltd. | Method for measuring a surface plasmon resonance and noble metal compound used for the same |
| JP2005291956A (en) * | 2004-03-31 | 2005-10-20 | Hitachi High-Technologies Corp | How to remove sugar |
| JPWO2006019116A1 (en) * | 2004-08-17 | 2008-05-08 | 国立大学法人富山大学 | Photoreactive compound, photoreactive polyamine and method for producing polyamine sheet |
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| Publication number | Publication date |
|---|---|
| JP3746181B2 (en) | 2006-02-15 |
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