JP7479667B2 - Compound having a sulfonylaniline skeleton or a salt thereof, or organic fluorescent material having the same - Google Patents
Compound having a sulfonylaniline skeleton or a salt thereof, or organic fluorescent material having the same Download PDFInfo
- Publication number
- JP7479667B2 JP7479667B2 JP2020021477A JP2020021477A JP7479667B2 JP 7479667 B2 JP7479667 B2 JP 7479667B2 JP 2020021477 A JP2020021477 A JP 2020021477A JP 2020021477 A JP2020021477 A JP 2020021477A JP 7479667 B2 JP7479667 B2 JP 7479667B2
- Authority
- JP
- Japan
- Prior art keywords
- group
- sulfonylaniline
- halogen atom
- substituted
- bmes
- 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.)
- Active
Links
- QLRBNEZOQPLERN-UHFFFAOYSA-N (sulfonylamino)benzene Chemical group O=S(=O)=NC1=CC=CC=C1 QLRBNEZOQPLERN-UHFFFAOYSA-N 0.000 title claims description 48
- 150000001875 compounds Chemical class 0.000 title claims description 33
- 150000003839 salts Chemical class 0.000 title claims description 24
- 239000000463 material Substances 0.000 title claims description 11
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 37
- 125000005843 halogen group Chemical group 0.000 claims description 29
- 125000003277 amino group Chemical group 0.000 claims description 15
- 125000004076 pyridyl group Chemical group 0.000 claims description 14
- 125000000335 thiazolyl group Chemical group 0.000 claims description 14
- 125000001544 thienyl group Chemical group 0.000 claims description 14
- 125000004432 carbon atom Chemical group C* 0.000 claims description 13
- 125000000217 alkyl group Chemical group 0.000 claims description 9
- NAWXUBYGYWOOIX-SFHVURJKSA-N (2s)-2-[[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]amino]-4-methylidenepentanedioic acid Chemical compound C1=CC2=NC(N)=NC(N)=C2C=C1CCC1=CC=C(C(=O)N[C@@H](CC(=C)C(O)=O)C(O)=O)C=C1 NAWXUBYGYWOOIX-SFHVURJKSA-N 0.000 claims description 8
- 125000003545 alkoxy group Chemical group 0.000 claims description 8
- 125000002947 alkylene group Chemical group 0.000 claims description 8
- 125000004122 cyclic group Chemical group 0.000 claims description 8
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 8
- 125000001424 substituent group Chemical group 0.000 claims description 8
- 125000003709 fluoroalkyl group Chemical group 0.000 claims description 7
- 125000001624 naphthyl group Chemical group 0.000 claims description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 36
- 239000000975 dye Substances 0.000 description 32
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 18
- 239000007787 solid Substances 0.000 description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 12
- 239000007850 fluorescent dye Substances 0.000 description 12
- 238000010791 quenching Methods 0.000 description 12
- 230000000171 quenching effect Effects 0.000 description 12
- 238000003786 synthesis reaction Methods 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000002372 labelling Methods 0.000 description 9
- 235000017557 sodium bicarbonate Nutrition 0.000 description 9
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 9
- 238000005160 1H NMR spectroscopy Methods 0.000 description 8
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 8
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 230000005284 excitation Effects 0.000 description 8
- 239000012044 organic layer Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000002189 fluorescence spectrum Methods 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 230000007935 neutral effect Effects 0.000 description 7
- 238000010898 silica gel chromatography Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 239000010410 layer Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 5
- VEZXCJBBBCKRPI-UHFFFAOYSA-N beta-propiolactone Chemical compound O=C1CCO1 VEZXCJBBBCKRPI-UHFFFAOYSA-N 0.000 description 5
- 239000000460 chlorine Substances 0.000 description 5
- 238000001215 fluorescent labelling Methods 0.000 description 5
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 229960000380 propiolactone Drugs 0.000 description 5
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 5
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 4
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 4
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 4
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 4
- 229910052794 bromium Inorganic materials 0.000 description 4
- 229910052801 chlorine Inorganic materials 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 239000011737 fluorine Substances 0.000 description 4
- 229910052731 fluorine Inorganic materials 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 239000013067 intermediate product Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- UNILWMWFPHPYOR-KXEYIPSPSA-M 1-[6-[2-[3-[3-[3-[2-[2-[3-[[2-[2-[[(2r)-1-[[2-[[(2r)-1-[3-[2-[2-[3-[[2-(2-amino-2-oxoethoxy)acetyl]amino]propoxy]ethoxy]ethoxy]propylamino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-1-oxopropan-2-yl Chemical compound O=C1C(SCCC(=O)NCCCOCCOCCOCCCNC(=O)COCC(=O)N[C@@H](CSC[C@@H](COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC)C(=O)NCC(=O)N[C@H](CO)C(=O)NCCCOCCOCCOCCCNC(=O)COCC(N)=O)CC(=O)N1CCNC(=O)CCCCCN\1C2=CC=C(S([O-])(=O)=O)C=C2CC/1=C/C=C/C=C/C1=[N+](CC)C2=CC=C(S([O-])(=O)=O)C=C2C1 UNILWMWFPHPYOR-KXEYIPSPSA-M 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000000695 excitation spectrum Methods 0.000 description 3
- 230000005281 excited state Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 230000005283 ground state Effects 0.000 description 3
- 239000011630 iodine Substances 0.000 description 3
- 229910052740 iodine Inorganic materials 0.000 description 3
- 150000002596 lactones Chemical class 0.000 description 3
- 238000004020 luminiscence type Methods 0.000 description 3
- SWVMLNPDTIFDDY-FVGYRXGTSA-N methyl (2s)-2-amino-3-phenylpropanoate;hydrochloride Chemical compound Cl.COC(=O)[C@@H](N)CC1=CC=CC=C1 SWVMLNPDTIFDDY-FVGYRXGTSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000008213 purified water Substances 0.000 description 3
- 238000010992 reflux Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- -1 succinyl ester Chemical class 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 150000001345 alkine derivatives Chemical group 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 239000013065 commercial product Substances 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 125000005439 maleimidyl group Chemical class C1(C=CC(N1*)=O)=O 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000003077 quantum chemistry computational method Methods 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229910052724 xenon Inorganic materials 0.000 description 2
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 2
- PBKAXANOUZYEDU-UHFFFAOYSA-N 2,5-bis(methylsulfonyl)benzene-1,4-diamine Chemical compound CS(=O)(=O)C1=C(C=C(C(=C1)N)S(=O)(=O)C)N PBKAXANOUZYEDU-UHFFFAOYSA-N 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 238000004057 DFT-B3LYP calculation Methods 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 238000012650 click reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- OVTCUIZCVUGJHS-UHFFFAOYSA-N dipyrrin Chemical compound C=1C=CNC=1C=C1C=CC=N1 OVTCUIZCVUGJHS-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical group 0.000 description 1
- 230000020169 heat generation Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical class NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- KQEVIFKPZOGBMZ-UHFFFAOYSA-N methyl 3-bromopropanoate Chemical compound COC(=O)CCBr KQEVIFKPZOGBMZ-UHFFFAOYSA-N 0.000 description 1
- VSDUZFOSJDMAFZ-VIFPVBQESA-N methyl L-phenylalaninate Chemical compound COC(=O)[C@@H](N)CC1=CC=CC=C1 VSDUZFOSJDMAFZ-VIFPVBQESA-N 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 125000006353 oxyethylene group Chemical group 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000001443 photoexcitation Effects 0.000 description 1
- CHKVPAROMQMJNQ-UHFFFAOYSA-M potassium bisulfate Chemical compound [K+].OS([O-])(=O)=O CHKVPAROMQMJNQ-UHFFFAOYSA-M 0.000 description 1
- 229910000343 potassium bisulfate Inorganic materials 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical class O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Landscapes
- Pyrrole Compounds (AREA)
Description
本発明は、スルホニルアニリン骨格を有する有機蛍光材料に関する。 The present invention relates to an organic fluorescent material having a sulfonylaniline skeleton.
蛍光色素を用いて目的のタンパク質や分子を可視化し、生命現象の空間や時間を捉える蛍光イメージングは、生化学・生物学だけでなく医学の分野も含めたライフサイエンスにおける重要技術である。生体機能や疾病メカニズムの解明につながることから、予防・診断・治療といった医療技術への糸口となることが期待されている。特に近年、蛍光標識糖鎖・蛍光標識ペプチドは、蛍光プローブの開発や蛍光イメージングといったライフサイエンス研究を支える重要な基幹ツールとして顧客ニーズが多様化している。 Fluorescence imaging, which uses fluorescent dyes to visualize target proteins and molecules and capture the space and time of biological phenomena, is an important technology in life sciences, including not only biochemistry and biology but also medicine. As it leads to elucidation of biological functions and disease mechanisms, it is expected to provide clues for medical technologies such as prevention, diagnosis, and treatment. In particular, in recent years, customer needs have become more diverse for fluorescently labeled glycans and peptides, which serve as important core tools supporting life science research such as the development of fluorescent probes and fluorescent imaging.
しかしながら、ＧＦＰ（緑色蛍光タンパク質）に代表される蛍光タンパク質はもちろんのこと、しばしばフルオレセインやローダミンといった低分子系の汎用性蛍光色素であっても、分子サイズが大きく、複雑なターゲットの機能を阻害し、ターゲットへの導入が困難な場合がある。また、励起光と発光との差（ストークスシフト）が小さく、多数の発光シグナルを同時に可視化するマルチカラーイメージングに向かないという大きな課題がある。 However, fluorescent proteins such as GFP (green fluorescent protein) and even general-purpose low-molecular-weight fluorescent dyes such as fluorescein and rhodamine often have large molecular sizes that can inhibit the function of complex targets and make them difficult to introduce into targets. In addition, there is a major issue in that the difference between excitation light and emission light (Stokes shift) is small, making them unsuitable for multi-color imaging that visualizes multiple emission signals simultaneously.
ストークスシフトの大きな標識剤は、ストークスシフトの小さな多くの従来型蛍光標識剤に比べて、励起光の検出波長領域への重なりを抑えることができ、高感度化や高精度化にとっても大きな利点がある。そのため、小さな分子構造と大きなストークスシフトを持つ蛍光色素が必要である。 Compared to many conventional fluorescent labeling agents with small Stokes shifts, labeling agents with large Stokes shifts can reduce overlap with the detection wavelength range of excitation light, and have the great advantage of enabling high sensitivity and high precision. For this reason, fluorescent dyes with small molecular structures and large Stokes shifts are required.
優れた蛍光特性の発現には拡張されたπ共役系が有利なことから、芳香族骨格を拡張した分子設計が広く用いられてきた。しかし、そのような分子設計による材料の多くは高い平面性と剛直な骨格とを持つことから、分子サイズが大きく、安定性が低く、水溶性が低く、ストークスシフトが小さく、さらには強い分子間相互作用が濃度消光を引き起こすという問題があった。 Because extended π-conjugated systems are advantageous for expressing excellent fluorescent properties, molecular designs with extended aromatic skeletons have been widely used. However, many of the materials produced by such molecular designs have high planarity and rigid skeletons, leading to problems such as large molecular size, low stability, low water solubility, small Stokes shift, and strong intermolecular interactions that cause concentration quenching.
本発明者らは、１つのベンゼン環に複数のアミノ基とスルホニル基とを有するスルホニルアニリン系色素が、（１）分子サイズが極めて小さい、（２）Ｐｕｓｈ－Ｐｕｌｌ効果により吸収波長が長波長化する、（３）スルホニル基が持つ折れ曲がり構造により高い溶解性を示すため、分子が会合し、固体や凝集状態で発光する固体蛍光性を示す、（４）アミノ基‐スルホニル基間の水素結合がアミノ基の自由回転を抑制し、蛍光量子効率や安定性が向上する等、従来の有機蛍光材料よりも優れた特性を示すことを報告した（非特許文献１、特許文献１）。
The present inventors have reported that sulfonylaniline dyes, which have multiple amino groups and sulfonyl groups on one benzene ring, have superior properties to conventional organic fluorescent materials, such as (1) extremely small molecular size, (2) a longer absorption wavelength due to the push-pull effect, (3) high solubility due to the bent structure of the sulfonyl group, which allows the molecules to associate and emit light in a solid or aggregated state, and (4) hydrogen bonds between the amino group and sulfonyl group suppress the free rotation of the amino group, improving the fluorescence quantum efficiency and stability (Non-Patent
本発明では、前記スルホニルアニリン系色素をさらに改良するとともに、これらに生体分子結合基を導入することにより、蛍光標識薬に好適な材料を開発することを目的とする。 The present invention aims to further improve the sulfonylaniline dyes and introduce biomolecule binding groups into them to develop materials suitable for fluorescent labeling drugs.
本発明は以下の事項からなる。
本発明は、下記一般式（１）～（５）で表されるスルホニルアニリン骨格を有する化合物又はその塩にある。
The present invention relates to compounds having a sulfonylaniline skeleton represented by the following general formulas (1) to (5) or salts thereof.
ここで、一般式（１）～（５）中、Ｒ1及びＲ2は、それぞれ独立に、アルキル基、フルオロアルキル基、フェニル基、ハロゲン原子で置換されたフェニル基、アミノ基で置換されたフェニル基、アルコキシ基で置換されたフェニル基、ナフチル基、チエニル基、ハロゲン原子で置換されたチエニル基、チアゾリル基、ハロゲン原子で置換されたチアゾリル基、ピリジル基、又は、ハロゲン原子で置換されたピリジル基であり、Ｓ１は下記一般式（１０）で表される置換基である。 In the general formulas (1) to (5), R 1 and R 2 each independently represent an alkyl group, a fluoroalkyl group, a phenyl group, a phenyl group substituted with a halogen atom, a phenyl group substituted with an amino group, a phenyl group substituted with an alkoxy group, a naphthyl group, a thienyl group, a thienyl group substituted with a halogen atom, a thiazolyl group, a thiazolyl group substituted with a halogen atom, a pyridyl group, or a pyridyl group substituted with a halogen atom, and S1 is a substituent represented by the following general formula (10):
一般式（１０）中、Ｒは鎖中に一以上の酸素原子を含んでもよい、炭素原子数１～１８の直鎖状、分岐状又は環状のアルキレン基である。 In general formula (10), R is a linear, branched, or cyclic alkylene group having 1 to 18 carbon atoms, which may contain one or more oxygen atoms in the chain.
本発明は、下記一般式（６）～（９）で表されるスルホニルアニリン骨格を有する化合物又はその塩にある。
ここで、一般式（６）～（９）中、Ｒ1及びＲ2は、それぞれ独立に、アルキル基、フルオロアルキル基、フェニル基、ハロゲン原子で置換されたフェニル基、アミノ基で置換されたフェニル基、アルコキシ基で置換されたフェニル基、ナフチル基、チエニル基、ハロゲン原子で置換されたチエニル基、チアゾリル基、ハロゲン原子で置換されたチアゾリル基、ピリジル基、又は、ハロゲン原子で置換されたピリジル基であり、Ｓ２は下記一般式（１１）で表される置換基である。 In the general formulas (6) to (9), R1 and R2 each independently represent an alkyl group, a fluoroalkyl group, a phenyl group, a phenyl group substituted with a halogen atom, a phenyl group substituted with an amino group, a phenyl group substituted with an alkoxy group, a naphthyl group, a thienyl group, a thienyl group substituted with a halogen atom, a thiazolyl group, a thiazolyl group substituted with a halogen atom, a pyridyl group, or a pyridyl group substituted with a halogen atom, and S2 is a substituent represented by the following general formula (11):
本発明の有機蛍光材料は、前記スルホニルアニリン骨格を有する化合物又はその塩からなることを特徴とする。 The organic fluorescent material of the present invention is characterized by being composed of a compound having the above-mentioned sulfonylaniline skeleton or a salt thereof.
本発明のスルホニルアニリン骨格を有する化合物又はその塩は、最も小さな汎用性色素のＢＯＤＩＰＹ（登録商標）と同等の分子サイズを持ち、また、大きなストークスシフト、高い安定性、高い量子効率、及び水溶性を持つ。また、前記スルホニルアニリン骨格を有する化合物又はその塩からなる有機蛍光材料は、従来の蛍光試薬とは大きく異なり、濃度消光を示すことがなく、ｐＨや極性といった周囲の環境に依存しない安定な発光特性を示す。 The compound having a sulfonylaniline skeleton or its salt of the present invention has a molecular size equivalent to that of BODIPY (registered trademark), the smallest versatile dye, and also has a large Stokes shift, high stability, high quantum efficiency, and water solubility. Furthermore, the organic fluorescent material made of the compound having a sulfonylaniline skeleton or its salt is significantly different from conventional fluorescent reagents in that it does not exhibit concentration quenching and exhibits stable luminescence characteristics that are not dependent on the surrounding environment, such as pH or polarity.
以下、本発明について、詳細に説明する。
［スルホニルアニリン骨格を有する化合物又はその塩］
本発明は、一般式（１）～（５）で表されるスルホニルアニリン骨格を有する化合物又はその塩にある。
[Compound having a sulfonylaniline skeleton or a salt thereof]
The present invention resides in compounds having a sulfonylaniline skeleton represented by general formulas (1) to (5) or salts thereof.
一般式（１）～（５）中、Ｒ1及びＲ2は、それぞれ独立に、アルキル基、フルオロアルキル基、フェニル基、ハロゲン原子で置換されたフェニル基、アミノ基で置換されたフェニル基、アルコキシ基で置換されたフェニル基、ナフチル基、チエニル基、ハロゲン原子で置換されたチエニル基、チアゾリル基、ハロゲン原子で置換されたチアゾリル基、ピリジル基、又は、ハロゲン原子で置換されたピリジル基であり、Ｓ１は下記一般式（１０）で表される置換基である。 In general formulas (1) to (5), R1 and R2 each independently represent an alkyl group, a fluoroalkyl group, a phenyl group, a phenyl group substituted with a halogen atom, a phenyl group substituted with an amino group, a phenyl group substituted with an alkoxy group, a naphthyl group, a thienyl group, a thienyl group substituted with a halogen atom, a thiazolyl group, a thiazolyl group substituted with a halogen atom, a pyridyl group, or a pyridyl group substituted with a halogen atom, and S1 is a substituent represented by the following general formula (10):
アルキル基及びフルオロアルキル基を構成するアルキル基は、具体的には、炭素原子数１～１８、好ましくは炭素原子数１～１２、より好ましくは炭素原子数１～６の直鎖又は分岐を有していてもよいアルキル基である。
ハロゲン原子で置換されたフェニル基とは、フェニル基の水素原子の一部又は全部がハロゲン原子（フッ素、塩素、臭素、ヨウ素）で置換された基をいう。
アミノ基で置換されたフェニル基とは、フェニル基の水素原子の一部又は全部が、１級アミノ基又は２級アミノ基で置換された基をいう。
アルコキシ基で置換されたフェニル基を構成するアルコキシ基は、具体的には、炭素原子数１～１８、好ましくは炭素原子数１～１２、より好ましくは炭素原子数１～６の直鎖又は分岐を有していてもよいアルコキシ基である。
ハロゲン原子で置換されたチエニル基とは、チエニル基の水素原子の一部又は全部がハロゲン（フッ素、塩素、臭素又はヨウ素）原子で置換された基をいう。
ハロゲン原子で置換されたチアゾリル基は、チアゾリル基の水素原子の一部又は全部がハロゲン原子（フッ素、塩素、臭素、ヨウ素）で置換された基をいう。
ハロゲン原子で置換されたピリジル基は、ピリジル基の水素原子の一部又は全部がハロゲン原子（フッ素、塩素、臭素、ヨウ素）で置換された基をいう。
The alkyl group and the alkyl group constituting the fluoroalkyl group are specifically alkyl groups having 1 to 18 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms, which may be linear or branched.
The term "phenyl group substituted with a halogen atom" refers to a phenyl group in which some or all of the hydrogen atoms have been substituted with a halogen atom (fluorine, chlorine, bromine, iodine).
The phenyl group substituted with an amino group refers to a phenyl group in which some or all of the hydrogen atoms have been substituted with a primary amino group or a secondary amino group.
The alkoxy group constituting the alkoxy-substituted phenyl group is specifically an alkoxy group having 1 to 18 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms, which may be linear or branched.
The term "halogen-substituted thienyl group" refers to a thienyl group in which some or all of the hydrogen atoms have been substituted with halogen (fluorine, chlorine, bromine or iodine) atoms.
The halogen-substituted thiazolyl group refers to a thiazolyl group in which some or all of the hydrogen atoms have been substituted with halogen atoms (fluorine, chlorine, bromine, iodine).
The pyridyl group substituted with a halogen atom refers to a pyridyl group in which some or all of the hydrogen atoms have been substituted with a halogen atom (fluorine, chlorine, bromine, iodine).
一般式（１０）中、Ｒは鎖中に一以上の酸素原子を含んでもよい、炭素原子数１～１８の直鎖状、分岐状又は環状のアルキレン基である。鎖中に一以上の酸素原子を含むアルキレン基の例としては、オキシエチレン基（－ＯＣＨ2ＣＨ2－又は－ＣＨ2ＣＨ2Ｏ－）が挙げられる。
一般式（１０）で表される置換基は、蛍光標識のための反応性基である。反応性基には、下記構造式に示すように、スクシミジルエステル体、マレイミド体、イソチオシアネート体、テトラフルオロフェニル（ＴＦＰ）エステル体、及びヨードアセトアミド体のほか、アルキン部位を持つ基であって、生体分子に組み込まれたアジド基と、アルキンとを結合させ、炭素－ヘテロ原子結合を形成するクリック反応を利用した反応性基が挙げられる。
これらのうち、化合物の安定性と操作の簡便性の点から、スクシミジルエステル体及びマレイミド体が好ましい。
In general formula (10), R is a linear, branched or cyclic alkylene group having 1 to 18 carbon atoms which may contain one or more oxygen atoms in the chain. Examples of alkylene groups containing one or more oxygen atoms in the chain include oxyethylene groups ( -OCH2CH2- or -CH2CH2O- ).
The substituent represented by the general formula (10) is a reactive group for fluorescent labeling. Examples of the reactive group include succinidyl esters, maleimides, isothiocyanates, tetrafluorophenyl (TFP) esters, and iodoacetamides, as well as groups having an alkyne moiety, which utilize a click reaction to bond an azide group incorporated in a biomolecule with an alkyne to form a carbon-heteroatom bond, as shown in the following structural formula:
Among these, succinimide esters and maleimides are preferred from the viewpoints of compound stability and ease of handling.
また、一般式（６）～（９）で表されるスルホニルアニリン骨格を有する化合物又はその塩も、本発明の実施形態に含まれる。
一般式（６）～（９）中、Ｒ1及びＲ2は、それぞれ独立に、アルキル基、フルオロアルキル基、フェニル基、ハロゲン原子で置換されたフェニル基、アミノ基で置換されたフェニル基、アルコキシ基で置換されたフェニル基、ナフチル基、チエニル基、ハロゲン原子で置換されたチエニル基、チアゾリル基、ハロゲン原子で置換されたチアゾリル基、ピリジル基、又は、ハロゲン原子で置換されたピリジル基であり、Ｓ２は下記一般式（１１）で表される置換基である。
一般式（１１）中、Ｒは鎖中に一以上の酸素原子を含んでもよい、炭素原子数１～１８の直鎖状、分岐状又は環状のアルキレン基であり、好ましくは－ＣＨ2ＣＨ2－である。
In general formulas (6) to (9), R1 and R2 each independently represent an alkyl group, a fluoroalkyl group, a phenyl group, a phenyl group substituted with a halogen atom, a phenyl group substituted with an amino group, a phenyl group substituted with an alkoxy group, a naphthyl group, a thienyl group, a thienyl group substituted with a halogen atom, a thiazolyl group, a thiazolyl group substituted with a halogen atom, a pyridyl group, or a pyridyl group substituted with a halogen atom, and S2 is a substituent represented by the following general formula (11):
In formula (11), R is a straight-chain, branched or cyclic alkylene group having 1 to 18 carbon atoms which may contain one or more oxygen atoms in the chain, and is preferably --CH 2 CH 2 --.
なお、一般式（６）～（９）で表されるスルホニルアニリン骨格を有する化合物は、前記一般式（１）及び（２）で表されるスルホニルアニリン骨格を有する化合物のうち、反応性基がスクシミジルエステル体であるものを合成する際の中間生成物である。 The compounds having a sulfonylaniline skeleton represented by the general formulas (6) to (9) are intermediate products in the synthesis of compounds having a sulfonylaniline skeleton represented by the general formulas (1) and (2) in which the reactive group is a succinyl ester.
一般式（１）～（９）で表されるスルホニルアニリン骨格を有する化合物は、そのイオン性親水性基（例えば、アミノ基）が塩酸塩又は硫酸塩などの塩を形成していてもよい。 In the compounds having a sulfonylaniline skeleton represented by the general formulas (1) to (9), the ionic hydrophilic group (e.g., amino group) may form a salt such as a hydrochloride or sulfate.
一般式（１）～（９）で表されるスルホニルアニリン骨格を有する化合物又はその塩は、下記構造式を有する化合物（２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒ、２，６－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒ、２，５－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨ、及び２，６－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨ）であることが特に好ましい。
本発明のスルホニルアニリン骨格を有する化合物又はその塩は、以下に示すように、ラクトンの開環反応を用いて、カルボン酸を有する中間生成物を経由することにより合成することができる。これまで、ラクトンの少なくとも一部が、スルホニルアニリン骨格を有する化合物にある２箇所のアミノ基と反応するために、副生成物が生成し、目的物が高い収率で得られなかった。本発明では、反応温度及び濃度を最適化するとともに、縮合剤としてジシクロヘキシルカルボジイミド（ＤＣＣ）を選択することで、４位のアミノ基に高選択的にラクトンが反応することを見出し、目的物の収率の改善に成功した。 The compound having a sulfonylaniline skeleton or a salt thereof of the present invention can be synthesized by using a ring-opening reaction of lactone via an intermediate product having a carboxylic acid, as shown below. Until now, at least a part of the lactone reacts with two amino groups in the compound having a sulfonylaniline skeleton, resulting in the generation of by-products and making it difficult to obtain the target product in high yield. In the present invention, by optimizing the reaction temperature and concentration and selecting dicyclohexylcarbodiimide (DCC) as a condensing agent, it has been found that the lactone reacts with the amino group at the 4th position with high selectivity, and the yield of the target product has been successfully improved.
一例として、２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒの合成方法を示す。
２，５－ＭＢｅＳ－ｐ－Ａにβ－プロピオラクトンを添加し、アセトニトリル中で４日間、加熱還流して、中間生成物である２，５－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨを収率４０％で得る。
次いで、２，５－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨに、Ｎ－ヒドロキシスクシンイミド（ＮＨＳ）及びＤＣＣを添加し、ジクロロメタン中で室温下に２０時間、攪拌することにより、２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒを収率８０％で得る。
ただし、本発明のスルホニルアニリン骨格を有する化合物又はその塩は、前記した方法に限られず、種々の公知の方法で製造することができる。
β-propiolactone is added to 2,5-MBeS-p-A and the mixture is heated to reflux in acetonitrile for 4 days to obtain the intermediate product 2,5-BMeS-p-A-COOH in a 40% yield.
Next, N-hydroxysuccinimide (NHS) and DCC are added to 2,5-BMeS-p-A-COOH, and the mixture is stirred in dichloromethane at room temperature for 20 hours to obtain 2,5-BMeS-p-A-NHS-ester in an 80% yield.
However, the compound having a sulfonylaniline skeleton or a salt thereof of the present invention can be produced by various known methods without being limited to the above-mentioned methods.
本発明の有機蛍光材料は、一般式（１）～（９）で表されるスルホニルアニリン骨格を有する化合物又はその塩からなる。
前記スルホニルアニリン骨格を有する化合物又はその塩のクロロホルム溶液の励起／蛍光スペクトルを測定結果から、大きなストークスシフトを示すことがわかる。発光のエネルギーは吸収のエネルギーよりも低く、発光スペクトルは吸収スペクトルより長波長になる。ストークスシフトはこの吸収及び波長スペクトルの極大波長の差をいう。前記スルホニルアニリン骨格を有する化合物又はその塩において、大きなストークスシフトは光励起による基底状態の構造と励起状態の構造が大きく異なることに起因して起こると考えられる。
本発明のスルホニルアニリン骨格を有する化合物又はその塩は、大きなストークスシフトを示すため、複数の観測対象分子を同時にモニタリングできる蛍光プローブ等への応用が期待できる。
The organic fluorescent material of the present invention comprises a compound having a sulfonylaniline skeleton represented by any one of the general formulas (1) to (9) or a salt thereof.
The measurement results of the excitation/fluorescence spectrum of a chloroform solution of the compound having a sulfonylaniline skeleton or a salt thereof show that it exhibits a large Stokes shift. The emission energy is lower than the absorption energy, and the emission spectrum has a longer wavelength than the absorption spectrum. The Stokes shift refers to the difference in the maximum wavelength of this absorption and wavelength spectrum. In the compound having a sulfonylaniline skeleton or a salt thereof, the large Stokes shift is considered to occur due to the large difference between the structure of the ground state and the structure of the excited state due to photoexcitation.
The compound having a sulfonylaniline skeleton or a salt thereof of the present invention exhibits a large Stokes shift and is therefore expected to be applicable to fluorescent probes and the like capable of simultaneously monitoring a plurality of target molecules.
前記スルホニルアニリン骨格を有する化合物又はその塩について、量子化学計算によって最適化構造を求めると、分子サイズは２５０～３００Å3（例えば、２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒでは２７９Å3であり、２，６－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒでは２８８Å3である。）、最も分子サイズが小さいＢＯＤＩＰＹ（登録商標、ボロンジピロメテン）と同等の分子サイズである。したがって、本発明のスルホニルアニリン骨格を有する化合物又はその塩は、複雑構造を持つ糖鎖やペプチド、あるいはタンパク質などの生体高分子を標識する際に立体障害が小さいこと、また、ターゲット分子の機能を阻害しないという利点がある。 When the optimized structure of the compound having a sulfonylaniline skeleton or a salt thereof is determined by quantum chemical calculation, the molecular size is 250 to 300 Å 3 (for example, 279 Å 3 for 2,5-BMeS-p-A-NHS-ester and 288 Å 3 for 2,6-BMeS-p-A-NHS-ester), which is equivalent to that of BODIPY (registered trademark, boron dipyrromethene), which has the smallest molecular size. Therefore, the compound having a sulfonylaniline skeleton or a salt thereof of the present invention has the advantages of small steric hindrance when labeling biopolymers such as sugar chains, peptides, or proteins having complex structures, and of not inhibiting the function of the target molecule.
前記スルホニルアニリン骨格を有する化合物は、ＮＨＳエステル結合を有するため、極性が高く、高い水溶性を持つ。また、イオン性官能基を持たないため、環境（ｐＨ、極性又は紫外線など）によって蛍光強度や波長が変化することがなく、安定な発光特性を示す。 The compound having the sulfonylaniline skeleton has high polarity and high water solubility due to the presence of an NHS ester bond. In addition, since it does not have an ionic functional group, the fluorescence intensity or wavelength does not change depending on the environment (pH, polarity, ultraviolet light, etc.), and it exhibits stable luminescence characteristics.
さらに、従来の汎用色素のほとんどは、その構造中に剛直な拡張π共役系を持つため、色素が集積してある濃度になると発光強度が低下する濃度消光の問題があった。一方、本発明のスルホニルアニリン骨格を有する化合物又はその塩は、スルホニル基の折れ曲がり構造に起因して、全く濃度消光を示さない。 Furthermore, most conventional general-purpose dyes have a rigid extended π-conjugated system in their structure, which causes a problem of concentration quenching, where the emission intensity decreases when the dye accumulates to a certain concentration. On the other hand, the compound having a sulfonylaniline skeleton or a salt thereof of the present invention does not exhibit concentration quenching at all due to the bent structure of the sulfonyl group.
前記スルホニルアニリン骨格を有する化合物又はその塩は、その構造中で、生体分子との結合基となるＮＨＳエステルを形成している。ＮＨＳエステルは有機カルボン酸の活性エステルであり、生体分子のアミノ基と速やかにかつ選択的に反応して安定的なアミド結合を形成する。そのため、糖鎖やペプチド類の標識に広く用いられる。例えば、２，６－ＢＭｅＳ－ｐ－Ａ（青色）又は２，５－ＢＭｅＳ－ｐ－Ａ（緑色）にＮＨＳを導入した２，６－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒ及び２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒは、青色及び緑色の蛍光標識試薬となる。 The compound or salt thereof having the sulfonylaniline skeleton forms an NHS ester in its structure, which serves as a binding group for biomolecules. NHS esters are active esters of organic carboxylic acids, and react quickly and selectively with amino groups in biomolecules to form stable amide bonds. For this reason, they are widely used to label glycans and peptides. For example, 2,6-BMeS-p-A-NHS-ester and 2,5-BMeS-p-A-NHS-ester, which are obtained by introducing NHS into 2,6-BMeS-p-A (blue) or 2,5-BMeS-p-A (green), respectively, become blue and green fluorescent labeling reagents.
以下、本発明を実施例に基づいてさらに具体的に説明するが、本発明は下記実施例により制限されるものではない。
［スルホニルアニリン骨格を有する化合物の合成］
The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.
[Synthesis of compounds having a sulfonylaniline skeleton]
［実施例１］
〔２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒの合成〕
（ｉ）２，５－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨの合成
[Synthesis of 2,5-BMeS-p-A-NHS-ester]
(i) Synthesis of 2,5-BMeS-p-A-COOH
１００ｍＬ三口フラスコと滴下ロートを窒素置換し、滴下ロートに乾燥アセトニトリルを１０ｍＬとβ－プロピオラクトン１．３ｍＬ（１９．７ｍｍｏｌ）を添加、三口フラスコに乾燥アセトニトリルを９０ｍＬとＢＭｅＳ－ｐ－Ａ５１０．３ｍｇ（１．９３ｍｍｏｌ）を加えて還流するまで攪拌した。還流後、撹拌速度を素早くし、β－プロピオラクトンをゆっくりと滴下した。
４日後、アセトニトリルを濃縮して黄色固体と茶色油状物を１．７９ｇ得た。そこに、２Ｍの水酸化ナトリウム水溶液を１００ｍＬ加え、３５℃で２４時間撹拌した。それを一度吸引ろ過し、ろ液を酢酸エチルで分液した。水層を６Ｍ塩酸でｐＨ４付近になるように調整し、酢酸エチル１００ｍＬで３回抽出した。得られた有機層を飽和食塩水８０ｍＬで洗浄、濃縮を行い、黄色固体を３７９．１ｍｇ得た。最後に、中性シリカゲルカラムクロマトグラフィー（ジクロロメタン：メタノール＝１０：１）で精製し、黄色固体を２５９．７ｍｇ（０．７７２ｍｍｏｌ）得た（収率４０％）。
1ＨＮＭＲ及び13ＣＮＭＲの測定結果を示す。
1Ｈ ＮＭＲ（ＤＭＳＯ－ｄ6，５００ＭＨｚ）：δ＝１２．３２（ｂｒ ｓ，１Ｈ －ＯＨ），７．４２（ｓ，１Ｈ，Ａｒ－Ｈ），７．０６（ｓ，１Ｈ，Ａｒ－Ｈ），５．６４（ｓ，２Ｈ，－ＮＨ
2），５．４９（ｔ，Ｊ＝５．６Ｈｚ，１Ｈ，－ＮＨ），３．４８（ｄｔ，Ｊ＝６．１Ｈｚ，２Ｈ，－ＣＨ
2），３．１８（ｓ，３Ｈ，－ＣＨ
3），３．１７（ｓ，３Ｈ，－ＣＨ
3），２．５８（ｔ，Ｊ＝６．５Ｈｚ，２Ｈ，－ＣＨ
2）
13Ｃ ＮＭＲ（ＤＭＳＯ－ｄ6，１２５ＭＨｚ）：δ＝１７３．１１，１３７．５８，１３６．１２，１２９．６４，１２６．９０，１１８．６８，１１２．１２，４１．２２，４１．２０，３９．０６，３３．２３
The 100 mL three-neck flask and the dropping funnel were replaced with nitrogen, 10 mL of dry acetonitrile and 1.3 mL (19.7 mmol) of β-propiolactone were added to the dropping funnel, and 90 mL of dry acetonitrile and 510.3 mg (1.93 mmol) of BMeS-p-A were added to the three-neck flask and stirred until refluxed. After refluxing, the stirring speed was increased to high speed, and β-propiolactone was slowly added dropwise.
After 4 days, acetonitrile was concentrated to obtain 1.79 g of a yellow solid and a brown oil. 100 mL of 2 M aqueous sodium hydroxide was added thereto and stirred at 35 ° C. for 24 hours. It was filtered once by suction, and the filtrate was separated with ethyl acetate. The aqueous layer was adjusted to about pH 4 with 6 M hydrochloric acid, and extracted three times with 100 mL of ethyl acetate. The obtained organic layer was washed with 80 mL of saturated saline and concentrated to obtain 379.1 mg of a yellow solid. Finally, it was purified by neutral silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain 259.7 mg (0.772 mmol) of a yellow solid (yield 40%).
The results of 1 H NMR and 13 C NMR measurements are shown below.
1H NMR (DMSO- d6 , 500MHz): δ = 12.32 (br s, 1H -OH ), 7.42 (s, 1H, Ar- H ), 7.06 (s, 1H, Ar - H ), 5.64 (s, 2H, -NH2 ), 5.49 (t, J = 5.6Hz, 1H, -NH ), 3.48 (dt, J = 6.1Hz, 2H, -CH2 ), 3.18 (s, 3H, -CH3 ), 3.17 (s, 3H , -CH3 ) , 2.58 (t, J = 6.5Hz, 2H, -CH2 ) .
13C NMR (DMSO- d6 , 125MHz): δ = 173.11, 137.58, 136.12, 129.64, 126.90, 118.68, 112.12, 41.22, 41.20, 39.06, 33.23
（ii）２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒの合成
２０時間後、０℃に冷却し吸引ろ過を行った。ろ液のジクロロメタンの一部を留去し吸引ろ過する作業を２回行い、ジクロロメタンを完全に留去することで黄色固体を１３３．２ｍｇ得た。得られた固体を中性シリカゲルカラムクロマトグラフィー（ジクロロメタン：メタノール＝１０：１）で精製し、濃縮、エタノール洗浄することで黄色固体１０７．５ｍｇ（０．２４８ｍｍｏｌ）を得た（収率８０％）。
1ＨＮＭＲ及び13ＣＮＭＲの測定結果を示す。
1Ｈ ＮＭＲ（ＤＭＳＯ－ｄ6，５００ＭＨｚ）：δ＝７．４４（ｓ，１Ｈ，Ａｒ－Ｈ），７．０８（ｓ，１Ｈ，Ａｒ－Ｈ），５.６９（ｓ，２Ｈ，－ＮＨ
2），５．３６（ｔ，Ｊ＝５．９Ｈｚ，１Ｈ，－ＮＨ），３．４８（ｄｔ，Ｊ＝６．２Ｈｚ，２Ｈ，－ＣＨ
2），３．１９（ｓ，３Ｈ，－ＣＨ
3），３．１６（ｓ，３Ｈ，－ＣＨ
3），３．０７（ｔ，Ｊ＝６．３Ｈｚ，２Ｈ，－ＣＨ
2），２．８１（ｓ，４Ｈ，－ＣＨ
2）
13Ｃ ＮＭＲ（ＤＭＳＯ－ｄ6，１２５ＭＨｚ）：δ＝１７０．００，１６７．８０，１３８．０３，１３５．５５，１３０．１３，１２６．６６，１１８．７７，１１２．３０，４１．２７，４１．２５，３８．７１，３０．３７，２５．３６
(ii) Synthesis of 2,5-BMeS-p-A-NHS-ester
After 20 hours, the mixture was cooled to 0°C and suction filtered. The dichloromethane in the filtrate was partially removed by distillation and suction filtered twice, and 133.2 mg of a yellow solid was obtained by completely removing the dichloromethane. The obtained solid was purified by neutral silica gel column chromatography (dichloromethane:methanol=10:1), concentrated, and washed with ethanol to obtain 107.5 mg (0.248 mmol) of a yellow solid (yield 80%).
The results of 1 H NMR and 13 C NMR measurements are shown below.
1H NMR (DMSO- d6 , 500MHz): δ = 7.44 (s, 1H, Ar- H ), 7.08 (s, 1H, Ar- H ), 5.69 (s, 2H, -NH2 ), 5.36 (t, J = 5.9Hz, 1H, -NH ) , 3.48 (dt, J = 6.2Hz, 2H, -CH2 ), 3.19 (s, 3H , -CH3 ) , 3.16 (s, 3H, -CH3 ), 3.07 (t, J = 6.3Hz, 2H, -CH2 ) , 2.81 (s, 4H, -CH2 ) .
13C NMR (DMSO- d6 , 125MHz): δ = 170.00, 167.80, 138.03, 135.55, 130.13, 126.66, 118.77, 112.30, 41.27, 41.25, 38.71, 30.37, 25.36
〔標識試薬の作製〕
標識試薬としての機能を確認するため、以下の構造式で表される２，５－ＢＭｅＳ－ｐ－Ａ－Ｐｈｅ－ＯＭｅを作製した。
In order to confirm its function as a labeling reagent, 2,5-BMeS-p-A-Phe-OMe, represented by the following structural formula, was prepared.
炭酸水素ナトリウム８４０．１ｍｇ（０．１ｍｏｌ）を量り取り、純水１００ｍＬに溶解させ、ｐＨ８．３の０．１Ｍ炭酸水素ナトリウム緩衝液を調製した。
試験管１にＰｈｅ－ＯＭｅ・ＨＣｌ１０．２ｍｇ（４７．１μｍｏｌ）を量り取り、１ｍＬの０．１Ｍ炭酸水素ナトリウム緩衝液を加え溶解させた。
試験管２に２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒ１０．３ｍｇ（２３．７μｍｏｌ）を量り取り、１ｍＬの乾燥ＤＭＳＯを加え溶解させた。
試験管１中の炭酸水素ナトリウム水溶液を撹拌しながら、ガラスシリンジを用いて、試験管２のＤＭＳＯ溶液を試験管１中に１滴ずつゆっくりと滴下した。
撹拌しながら、１時間反応させた。
ジクロロメタンを用いて抽出作業を行った。反応は定量的に進行し、黄色油状物を収率１００％で得た。
1ＨＮＭＲの測定結果を示す。
1Ｈ ＮＭＲ（５００ＭＨｚ，ＤＭＳＯ－ｄ6，２５℃）：δ＝８．４１－８．４３（ｄ，Ｈ，－ＮＨ），７．４１（ｓ，１Ｈ，ＡｒＨ），７．１５－７．２８（ｍ，５Ｈ，ＡｒＨ），７．０２（ｓ，１Ｈ，ＡｒＨ），５．６２（ｓ，２Ｈ，ＮＨ
２），５．４２（ｔ，１Ｈ，ＮＨ），４．４５－４．５１（ｍ，１Ｈ，－ＣＨ），３．５８（ｓ，３Ｈ，ＣＨ
3），３．１８（ｓ，３Ｈ，ＣＨ
3），３．０９（ｓ，３Ｈ，ＣＨ
3），２．７５－３．０３（ｍ，４Ｈ，ＣＨ
2），１．４９（ｔ，２Ｈ，ＣＨ
2）
840.1 mg (0.1 mol) of sodium hydrogen carbonate was weighed out and dissolved in 100 mL of pure water to prepare a 0.1 M sodium hydrogen carbonate buffer solution with a pH of 8.3.
10.2 mg (47.1 μmol) of Phe-OMe·HCl was weighed out into
10.3 mg (23.7 μmol) of 2,5-BMeS-p-A-NHS-ester was weighed out into
While stirring the aqueous sodium hydrogen carbonate solution in
The reaction was carried out for 1 hour with stirring.
The reaction was quantitatively carried out and a yellow oil was obtained in 100% yield.
The results of 1 HNMR measurements are shown below.
1H NMR (500MHz, DMSO- d6 , 25°C): δ = 8.41-8.43 (d, H, -NH ), 7.41 (s, 1H, ArH ), 7.15-7.28 (m, 5H, ArH ), 7.02 (s, 1H, ArH ), 5.62 (s, 2H, NH2 ), 5.42 (t, 1H, NH ), 4.45-4.51 (m, 1H , -CH ) , 3.58 (s, 3H, CH3 ) , 3.18 (s, 3H, CH3 ) , 3.09 (s, 3H, CH3 ), 2.75-3.03 (m, 4H, CH2 ), 1.49 (t, 2H, CH H2 )
［実施例２］
［２，６－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒの合成］
（ｉ）化合物１３の合成
[Synthesis of 2,6-BMeS-p-A-NHS-ester]
(i) Synthesis of Compound 13
窒素置換した１０ｍＬナシフラスコに２，６－ＢＭｅＳ－ｐ－Ａ ３６１．５ｍｇ（１．３７ｍｍｏｌ）及び３－ブロモプロピオン酸メチル（スキーム中、１２と表示）２９０μＬ（２．６６ｍｍｏｌ）、Ｎ，Ｎ－ジメチルホルムアミド７．５ｍＬを加え、室温で６日間攪拌した。その後、反応溶液を分液ロートに移し，酢酸エチル５０ｍＬと精製水５０ｍＬを加えて有機層と水層を分離した。この水層を酢酸エチル５０ｍＬにより６回抽出し，先の有機層と合一した。この有機層を精製水により洗浄、無水硫酸ナトリウム乾燥，濃縮及び真空乾燥を行い、褐色油状物を６４９．５ｍｇ得た。続いて、中性シリカゲルカラムクロマトグラフィー（ジクロロメタン：メタノール＝１０：１）を行い、化合物１３を黄色固体として２０４．３ｍｇ得た（収率４３％）。
1ＨＮＭＲの測定結果を示す。
1Ｈ ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ3，２５℃）：δ＝７．３７（ｓ，２Ｈ，ｐｈｅｎｙｌ），５．９２（ｓ，２Ｈ，－ＮＨ
2），３．７２（ｓ，３Ｈ，－ＣＨ
3），３．４２（ｔ，Ｊ＝６．５，２Ｈ，－ＣＨ
2），３．１０（ｓ，６Ｈ，－ＣＨ
3），２．６３（ｔ，Ｊ＝６．５，２Ｈ，－ＣＨ
2）.
361.5 mg (1.37 mmol) of 2,6-BMeS-p-A, 290 μL (2.66 mmol) of methyl 3-bromopropionate (in the scheme, designated as 12), and 7.5 mL of N,N-dimethylformamide were added to a nitrogen-substituted 10 mL pear flask, and the mixture was stirred at room temperature for 6 days. The reaction solution was then transferred to a separatory funnel, and 50 mL of ethyl acetate and 50 mL of purified water were added to separate the organic layer and the aqueous layer. This aqueous layer was extracted six times with 50 mL of ethyl acetate and combined with the previous organic layer. This organic layer was washed with purified water, dried with anhydrous sodium sulfate, concentrated, and dried in vacuum to obtain 649.5 mg of a brown oily product. Subsequently, neutral silica gel column chromatography (dichloromethane:methanol=10:1) was performed to obtain 204.3 mg of compound 13 as a yellow solid (yield 43%).
The results of 1 HNMR measurements are shown below.
1H NMR (500MHz, CDCl3 , 25°C): δ = 7.37 (s, 2H, phenyl), 5.92 (s, 2H, -NH2 ) , 3.72 (s, 3H, -CH3), 3.42 (t, J = 6.5, 2H, -CH2 ) , 3.10 ( s, 6H, -CH3 ), 2.63 (t, J = 6.5, 2H , -CH2 ).
（ii）２，６－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨの合成
窒素置換した５０ｍＬ三口フラスコに化合物１３を９６．４ｍｇ（０．２８ｍｍｏｌ）、メタノール２８ｍＬ及び４０％水酸化ナトリウム水溶液７ｍＬを入れ、室温で３時間撹拌した。その後メタノールを除去し、そこに飽和硫酸水素カリウム水溶液をｐＨ＝３になるように加えた。この溶液を分液ロートに移し、酢酸エチル５０ｍＬと精製水５０ｍＬを加えて有機層と水層を分離した。この水層を酢酸エチル５０ｍＬにより６回抽出し、先の有機層と合一した。得られた有機層を無水硫酸ナトリウム乾燥及び濃縮、真空乾燥し、茶色固体を９５．７ｍｇ得た。最後に、中性シリカゲルカラムクロマトグラフィー（ＴＨＦ：酢酸エチル＝１：２０＋酢酸１ｖｏｌ％）を行い、２，６－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨを黄色固体として６０．９ｍｇ得た（収率６６％）。
1ＨＮＭＲの測定結果を示す。
1Ｈ ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ3，２５℃）：δ＝７．３７（ｓ，２Ｈ，ｐｈｅｎｙｌ），５．９３（ｓ，２Ｈ，－ＮＨ
2），３．４５（ｔ，Ｊ＝６．０，２Ｈ，－ＣＨ
2 ），３．１１（ｓ，６Ｈ，－ＣＨ
3），２．６８（ｔ，Ｊ＝６．０，２Ｈ，－ＣＨ
2）.
In a 50 mL three-neck flask substituted with nitrogen, 96.4 mg (0.28 mmol) of compound 13, 28 mL of methanol, and 7 mL of 40% aqueous sodium hydroxide solution were placed and stirred at room temperature for 3 hours. Then, methanol was removed, and saturated aqueous potassium hydrogen sulfate solution was added thereto so that the pH was 3. This solution was transferred to a separatory funnel, and 50 mL of ethyl acetate and 50 mL of purified water were added thereto to separate the organic layer and the aqueous layer. This aqueous layer was extracted six times with 50 mL of ethyl acetate and combined with the previous organic layer. The obtained organic layer was dried with anhydrous sodium sulfate, concentrated, and vacuum dried to obtain 95.7 mg of a brown solid. Finally, neutral silica gel column chromatography (THF:ethyl acetate=1:20 +
The results of 1 HNMR measurements are shown below.
1H NMR (500MHz, CDCl3, 25 °C): δ = 7.37 (s, 2H, phenyl), 5.93 (s, 2H, -NH2 ) , 3.45 (t, J = 6.0, 2H , -CH2 ), 3.11 (s, 6H, -CH3 ) , 2.68 (t, J = 6.0, 2H , -CH2 ).
（iii）２，６－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒの合成
1ＨＮＭＲの測定結果を示す。
1Ｈ ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ3，２５℃）：δ＝７．２９（ｓ，２Ｈ，ｐｈｅｎｙｌ），６．０１（ｔ，Ｊ＝６．０，Ｈ，－ＮＨ），５．０１（ｓ，２Ｈ，－ＮＨ
2），３．２３（ｓ，６Ｈ，－ＣＨ
3），２．９５（ｔ，Ｊ＝６．５，２Ｈ，－ＣＨ
2），２．８２（ｓ，４Ｈ，ＣＨ
2）.
(iii) Synthesis of 2,6-BMeS-p-A-NHS-ester
The results of 1 HNMR measurements are shown below.
1H NMR (500MHz, CDCl3, 25 °C): δ = 7.29 (s, 2H, phenyl), 6.01 (t, J = 6.0, H , -NH ), 5.01 (s, 2H, -NH2 ), 3.23 (s, 6H, -CH3 ), 2.95 (t, J = 6.5, 2H , -CH2 ), 2.82 (s, 4H, CH2 ) .
［標識試薬の作製］
標識試薬としての機能を確認するため、以下の構造式で表される２，６－ＢＭｅＳ－ｐ－Ａ－Ｐｈｅ－ＯＭｅを作製した。
In order to confirm its function as a labeling reagent, 2,6-BMeS-p-A-Phe-OMe, represented by the following structural formula, was prepared.
炭酸水素ナトリウム８４０．１ｍｇ（１０．０ｍｍｏｌ）を量り取り、純水に溶解させ、ｐＨ８．３の０．１Ｍ炭酸水素ナトリウム緩衝液を調製した。次に、試験管１にＰｈｅ－ＯＭｅ・ＨＣｌ ２１．５ｍｇ（９９．７μｍｏｌ）を量り取り、１ｍＬの０．１Ｍ炭酸水素ナトリウム緩衝液を加え溶解させた。
試験管２に２，６－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒ２１．１ｍｇ（４８．７μｍｏｌ）を量り取り、１ｍＬの乾燥ＤＭＳＯを加え溶解させた。
試験管１の炭酸水素ナトリウム水溶液を撹拌しながら、ガラスシリンジを用いて、試験管２のＤＭＳＯ溶液を試験管１中に１滴ずつゆっくりと滴下した。
撹拌しながら、１時間反応させた後、ジクロロメタンを用いて抽出作業を行い、黄色油状物を得た。中性シリカゲルカラムクロマトグラフィー（酢酸エチル）を行い、黄色油状物を収率１００％で得た。反応は定量的に進行した。
1ＨＮＭＲの結果を示す。
1Ｈ ＮＭＲ（５００ＭＨｚ，ＤＭＳＯ－ｄ6，２５℃）：δ＝８．４１－８．４３（ｄ，Ｈ，－ＮＨ），７．１９－７．２８（ｍ，７Ｈ，ｐｈｅｎｙｌ），５．７６－５．７９（ｍ，３Ｈ，－ＮＨ，－ＮＨ
2），４．４６－４．５１（ｍ，Ｈ，－ＣＨ），３．５９（ｓ，３Ｈ，ＣＨ
3），３．２１（ｓ，６Ｈ，ＣＨ
3），３．０９－３．１３（ｑ，２Ｈ，－ＣＨ
2），２．８６－２．９１，３．００－３．０３（ｑ，２Ｈ，ＣＨ
2），２．３４－２．３７（ｔ，２Ｈ，ＣＨ
2）．
840.1 mg (10.0 mmol) of sodium bicarbonate was weighed out and dissolved in pure water to prepare a 0.1 M sodium bicarbonate buffer solution with a pH of 8.3. Next, 21.5 mg (99.7 μmol) of Phe-OMe.HCl was weighed out into
21.1 mg (48.7 μmol) of 2,6-BMeS-p-A-NHS-ester was weighed out into
While stirring the aqueous sodium hydrogen carbonate solution in
After reacting for 1 hour with stirring, extraction was performed using dichloromethane to obtain a yellow oily product. Neutral silica gel column chromatography (ethyl acetate) was performed to obtain a yellow oily product in 100% yield. The reaction proceeded quantitatively.
The results of 1 HNMR are shown below.
1H NMR (500MHz, DMSO- d6 , 25°C): δ = 8.41-8.43 (d, H, -NH ), 7.19-7.28 (m, 7H, phenyl), 5.76-5.79 (m, 3H, -NH , -NH2 ), 4.46-4.51 (m, H, -CH ), 3.59 ( s , 3H, CH3 ) , 3.21 (s, 6H, CH3 ), 3.09-3.13 (q, 2H, -CH2 ), 2.86-2.91 , 3.00-3.03 (q, 2H , CH2 ), 2.34-2.37 (t, 2H , CH2 ) .
［実施例３］
実施例１の２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒを合成する際の中間生成物である２，５－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨを使用した。
2,5-BMeS-p-A-COOH, which is an intermediate product in the synthesis of 2,5-BMeS-p-A-NHS-ester in Example 1, was used.
［実施例４］
［２，６－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨの合成］
２日後、アセトニトリルを濃縮して黄色固体と茶色油状物を１．２３ｇ得た。そこに、２Ｍの水酸化ナトリウム水溶液を１００ｍＬ加え３５℃で２４時間撹拌した。それを一度吸引ろ過し、ろ液を酢酸エチルで分液した。水層を６Ｍ塩酸でｐＨ４付近になるように調整し、酢酸エチル１００ｍＬで３回抽出した。得られた有機層を飽和食塩水８０ｍＬで洗浄、濃縮を行い、黄色固体を３３０．６ｍｇ得た。最後に、中性シリカゲルカラムクロマトグラフィー（ＴＨＦ：酢酸エチル＝１：２０＋酢酸１ｖｏｌ％）で精製し、黄色固体を１２７．８ ｍｇ（０．３８ ｍｍｏｌ）得た（収率２０％）。
1ＨＮＭＲの結果を示す。
1Ｈ ＮＭＲ（５００ＭＨｚ，ＣＤＣｌ3，２５℃）：δ＝７．３７（ｓ，２Ｈ，ｐｈｅｎｙｌ），５．９３（ｓ，２Ｈ，－ＮＨ
2），３．４５（ｔ，Ｊ＝６．０，２Ｈ，－ＣＨ
2），３．１１（ｓ，６Ｈ，－ＣＨ
3），２．６８（ｔ，Ｊ＝６．０，２Ｈ，－ＣＨ
2）.
[Example 4]
[Synthesis of 2,6-BMeS-p-A-COOH]
After 2 days, acetonitrile was concentrated to obtain 1.23 g of a yellow solid and a brown oil. 100 mL of 2M aqueous sodium hydroxide was added thereto and stirred at 35° C. for 24 hours. It was filtered once by suction, and the filtrate was separated with ethyl acetate. The aqueous layer was adjusted to about pH 4 with 6M hydrochloric acid, and extracted three times with 100 mL of ethyl acetate. The obtained organic layer was washed with 80 mL of saturated saline and concentrated to obtain 330.6 mg of a yellow solid. Finally, it was purified by neutral silica gel column chromatography (THF:ethyl acetate=1:20 +
The results of 1 HNMR are shown below.
1H NMR (500MHz, CDCl3, 25 °C): δ = 7.37 (s, 2H, phenyl), 5.93 (s, 2H, -NH2 ) , 3.45 (t, J = 6.0, 2H , -CH2 ), 3.11 (s, 6H, -CH3 ) , 2.68 (t, J = 6.0, 2H , -CH2 ).
なお、２，６－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨは、実施例２の「（ii）２，６－ＢＭｅＳ－ｐ－Ａ－ＣＯＯＨの合成」に記載された方法によって合成してもよい。 2,6-BMeS-p-A-COOH may be synthesized by the method described in "(ii) Synthesis of 2,6-BMeS-p-A-COOH" in Example 2.
［比較例１］フルオレセイン－ＮＨＳ－ｅｓｔｅｒ
東京化成工業（株）からの市販品を用いた。
A commercial product from Tokyo Chemical Industry Co., Ltd. was used.
［比較例２］ＢＯＤＩＰＹ－ＮＨＳ－ｅｓｔｅｒ
東京化成工業（株）からの市販品を用いた。
A commercial product from Tokyo Chemical Industry Co., Ltd. was used.
［有機蛍光材料としての評価］
以下の項目について、実施例１、２で得られたスルホニルアニリン系色素と、比較例１、２の汎用色素との比較を行った。
〔光学特性〕
実施例１、２のスルホニルアニリン系色素の光学特性を調査するために、細胞イメージングを考慮して水を用いた吸収スペクトル（（株）日立ハイテクサイエンス製ダブルビーム分光光度計 Ｕ－２８１０）及び励起／蛍光スペクトル（（株）島津製作所製 分光蛍光光度計 ＲＦ－６０００）を測定した。
励起／発光波長は蛍光分光光度計を用いて測定した。
量子効率（水溶液）は、蛍光分光光度計を用いて、２，５－ＢＭｅＳ－ｐ－ＡのＤＭＳＯ溶液（１．０×１０-5Ｍ；φ＝０．７８）を標準試料としたときの相対量子効率である。一方、量子効率（固体）は、積分球内に試料を置いて測定した絶対発光量子効率である。
ストークスシフトは、励起／蛍光スペクトルのピークの位置の差から求めた。
[Evaluation as an organic fluorescent material]
The sulfonylaniline dyes obtained in Examples 1 and 2 were compared with the general-purpose dyes of Comparative Examples 1 and 2 with respect to the following items.
〔optical properties〕
In order to investigate the optical properties of the sulfonylaniline dyes of Examples 1 and 2, the absorption spectrum (double beam spectrophotometer U-2810, manufactured by Hitachi High-Tech Science Corp.) and the excitation/fluorescence spectrum (spectrofluorophotometer RF-6000, manufactured by Shimadzu Corp.) were measured using water in consideration of cell imaging.
The excitation/emission wavelengths were measured using a fluorescence spectrophotometer.
The quantum efficiency (aqueous solution) is the relative quantum efficiency when a DMSO solution of 2,5-BMeS-p-A (1.0×10 −5 M; φ=0.78) is used as a standard sample using a fluorescence spectrophotometer, while the quantum efficiency (solid state) is the absolute luminescence quantum efficiency measured by placing a sample in an integrating sphere.
The Stokes shift was determined from the difference in the positions of the peaks in the excitation/fluorescence spectra.
〔光安定性〕
各試料とも空気中で調製した。２，５－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒ、２，６－ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒ、ＢＤＰＦＬ ＮＨＳ Ｅｓｔｅｒ（ＢＯＤＩＰＹ ＦＬ色素）、及びフルオレセイン－ＮＨＳ－ｅｓｔｅｒをそれぞれ（ｐＨ＝７、リン酸緩衝溶液）に溶解させた１．０×１０-6Ｍ溶液を調製した。図１に各化合物の構造式を示す。
各試料とも調製直後に蛍光スペクトルを測定し（ｔ＝０分）、この溶液の入った石英セルに対して１０ｃｍの距離から１５０Ｗのキセノンランプからの白色光を照射した。なお、熱の発生を抑制するために、ランプと石英セルの間に水を置いた。この状態で４５分間照射し、一定時間ごとに蛍光スペクトルを測定した（ｔ＝１分、３分、５分、１０分、１５分、３０分、４５分）。
耐光性試験の結果を図１に示す。表１中、３０分経過しても、蛍光強度が５０％以上である場合を◎、２０～５０％となる場合を〇、１０分程度経過すると大きく減衰し始める場合を△とした。また、蛍光強度の時間による減衰を測定することで濃度消光の有無を判定した。表１中、５０％減衰以上の場合を「有」、５０％減衰未満の場合を「無」とした。
[Photostability]
Each sample was prepared in air. 2,5-BMeS-p-A-NHS-ester, 2,6-BMeS-p-A-NHS-ester, BDPFL NHS ester (BODIPY FL dye), and fluorescein-NHS-ester were dissolved in phosphate buffer solution (pH = 7) to prepare 1.0 x 10-6 M solutions. The structural formulas of each compound are shown in Figure 1.
The fluorescence spectrum of each sample was measured immediately after preparation (t = 0 min), and the solution was irradiated with white light from a 150 W xenon lamp from a distance of 10 cm into a quartz cell. Water was placed between the lamp and the quartz cell to suppress heat generation. Irradiation was continued in this state for 45 minutes, and the fluorescence spectrum was measured at regular intervals (t = 1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 45 min).
The results of the light resistance test are shown in Figure 1. In Table 1, the cases where the fluorescence intensity was 50% or more even after 30 minutes were marked with ◎, the cases where it was 20-50% were marked with ◯, and the cases where it started to decay significantly after about 10 minutes were marked with △. The presence or absence of concentration quenching was also determined by measuring the decay of the fluorescence intensity over time. In Table 1, the cases where the decay was 50% or more were marked with "Yes", and the cases where the decay was less than 50% were marked with "No".
〔濃度消光〕
実施例１、２のスルホニルアニリン系色素と比較例１、２の汎用色素のそれぞれの固体蛍光を測定して、蛍光量子効率が０．２以上のものを濃度消光（無）とした。
〔分子サイズ〕
量子化学計算Gaussian09（B3LYP/6-31g(d)）によって最適化した分子体積（Å3）を求めた。
〔環境依存性〕
吸収／蛍光スペクトルの波長や蛍光強度がｐＨや溶媒の極性などの因子によって変化するかどうかを観察した。ｐＫａの変動幅が１０ｎｍ以上の場合を「有」、１０ｎｍ未満の場合を「無」とした。
結果を表１に示す。
[Concentration quenching]
The solid-state fluorescence of each of the sulfonylaniline dyes of Examples 1 and 2 and the general-purpose dyes of Comparative Examples 1 and 2 was measured, and those having a fluorescence quantum efficiency of 0.2 or more were determined to have concentration quenching (none).
[Molecular size]
The optimized molecular volume (Å 3 ) was determined by quantum chemical calculations using Gaussian09 (B3LYP/6-31g(d)).
[Environmental Dependence]
We observed whether the wavelength and fluorescence intensity of the absorption/fluorescence spectrum changed depending on factors such as pH and solvent polarity. A pKa fluctuation range of 10 nm or more was judged as "present," and a pKa fluctuation range of less than 10 nm was judged as "absent."
The results are shown in Table 1.
（１）励起／発光波長
実施例１、２のスルホニルアニリン系色素は、環状構造がベンゼン環１つであるにもかかわらず、緑色蛍光を示した。これは分子内水素結合及びＰｕｓｈ－Ｐｕｌｌ構造に起因するものと考えられる。また、従来の色素と異なり、置換基の配置で発光波長が異なるために分子サイズが変化しないことも要因といえる。
(1) Excitation/Emission Wavelength The sulfonylaniline dyes of Examples 1 and 2 exhibited green fluorescence, even though the cyclic structure consisted of one benzene ring. This is believed to be due to intramolecular hydrogen bonds and a push-pull structure. Another factor is that, unlike conventional dyes, the molecular size does not change because the emission wavelength differs depending on the arrangement of the substituents.
（２）分子サイズ
実施例１、２のスルホニルアニリン系色素は、従来の小分子蛍光色素と比較して小さな分子サイズを持ち、その体積は、最も小さい分子サイズを持つ比較例２のＢＯＤＩＰＹの体積に匹敵する。したがって、本発明のスルホニルアニリン系色素は、複雑構造を持つ糖鎖やペプチド、あるいはタンパク質などの生体高分子を標識する際に立体障害が小さいという優位性を持つといえる。また、分子サイズが小さいため、ターゲット分子の機能を阻害しないという点においても優れている。
(2) Molecular Size The sulfonylaniline dyes of Examples 1 and 2 have a smaller molecular size than conventional small molecule fluorescent dyes, and their volume is comparable to that of BODIPY of Comparative Example 2, which has the smallest molecular size. Therefore, it can be said that the sulfonylaniline dyes of the present invention have the advantage of having less steric hindrance when labeling biopolymers such as sugar chains, peptides, or proteins that have complex structures. In addition, because of their small molecular size, they are also excellent in that they do not inhibit the function of target molecules.
（３）ストークスシフト
実施例１、２のスルホニルアニリン系色素では１３０ｎｍを超える極めて大きなストークスシフトが確認され、有機系低分子でありながら、量子ドットに匹敵する大きなストークスシフトを示した。これは、前記スルホニルアニリン系色素は、比較例１、２の汎用性色素のような従来の色素と比較して、剛直でないために、励起状態と基底状態では構造が異なることに起因する。比較例１のフルオレセイン-ＮＨＳ－ｅｓｔｅｒ（２７ｎｍ）や比較例２のＢＯＤＩＰＹ－ＮＨＳ－ｅｓｔｅｒ（６ｎｍ）と比較すると、明らかな優位性が認められた。
(3) Stokes shift The sulfonylaniline dyes of Examples 1 and 2 were confirmed to have an extremely large Stokes shift exceeding 130 nm, and showed a large Stokes shift comparable to that of quantum dots, despite being organic low-molecular-weight compounds. This is because the sulfonylaniline dyes are not as rigid as conventional dyes such as the general-purpose dyes of Comparative Examples 1 and 2, and therefore have different structures in the excited state and ground state. Compared with the fluorescein-NHS-ester (27 nm) of Comparative Example 1 and the BODIPY-NHS-ester (6 nm) of Comparative Example 2, a clear superiority was observed.
（４）量子効率
相対法で求めた量子効率（水溶液）は、実施例１では０．６９、実施例２では０．４６と高い数値を示した。この数値は、比較例１、２の汎用性色素と比較すると、０．１～０．２程度低いが、汎用性色素は、環境によって量子効率が変化するため、測定条件に依存する。これに対し、本発明のスルホニルアニリン系色素は安定した量子効率を示しており、十分に実用的な量子効率であるといえる。
また、固体状態でも蛍光を示したことから、濃度消光の影響が極めて小さいといえる。
(4) Quantum efficiency The quantum efficiency (aqueous solution) determined by the relative method was 0.69 in Example 1 and 0.46 in Example 2, which were high values. This value is about 0.1 to 0.2 lower than the general-purpose dyes of Comparative Examples 1 and 2, but the quantum efficiency of the general-purpose dyes varies depending on the environment, and therefore depends on the measurement conditions. In contrast, the sulfonylaniline dye of the present invention exhibits stable quantum efficiency, and can be said to have a sufficiently practical quantum efficiency.
In addition, since the compound exhibited fluorescence even in the solid state, it can be said that the effect of concentration quenching was extremely small.
（５）モル吸光係数
実施例１、２のスルホニルアニリン系色素とも、比較例１、２の汎用性色素と比べて、モル吸光係数は小さかった。
モル吸光係数が小さければ、蛍光の輝度も小さくなる。しかしながら、本発明の場合、大きなストークスシフトが可能にする大きな吸収領域での励起がこの欠点を十分に克服している。
(5) Molar Absorption Coefficient The sulfonylaniline dyes of Examples 1 and 2 had smaller molar absorption coefficients than the general-purpose dyes of Comparative Examples 1 and 2.
A small molar extinction coefficient leads to a small fluorescence intensity. However, in the present invention, excitation in a large absorption region made possible by a large Stokes shift sufficiently overcomes this drawback.
（６）耐光性
実施例１、２のスルホニルアニリン系色素とも、比較例１のフルオレセイン－ＮＨＳ－ｅｓｔｅｒの５０倍、比較例２のＢＯＤＩＰＹ－ＮＨＳ－ｅｓｔｅｒの１０倍の高い耐光性が認められた。
実施例１、２のスルホニルアニリン系色素の構造において、ベンゼン環をより安定な骨格であるナフタレン環に替えた場合、さらに耐光性が向上すると予測される。
(6) Lightfastness The sulfonylaniline dyes of Examples 1 and 2 were found to have
In the structures of the sulfonylaniline dyes of Examples 1 and 2, if the benzene ring is replaced with a naphthalene ring, which is a more stable skeleton, it is expected that the light resistance will be further improved.
（７）濃度消光
比較例１、２の汎用色素を含めて、ほとんどの汎用性色素は、剛直な共役系を持つため、濃度消光を示す。一方、実施例１、２のスルホニルアニリン系色素は、従来の拡張π共役系とは全く異なる独創的な分子構造を持つため、濃度消光を全く示さないどころか、固体蛍光性を示す。本性質はスルホニル基の折れ曲がり構造に起因する。
(7) Concentration quenching Most of the general-purpose dyes, including those of Comparative Examples 1 and 2, have a rigid conjugated system and therefore exhibit concentration quenching. On the other hand, the sulfonylaniline dyes of Examples 1 and 2 have an original molecular structure that is completely different from the conventional extended π-conjugated system, so they do not show concentration quenching at all, and in fact, they exhibit solid-state fluorescence. This property is due to the bent structure of the sulfonyl group.
（８）環境依存性
比較例１のフルオレセイン－ＮＨＳ－ｅｓｔｅｒのｐＫａは６．４であった。ｐＨ５～９ではその蛍光強度にｐＨ依存性がある。比較例２のＢＯＤＩＰＹ－ＮＨＳ－ｅｓｔｅｒも若干のスペクトル変化を伴っていた。一方、実施例１、２のスルホニルアニリン系色素はｐＨや極性によって蛍光強度と波長が変化しなかった。これは、イオン性官能基を持たないこと、励起状態と基底状態の双極子モーメントの差がゼロであることに起因する。このような色素は極めて珍しく、スルホニルアニリン系色素の優位な点である。
(8) Environmental Dependence The pKa of the fluorescein-NHS-ester of Comparative Example 1 was 6.4. At pH 5 to 9, its fluorescence intensity was pH dependent. The BODIPY-NHS-ester of Comparative Example 2 also exhibited slight spectral changes. On the other hand, the fluorescence intensity and wavelength of the sulfonylaniline dyes of Examples 1 and 2 did not change depending on pH or polarity. This is due to the fact that they do not have an ionic functional group and that the difference in dipole moment between the excited state and the ground state is zero. Such dyes are extremely rare, which is an advantage of the sulfonylaniline dyes.
［標識試薬としての機能評価］
アミノ酸であるフェニルアラニンのカルボン酸を保護したフェニルアラニンメチルエステル塩酸塩（Ｐｈｅ－ＯＭｅ・ＨＣｌ）を用いて、実施例１、２のスルホニルアニリン系色素のアミンとの反応性を調査した。
たんぱく質とのコンジュゲートに推奨される溶媒及び手順を参考にして、下記スキームに示すように、反応緩衝液にはｐＨ８．３の０．１Ｍ炭酸水素ナトリウム緩衝液、溶媒にはＤＭＳＯを用いて、室温下で作用させた。
The reactivity of the sulfonylaniline dyes of Examples 1 and 2 with amines was investigated using phenylalanine methyl ester hydrochloride (Phe-OMe.HCl) in which the carboxylic acid of the amino acid phenylalanine is protected.
Referring to the solvent and procedure recommended for conjugation with proteins, as shown in the scheme below, 0.1 M sodium bicarbonate buffer at pH 8.3 was used as the reaction buffer, DMSO was used as the solvent, and the reaction was carried out at room temperature.
その結果、２，５－ＢＭｅＳ－ｐ－Ａ－Ｐｈｅ－ＯＭｅ及び２，６－ＢＭｅＳ－ｐ－Ａ－Ｐｈｅ－ＯＭｅを得ることに成功した。これにより、２，５－ＢＭｅＳ－ｐ－Ａ－Ｐｈｅ－ＯＭｅと２，６－ＢＭｅＳ－ｐ－Ａ－Ｐｈｅ－ＯＭｅは脂肪族アミノ基と選択的に反応を起こし、結合を形成することが明らかとなった。また、コンジュゲート後も、消光することなく蛍光特性を有していた。以上の結果から、ＢＭｅＳ－ｐ－Ａ－ＮＨＳ－ｅｓｔｅｒの蛍光標識試薬としての機能が確認された。 As a result, they succeeded in obtaining 2,5-BMeS-p-A-Phe-OMe and 2,6-BMeS-p-A-Phe-OMe. This revealed that 2,5-BMeS-p-A-Phe-OMe and 2,6-BMeS-p-A-Phe-OMe selectively react with aliphatic amino groups to form bonds. Furthermore, even after conjugation, they retained fluorescent properties without quenching. These results confirmed the function of BMeS-p-A-NHS-ester as a fluorescent labeling reagent.
本発明の有機蛍光材料は、糖鎖やペプチド鎖などの複雑構造を標識するのに好適な蛍光プローブとなりうる。それゆえ、マルチカラーイメージングや高精度イメージングを可能にすることから、新薬開発、臨床検査又はライフサイエンス研究など広範囲な領域への貢献が期待できる。 The organic fluorescent material of the present invention can be a suitable fluorescent probe for labeling complex structures such as glycans and peptide chains. As a result, it enables multicolor imaging and high-precision imaging, and is expected to contribute to a wide range of fields, such as new drug development, clinical testing, and life science research.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020021477A JP7479667B2 (en) | 2020-02-12 | 2020-02-12 | Compound having a sulfonylaniline skeleton or a salt thereof, or organic fluorescent material having the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020021477A JP7479667B2 (en) | 2020-02-12 | 2020-02-12 | Compound having a sulfonylaniline skeleton or a salt thereof, or organic fluorescent material having the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2021127305A JP2021127305A (en) | 2021-09-02 |
| JP7479667B2 true JP7479667B2 (en) | 2024-05-09 |
Family
ID=77487849
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2020021477A Active JP7479667B2 (en) | 2020-02-12 | 2020-02-12 | Compound having a sulfonylaniline skeleton or a salt thereof, or organic fluorescent material having the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7479667B2 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011506673A (en) | 2007-12-14 | 2011-03-03 | バイオティウム， インコーポレイテッド | Fluorescent compound |
| WO2010093726A8 (en) | 2009-02-11 | 2011-05-12 | Life Technologies Corporation | Large stokes shift dyes |
| JP2015054937A (en) | 2013-09-13 | 2015-03-23 | 国立大学法人山形大学 | Organic fluorescent material |
-
2020
- 2020-02-12 JP JP2020021477A patent/JP7479667B2/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011506673A (en) | 2007-12-14 | 2011-03-03 | バイオティウム， インコーポレイテッド | Fluorescent compound |
| WO2010093726A8 (en) | 2009-02-11 | 2011-05-12 | Life Technologies Corporation | Large stokes shift dyes |
| JP2015054937A (en) | 2013-09-13 | 2015-03-23 | 国立大学法人山形大学 | Organic fluorescent material |
Non-Patent Citations (2)
| Title |
|---|
| Angewandte Chemie International Edition，2015年，Vol. 54，pp. 7332-7335 |
| Zhang, Lei 他，Talanta，2019年，Vol. 198，pp. 472-479 |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2021127305A (en) | 2021-09-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108440475B (en) | Ratio type fluorescent probe for distinguishing lipid droplets with different polarities and preparation method and application thereof | |
| JP5090731B2 (en) | Fluorescent probe | |
| JP3383736B2 (en) | Novel pentacyclic compounds and their use as absorbing or fluorescent dyes | |
| CN108864056B (en) | Near infrared fluorescent compound and its preparation method and application with AIE performance | |
| JP6275689B2 (en) | Fluorescent probe | |
| US6403625B1 (en) | Fluorescent labeling reagents | |
| CN105017288A (en) | Blue fluorescent compound and preparation method thereof | |
| CN107253965A (en) | FRET molecule and its synthetic method of one class based on fluorine boron pyrroles and cumarin | |
| US7897787B2 (en) | Maleimide derivative | |
| EP3638659B1 (en) | New water soluble mono-branched and di-branched complexing agents, and corresponding lanthanide complexes | |
| JP7479667B2 (en) | Compound having a sulfonylaniline skeleton or a salt thereof, or organic fluorescent material having the same | |
| JP5319130B2 (en) | Keiko solvatochromic dye and method of use thereof | |
| CN116375692A (en) | Near infrared fluorescent molecular probe for detecting cysteine, preparation method and kit thereof | |
| WO2018181529A1 (en) | Phospha-rhodol compound, salt of same, and fluorescent dye using same | |
| CN113416196B (en) | benzothiadiazole-TB compound and synthesis method and application thereof | |
| CN112174988B (en) | Tertiary rare earth terbium trimer and its preparation method | |
| CN107286926B (en) | Heterozygosis fluorescent dye and its preparation method and application based on cumarin and fluorescein | |
| WO2018043579A1 (en) | Phospha-rhodamine compound or salt thereof, and fluorescent dye using same | |
| CN112300164A (en) | Rare earth samarium cryptand ether fluorescent complex and preparation method thereof | |
| JP5437615B2 (en) | Kaiko solvatochromic dye | |
| JP2023092930A (en) | Diaminobenzene compounds comprising sulfinyl or sulfonyl groups or salts thereof, and organic fluorescent materials having them | |
| CN111533761B (en) | Ratio type pH probe with organelle or protein targeting function and application thereof | |
| CN113387865B (en) | Fluorescent probe for detecting methylglyoxal and preparation method and application thereof | |
| WO2017098180A1 (en) | Water-soluble triazapyridinophane-based complexing agents and corresponding fluorescent lanthanide complexes | |
| WO2021087897A1 (en) | Bodipy-based high-efficiency real-time biological thiol fluorescent detection probe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20230203 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20231214 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20231222 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20240126 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20240327 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20240417 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7479667 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |