WO2016157937A1 - pH感受性蛍光プローブ - Google Patents
pH感受性蛍光プローブ Download PDFInfo
- Publication number
- WO2016157937A1 WO2016157937A1 PCT/JP2016/051119 JP2016051119W WO2016157937A1 WO 2016157937 A1 WO2016157937 A1 WO 2016157937A1 JP 2016051119 W JP2016051119 W JP 2016051119W WO 2016157937 A1 WO2016157937 A1 WO 2016157937A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- group
- carbon atoms
- atom
- salt
- compound
- Prior art date
Links
- 239000007850 fluorescent dye Substances 0.000 title claims description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 90
- 150000003839 salts Chemical class 0.000 claims abstract description 89
- 230000002378 acidificating effect Effects 0.000 claims abstract description 29
- 125000004432 carbon atom Chemical group C* 0.000 claims description 129
- 125000000217 alkyl group Chemical group 0.000 claims description 108
- 125000001424 substituent group Chemical group 0.000 claims description 66
- 125000005843 halogen group Chemical group 0.000 claims description 59
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 46
- 210000004027 cell Anatomy 0.000 claims description 45
- 238000009825 accumulation Methods 0.000 claims description 43
- 125000000524 functional group Chemical group 0.000 claims description 40
- 125000003277 amino group Chemical group 0.000 claims description 38
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 36
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 36
- 239000001257 hydrogen Substances 0.000 claims description 35
- 229910052739 hydrogen Inorganic materials 0.000 claims description 35
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 35
- 125000003545 alkoxy group Chemical group 0.000 claims description 29
- 125000001072 heteroaryl group Chemical group 0.000 claims description 29
- 125000003118 aryl group Chemical group 0.000 claims description 28
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 25
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 22
- 125000000304 alkynyl group Chemical group 0.000 claims description 18
- 125000003368 amide group Chemical group 0.000 claims description 17
- 125000000623 heterocyclic group Chemical group 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- 210000003463 organelle Anatomy 0.000 claims description 16
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 claims description 16
- 230000003834 intracellular effect Effects 0.000 claims description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims description 15
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 15
- 125000004437 phosphorous atom Chemical group 0.000 claims description 15
- 229910052698 phosphorus Inorganic materials 0.000 claims description 15
- 229910052799 carbon Inorganic materials 0.000 claims description 14
- 125000001153 fluoro group Chemical group F* 0.000 claims description 12
- 229910052717 sulfur Inorganic materials 0.000 claims description 12
- 125000003342 alkenyl group Chemical group 0.000 claims description 11
- 229910052731 fluorine Inorganic materials 0.000 claims description 10
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical group [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 9
- 238000002372 labelling Methods 0.000 claims description 9
- 229910052710 silicon Inorganic materials 0.000 claims description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052732 germanium Inorganic materials 0.000 claims description 8
- GNPVGFCGXDBREM-UHFFFAOYSA-N germanium atom Chemical group [Ge] GNPVGFCGXDBREM-UHFFFAOYSA-N 0.000 claims description 8
- 125000001188 haloalkyl group Chemical group 0.000 claims description 8
- 125000005842 heteroatom Chemical group 0.000 claims description 8
- 125000004434 sulfur atom Chemical group 0.000 claims description 8
- 125000003282 alkyl amino group Chemical group 0.000 claims description 7
- 125000005907 alkyl ester group Chemical group 0.000 claims description 7
- 125000003710 aryl alkyl group Chemical group 0.000 claims description 7
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims description 7
- 150000001721 carbon Chemical group 0.000 claims description 7
- 125000004185 ester group Chemical group 0.000 claims description 7
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N isothiocyanate group Chemical group [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 claims description 7
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical group ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 claims description 7
- 229920001222 biopolymer Polymers 0.000 claims description 5
- 150000002431 hydrogen Chemical class 0.000 claims description 5
- YZCKVEUIGOORGS-IGMARMGPSA-N Protium Chemical compound [1H] YZCKVEUIGOORGS-IGMARMGPSA-N 0.000 claims description 3
- 239000000523 sample Substances 0.000 abstract description 49
- 238000006862 quantum yield reaction Methods 0.000 abstract description 13
- 238000012800 visualization Methods 0.000 abstract description 5
- 230000007935 neutral effect Effects 0.000 abstract description 3
- 238000002845 discoloration Methods 0.000 abstract 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 60
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 51
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 48
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 34
- 239000000243 solution Substances 0.000 description 33
- 239000000203 mixture Substances 0.000 description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 28
- 238000006243 chemical reaction Methods 0.000 description 27
- 239000002904 solvent Substances 0.000 description 19
- 102000004169 proteins and genes Human genes 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 18
- 239000012064 sodium phosphate buffer Substances 0.000 description 18
- 238000000695 excitation spectrum Methods 0.000 description 16
- 229920001223 polyethylene glycol Chemical group 0.000 description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 230000008859 change Effects 0.000 description 15
- 210000001163 endosome Anatomy 0.000 description 15
- -1 for example Chemical group 0.000 description 15
- 238000000862 absorption spectrum Methods 0.000 description 14
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 14
- 238000001139 pH measurement Methods 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 230000005284 excitation Effects 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 10
- 239000003480 eluent Substances 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 239000012044 organic layer Substances 0.000 description 10
- 238000004064 recycling Methods 0.000 description 10
- 239000011734 sodium Substances 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 9
- 238000004132 cross linking Methods 0.000 description 9
- 125000006575 electron-withdrawing group Chemical group 0.000 description 9
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- 238000002189 fluorescence spectrum Methods 0.000 description 8
- 210000003712 lysosome Anatomy 0.000 description 8
- 230000001868 lysosomic effect Effects 0.000 description 8
- 125000004193 piperazinyl group Chemical group 0.000 description 8
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 7
- 238000003384 imaging method Methods 0.000 description 7
- 230000003287 optical effect Effects 0.000 description 7
- 229920002307 Dextran Polymers 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 238000011088 calibration curve Methods 0.000 description 6
- 230000002132 lysosomal effect Effects 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 125000001309 chloro group Chemical group Cl* 0.000 description 5
- 210000000805 cytoplasm Anatomy 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 239000002202 Polyethylene glycol Chemical group 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical class C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000005842 biochemical reaction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- 239000011541 reaction mixture Substances 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 230000003595 spectral effect Effects 0.000 description 4
- 125000001494 2-propynyl group Chemical group [H]C#CC([H])([H])* 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 description 3
- 125000004453 alkoxycarbonyl group Chemical group 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000012267 brine Substances 0.000 description 3
- 125000004181 carboxyalkyl group Chemical group 0.000 description 3
- 229910052801 chlorine Inorganic materials 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 229940125782 compound 2 Drugs 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 125000003827 glycol group Chemical group 0.000 description 3
- 125000002768 hydroxyalkyl group Chemical group 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 125000005647 linker group Chemical group 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- RFIOZSIHFNEKFF-UHFFFAOYSA-M piperazine-1-carboxylate Chemical compound [O-]C(=O)N1CCNCC1 RFIOZSIHFNEKFF-UHFFFAOYSA-M 0.000 description 3
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical group [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 3
- DYPYMMHZGRPOCK-UHFFFAOYSA-N seminaphtharhodafluor Chemical compound O1C(=O)C2=CC=CC=C2C21C(C=CC=1C3=CC=C(O)C=1)=C3OC1=CC(N)=CC=C21 DYPYMMHZGRPOCK-UHFFFAOYSA-N 0.000 description 3
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 3
- UGNWTBMOAKPKBL-UHFFFAOYSA-N tetrachloro-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(Cl)=C(Cl)C1=O UGNWTBMOAKPKBL-UHFFFAOYSA-N 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 108700012359 toxins Proteins 0.000 description 3
- 239000012581 transferrin Substances 0.000 description 3
- XWNJMSJGJFSGRY-UHFFFAOYSA-N 2-(benzylamino)-3,7-dihydropurin-6-one Chemical class N1C=2N=CNC=2C(=O)N=C1NCC1=CC=CC=C1 XWNJMSJGJFSGRY-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 2
- 229920001917 Ficoll Polymers 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108090001090 Lectins Proteins 0.000 description 2
- 102000004856 Lectins Human genes 0.000 description 2
- 108090001030 Lipoproteins Proteins 0.000 description 2
- 102000004895 Lipoproteins Human genes 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 2
- 206010057249 Phagocytosis Diseases 0.000 description 2
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 108090000901 Transferrin Proteins 0.000 description 2
- 102000004338 Transferrin Human genes 0.000 description 2
- 102000007238 Transferrin Receptors Human genes 0.000 description 2
- 108010033576 Transferrin Receptors Proteins 0.000 description 2
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 125000004448 alkyl carbonyl group Chemical group 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 150000003863 ammonium salts Chemical class 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 2
- 159000000007 calcium salts Chemical class 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 229940126214 compound 3 Drugs 0.000 description 2
- 125000001559 cyclopropyl group Chemical group [H]C1([H])C([H])([H])C1([H])* 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 150000002148 esters Chemical group 0.000 description 2
- 238000005562 fading Methods 0.000 description 2
- 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 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 108010045676 holotransferrin Proteins 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 150000004677 hydrates Chemical class 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 210000005061 intracellular organelle Anatomy 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 159000000003 magnesium salts Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 230000008782 phagocytosis Effects 0.000 description 2
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 2
- 230000005588 protonation Effects 0.000 description 2
- 125000004076 pyridyl group Chemical group 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- 239000012453 solvate Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 125000001174 sulfone group Chemical group 0.000 description 2
- 210000002504 synaptic vesicle Anatomy 0.000 description 2
- 229940095064 tartrate Drugs 0.000 description 2
- ASWQNRYSAGTXLK-UHFFFAOYSA-N tert-butyl 4-[3-bromo-4-[(6-bromo-1-methyl-2,3-dihydroindol-5-yl)methyl]phenyl]piperazine-1-carboxylate Chemical compound BrC=1C=C(C=CC=1CC=1C=C2CCN(C2=CC=1Br)C)N1CCN(CC1)C(=O)OC(C)(C)C ASWQNRYSAGTXLK-UHFFFAOYSA-N 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 0 **C*CC1NCCNCC1 Chemical compound **C*CC1NCCNCC1 0.000 description 1
- PVOAHINGSUIXLS-UHFFFAOYSA-N 1-Methylpiperazine Chemical group CN1CCNCC1 PVOAHINGSUIXLS-UHFFFAOYSA-N 0.000 description 1
- YMHOBZXQZVXHBM-UHFFFAOYSA-N 2,5-dimethoxy-4-bromophenethylamine Chemical compound COC1=CC(CCN)=C(OC)C=C1Br YMHOBZXQZVXHBM-UHFFFAOYSA-N 0.000 description 1
- PIINGYXNCHTJTF-UHFFFAOYSA-N 2-(2-azaniumylethylamino)acetate Chemical compound NCCNCC(O)=O PIINGYXNCHTJTF-UHFFFAOYSA-N 0.000 description 1
- OPZDXMCOWFPQPE-UHFFFAOYSA-N 2-bromo-4-fluorobenzaldehyde Chemical compound FC1=CC=C(C=O)C(Br)=C1 OPZDXMCOWFPQPE-UHFFFAOYSA-N 0.000 description 1
- 125000000143 2-carboxyethyl group Chemical group [H]OC(=O)C([H])([H])C([H])([H])* 0.000 description 1
- 125000000094 2-phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 description 1
- XUQZKSCQPMNDEY-UHFFFAOYSA-N 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanoic acid Chemical compound NCCOCCOCCOCCC(O)=O XUQZKSCQPMNDEY-UHFFFAOYSA-N 0.000 description 1
- PIKNVEVCWAAOMJ-UHFFFAOYSA-N 3-fluorobenzaldehyde Chemical compound FC1=CC=CC(C=O)=C1 PIKNVEVCWAAOMJ-UHFFFAOYSA-N 0.000 description 1
- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 description 1
- LHOUXFCFCBCKPY-UHFFFAOYSA-N 6-(benzylamino)-1h-pyrimidin-2-one Chemical class N1C(=O)N=CC=C1NCC1=CC=CC=C1 LHOUXFCFCBCKPY-UHFFFAOYSA-N 0.000 description 1
- KXJIMNJESWOULY-UHFFFAOYSA-N 6-bromo-1-methyl-2,3-dihydroindole Chemical compound C1=C(Br)C=C2N(C)CCC2=C1 KXJIMNJESWOULY-UHFFFAOYSA-N 0.000 description 1
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical group CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 229920001202 Inulin Polymers 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 229920000057 Mannan Polymers 0.000 description 1
- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108090000189 Neuropeptides Proteins 0.000 description 1
- ZCQWOFVYLHDMMC-UHFFFAOYSA-N Oxazole Chemical compound C1=COC=N1 ZCQWOFVYLHDMMC-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- WTKZEGDFNFYCGP-UHFFFAOYSA-N Pyrazole Chemical compound C=1C=NNC=1 WTKZEGDFNFYCGP-UHFFFAOYSA-N 0.000 description 1
- 101150060955 RAB11A gene Proteins 0.000 description 1
- 102100022873 Ras-related protein Rab-11A Human genes 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 101150072409 VAMP7 gene Proteins 0.000 description 1
- 241000545067 Venus Species 0.000 description 1
- 102000003786 Vesicle-associated membrane protein 2 Human genes 0.000 description 1
- 108090000169 Vesicle-associated membrane protein 2 Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 150000001345 alkine derivatives Chemical class 0.000 description 1
- 125000004103 aminoalkyl group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- ZSIQJIWKELUFRJ-UHFFFAOYSA-N azepane Chemical compound C1CCCNCC1 ZSIQJIWKELUFRJ-UHFFFAOYSA-N 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 125000000499 benzofuranyl group Chemical group O1C(=CC2=C1C=CC=C2)* 0.000 description 1
- 125000001164 benzothiazolyl group Chemical group S1C(=NC2=C1C=CC=C2)* 0.000 description 1
- 125000004196 benzothienyl group Chemical group S1C(=CC2=C1C=CC=C2)* 0.000 description 1
- WVDDGKGOMKODPV-UHFFFAOYSA-N benzyl alcohol Substances OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 1
- QHXLIQMGIGEHJP-UHFFFAOYSA-N boron;2-methylpyridine Chemical compound [B].CC1=CC=CC=N1 QHXLIQMGIGEHJP-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000004656 cell transport Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 150000001924 cycloalkanes Chemical class 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 125000004663 dialkyl amino group Chemical group 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical group OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- LIKFHECYJZWXFJ-UHFFFAOYSA-N dimethyldichlorosilane Chemical compound C[Si](C)(Cl)Cl LIKFHECYJZWXFJ-UHFFFAOYSA-N 0.000 description 1
- UUKHCUPMVISNFW-UHFFFAOYSA-L disodium;4-formylbenzene-1,3-disulfonate Chemical compound [Na+].[Na+].[O-]S(=O)(=O)C1=CC=C(C=O)C(S([O-])(=O)=O)=C1 UUKHCUPMVISNFW-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000004651 endocytosis pathway Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 125000002541 furyl group Chemical group 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 150000002430 hydrocarbons Chemical group 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
- 229940029339 inulin Drugs 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- WGOPGODQLGJZGL-UHFFFAOYSA-N lithium;butane Chemical compound [Li+].CC[CH-]C WGOPGODQLGJZGL-UHFFFAOYSA-N 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- DCQJQVFIEFMTTQ-UHFFFAOYSA-M magnesium;1,3-dimethylbenzene-2-ide;bromide Chemical compound [Mg+2].[Br-].CC1=CC=CC(C)=[C-]1 DCQJQVFIEFMTTQ-UHFFFAOYSA-M 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- MZRVEZGGRBJDDB-UHFFFAOYSA-N n-Butyllithium Substances [Li]CCCC MZRVEZGGRBJDDB-UHFFFAOYSA-N 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 230000030589 organelle localization Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical group 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 108060006184 phycobiliprotein Proteins 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 125000005412 pyrazyl group Chemical group 0.000 description 1
- 125000005495 pyridazyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- ADPUQRRLAAPXGT-UHFFFAOYSA-M sodium;2-formylbenzenesulfonate Chemical compound [Na+].[O-]S(=O)(=O)C1=CC=CC=C1C=O ADPUQRRLAAPXGT-UHFFFAOYSA-M 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 150000003408 sphingolipids Chemical class 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000004895 subcellular structure Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- LYHLHZACQVOWMX-UHFFFAOYSA-N tert-butyl 4-bromo-3,5-dimethylbenzoate Chemical compound CC1=CC(C(=O)OC(C)(C)C)=CC(C)=C1Br LYHLHZACQVOWMX-UHFFFAOYSA-N 0.000 description 1
- CWXPZXBSDSIRCS-UHFFFAOYSA-N tert-butyl piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCNCC1 CWXPZXBSDSIRCS-UHFFFAOYSA-N 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M thiocyanate group Chemical group [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 1
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical group OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 description 1
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B11/00—Diaryl- or thriarylmethane dyes
- C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
- C09B11/26—Triarylmethane dyes in which at least one of the aromatic nuclei is heterocyclic
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
- G01N21/80—Indicating pH value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/0803—Compounds with Si-C or Si-Si linkages
- C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
- C07F7/0812—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/10—Compounds having one or more C—Si linkages containing nitrogen having a Si-N linkage
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/12—Organo silicon halides
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B11/00—Diaryl- or thriarylmethane dyes
- C09B11/28—Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7769—Measurement method of reaction-produced change in sensor
- G01N2021/7786—Fluorescence
Definitions
- the present invention relates to a novel pH-sensitive fluorescent probe and a method for measuring an acidic region in a cell using the same.
- the cells maintain a vital function by performing various biochemical reactions such as metabolism of incorporated proteins and organic compounds, synthesis and transport of cell components, with high spatiotemporal resolution.
- biochemical reactions such as metabolism of incorporated proteins and organic compounds, synthesis and transport of cell components, with high spatiotemporal resolution.
- organelles organelles
- each organelle maintains a unique pH optimum for various biochemical reactions.
- the function of the organelle changes greatly if the pH of the organelle changes.
- the endosomal pH is 6.3 (early endosome) ⁇ 5.5 (late endosome) ⁇ 4.7 (lysosome), which is acidified according to endosomal maturation.
- the biochemical reaction occurring in the endosome also changes from protein selection (early endosome) to protein degradation (lysosome).
- the intracellular pH is greatly related to the chemical reaction occurring in the cell, measuring the intracellular pH is important for elucidating the life phenomenon occurring in the cell.
- a pH sensitive fluorescent probe (hereinafter referred to as a pH probe) based on an organic small molecule fluorescent dye or a fluorescent protein is used.
- the pH probe has a feature that the fluorescence characteristic changes greatly with the nearby pH change. By detecting this change in fluorescence characteristics with a device such as a fluorescence microscope or a plate reader, it is possible to easily measure the pH in cells that remain alive.
- the Off / On-type pH probe has an advantage that it can be used even in a relatively simple optical system because excitation and fluorescence detection are each performed with only one wavelength.
- the increase or decrease of the probe concentration such as cell contraction or probe leakage outside the cell is observed as an increase or decrease in fluorescence intensity, that is, a change in pH
- the pH inside the cell is adjusted using an Off / On type pH probe. It is difficult to measure quantitatively.
- the ratio type pH probe performs excitation or fluorescence detection at two wavelengths, calculates the ratio of the fluorescence intensities (ratio), and observes the change in the ratio value as a pH change. Therefore, in order to use the ratio type pH probe, an optical system suitable for the ratio measurement such as an excitation light switching device and a plurality of fluorescence detectors is required. On the other hand, as an excellent feature of the ratio type pH probe, the ratio value does not change even if the probe concentration fluctuates, so that measurement errors due to factors other than pH change can be reduced. Therefore, intracellular pH can be quantitatively measured by using a ratio type pH probe. Therefore, the ratio type pH probe has greatly contributed to the elucidation of the life phenomenon accompanied by the pH change.
- ratio-type pH probes that are widely used in biological research include semephathorhofluor (SNARF) s shown in FIG. 1 and 2 ′, 7′-Bis- (2-carboxyethyl) -5- (and-6-) carboxyfluorescein.
- SNARF semephathorhofluor
- BCECF carboxyfluorescein
- the problems with SNARF-1 are that the quantum yield of the fluorescence is relatively low (0.03 on the acidic side, 0.09 on the basic side), and that there is temperature and environmental sensitivity (when 25 ° C. ⁇ 37 ° C., the fluorescence is It is said that it decreases by 25%, and the fluorescence decreases even if it interacts with a protein), and is likely to cause photofading (Non-patent Document 2).
- the pK a to 6.3, a derivative of SNARF with reduced SNARF-4F are also used to acidification of the cytoplasm to visualize.
- BCECF shows a large absorbance / fluorescence quantum yield on the basic side, but a small absorbance / fluorescence quantum yield on the acidic side, and it is difficult to perform ratio imaging due to a large difference in luminance. Like some fluorescein, it is said to be easy to light fade. Further, BCECF derivatives having different pK a is not commercially available.
- the ratio-type pH probe has been developed on the basis of a semephathodohydrofluor or a fluorescein skeleton, which has a low fluorescence quantum yield, is dependent on temperature and environment, and easily causes photofading. Therefore, there are problems that imaging for a long time is difficult, and it is difficult to measure an accurate pH due to environmental factors such as a difference in ambient temperature and an organelle environment. Moreover, it is difficult to apply the structural modifications to the probe molecule, pH probe derivatives having various pK a by organic chemical modification was hardly developed.
- the present invention provides a pH probe having high fluorescence quantum yield, high resistance to photofading, and suitable for visualization of various pH environments in cells such as weakly basic, neutral, and weakly acidic environments.
- the purpose is to do.
- the present inventors have used a Si-based rhodamine skeleton having a high fluorescence quantum yield that has been developed by the present inventors in recent years and having high resistance to photofading.
- a ratio type pH probe we considered that it would be able to solve the problems of existing pH probes, and when various studies were conducted, a piperazine ring was introduced into the skeleton of an asymmetric Si-type rhodamine, and further on the piperazine ring amino group. It was found that the pKa of the pH probe can be easily adjusted by introducing an electron-withdrawing group into the present invention, thereby completing the present invention.
- R 1 represents a hydrogen atom or 1 to 3 identical or different monovalent substituents present on the benzene ring
- R 2a and R 2b each independently represent hydrogen or a monovalent substituent, provided that both R 2a and R 2b are not hydrogen
- R 3 and R 4 each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom
- R 5 and R 6 if present, each independently represents an alkyl group or aryl group having 1 to 6 carbon atoms,
- R 5 and R 6 when X is an oxygen atom, R 5 and R 6 do not exist, When X is a phosphorus atom, one of —R 5 and —R 6 may be ⁇ O
- R 7 and R 8 each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom
- R 9 and R 10 each independently represent a hydrogen atom or an alkyl group
- alkyl alkenyl having 2 to 6 carbon atoms, or alkynyl having 2 to 6 carbon atoms, aralkyl group having 6 to 10 carbon atoms, or alkyl-substituted alkenyl group having 6 to 10 carbon atoms
- Y represents a substituted or unsubstituted aryl group or heteroaryl group
- X represents a silicon atom, an oxygen atom, a carbon atom, a phosphorus atom or a germanium atom
- m is an integer of 0-6.
- a salt thereof [2] The compound or salt thereof according to [1], wherein Y is a substituted or unsubstituted phenyl group.
- At least one of R 1 is a carboxy group, an alkyl group having a carboxy group, an ester group, an alkyl ester group, an amino group, an amide group, an alkylamino group, an isothiocyanate group, a sulfonyl chloride group, a haloalkyl group, a halo
- At least one of R 1 is a carboxy group, an alkyl group having a carboxy group, an ester group, an alkyl ester group, an amino group, an amide group, an alkylamino group, an isothiocyanate group, a sulfonyl chloride group, a haloalkyl group, a halo
- the compound or a salt thereof according to any one of [10] to [13], which is selected from an acetamide group, an azide group, or an alkynyl group.
- the compound or salt thereof according to [14], wherein at least one of R 1 is a carboxy group, an alkyl group having a carboxyl group, an amino group, or an amide group.
- R 3 and R 4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom
- R 5 and R 6 if present, each independently represents an alkyl group or aryl group having 1 to 6 carbon atoms,
- X is an oxygen atom
- R 5 and R 6 do not exist
- X is a phosphorus atom
- one of —R 5 and —R 6 may be ⁇ O
- R 7 and R 8 each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom
- R 9 and R 10 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R 9 or R 10 together with R 3 or R 7 may form a 5- to 7-membered heterocyclyl or heteroaryl containing the nitrogen atom to which R 9 or R 10 is attached, It may contain 1 to 3 further heteroatoms selected
- Y represents a substituted or unsubstituted aryl group or heteroaryl group
- X represents a silicon atom, an oxygen atom, a carbon atom, a phosphorus atom or a germanium atom
- m is an integer from 0 to 6
- X ′ represents a structure after a functional group capable of introducing a label site or a target accumulation site is bonded to T
- T represents a bridging group, and the bridging group can be bonded to a functional group, a label site or a target accumulation site capable of introducing a label site or a target accumulation site at one or both ends thereof.
- R 1 ′ is hydrogen or the same or different monovalent substituent
- R 2a and R 2b each independently represent hydrogen or a monovalent substituent, provided that both R 2a and R 2b are not hydrogen, or (ii) one of R 2a and R 2b is X′-T and the other of R 2a and R 2b is a monovalent substituent
- n is an integer of 0 to 2
- R 3 and R 4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom
- R 5 and R 6 if present, each independently represents an alkyl group or aryl group having 1 to 6 carbon atoms,
- X is an oxygen atom
- R 5 and R 6 do not exist
- X is a phosphorus atom
- one of —R 5 and —R 6 may be ⁇ O
- R 7 and R 8 each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom
- R 9 and R 10 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R 9 or R 10 together with R 3 or R 7 may form a 5- to 7-membered heterocyclyl or heteroaryl containing the nitrogen atom to which R 9 or R 10 is attached, It may contain 1 to 3 further heteroatoms selected
- alkyl alkenyl having 2 to 6 carbon atoms, or alkynyl having 2 to 6 carbon atoms, aralkyl group having 6 to 10 carbon atoms, or alkyl-substituted alkenyl group having 6 to 10 carbon atoms
- Y represents a substituted or unsubstituted aryl group or heteroaryl group
- X represents a silicon atom, an oxygen atom, a carbon atom, a phosphorus atom or a germanium atom
- m is an integer from 0 to 6
- X ′ represents a structure after a functional group capable of introducing a biopolymer labeling site is bonded to T; T ′, if present, is the structure after the bridging group is attached to S; S.
- R 1 ′ is hydrogen or the same or different monovalent substituent
- R 2a and R 2b are (I) each independently represents hydrogen or a monovalent substituent, provided that both R 2a and R 2b are not hydrogen, or (ii) one of R 2a and R 2b is X ′-(T ′ ) -S and the other of R 2a and R 2b is a monovalent substituent
- n is an integer of 0 to 2
- [26] A method for measuring an acidic region in a cell, (A) introducing the compound or salt thereof according to any one of [1] to [24] into a cell, and (b) measuring fluorescence emitted from the compound or salt thereof in the cell. Including methods. [27] The method according to [26], wherein an acidic region where intracellular acidic organelles are present is measured. Is provided.
- a pH probe that has a high fluorescence quantum yield, has high resistance to light fading, and is suitable for visualization of the intracellular pH environment in a weakly acidic environment.
- the pKa can be further lowered or raised. It is possible to provide a group of pH probes suitable for visualization of various pH environments in cells such as weakly basic, neutral, and weakly acidic environments.
- an “alkyl group” or an alkyl part of a substituent containing an alkyl part (such as an alkoxy group), for example, unless otherwise specified, has, for example, 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, More preferably, it means an alkyl group composed of straight, branched, cyclic, or a combination thereof having about 1 to 3 carbon atoms.
- alkyl group for example, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, cyclopropyl
- methyl group, an n-pentyl group, an n-hexyl group and the like can be mentioned.
- halogen atom may be any of a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom, a chlorine atom, or a bromine atom.
- One embodiment of the present invention is a compound represented by the following general formula (I) or a salt thereof (hereinafter also referred to as “embodiment 1”).
- R 1 represents a hydrogen atom or 1 to 3 identical or different monovalent substituents present on the benzene ring.
- R 1 is introduced at three positions on the benzene ring other than the positions substituted by R 2a and R 2b .
- R 1 represents a monovalent substituent present on the benzene ring, it is preferable that about 1 to 2 substituents which are the same or different exist on the benzene ring.
- R 1 represents one or more monovalent substituents, the substituent can be substituted at any position on the benzene ring.
- R 1 represents a hydrogen atom (ie, all of R 1 are hydrogen atoms) or when one substituent is present (ie, one of R 1 is a monovalent substituent, otherwise Is a hydrogen atom).
- the type of monovalent substituent represented by R 1 is not particularly limited, and examples thereof include alkyl groups having 1 to 6 carbon atoms, alkenyl groups having 1 to 6 carbon atoms, alkynyl groups having 1 to 6 carbon atoms, carbon It is preferably selected from the group consisting of several to six alkoxy groups, hydroxyl groups, carboxy groups, sulfonyl groups, alkoxycarbonyl groups, halogen atoms, or amino groups. These monovalent substituents may further have one or more arbitrary substituents.
- the alkyl group represented by R 1 may have one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, and the like.
- the alkyl group represented by R 1 is a halogen atom.
- An alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or an aminoalkyl group may be used.
- the amino group represented by R 1 may be present one or two alkyl groups, an amino group represented by R 1 may be a monoalkylamino group or a dialkylamino group.
- examples of the case where the alkoxy group represented by R 1 has a substituent include a carboxy-substituted alkoxy group or an alkoxycarbonyl-substituted alkoxy group, and more specifically, a 4-carboxybutoxy group or 4-acetoxymethyloxy group.
- a carbonyl butoxy group etc. can be mentioned.
- R 1 is a monovalent substituent such as an alkyl group having 1 to 6 carbon atoms, and the substituent is present at the 3-position to 5-position on the benzene ring.
- R 1 is a functional group capable of introducing a label site or a target accumulation site (also referred to as “biopolymer labeling site”).
- the functional group capable of introducing a label site or a target accumulation site means a functional group capable of reacting with the label site or the target accumulation site, and examples thereof include a carboxyl group and an alkyl group having a carboxyl group. Ester group, alkyl ester group, amino group, amide group, alkylamino group, isothiocyanate group, sulfonyl chloride group, haloalkyl group, haloacetamide group, azide group, alkynyl group, etc.
- R 1 is a functional group capable of introducing a label site or a target accumulation site, the above monovalent substituent (the number of carbon atoms) even if other R 1 is hydrogen
- At least one of R 1 is a functional group capable of introducing a label site or a target accumulation site, and the functional group is present at the 3-position to the 5-position on the benzene ring.
- At least one of R 1 is a functional group capable of introducing a label site or a target accumulation site (particularly preferably, a carboxyl group, an alkyl group having a carboxyl group, an amino group, an amide group). And the other R 1 is hydrogen.
- R 2a and R 2b each independently represent hydrogen or a monovalent substituent. However, both R 2a and R 2b are not hydrogen.
- the type of the monovalent substituent represented by R 2a and R 2b is not particularly limited, but for example, as in R 1 , for example, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, It is selected from the group consisting of 1 to 6 alkynyl groups, alkoxy groups having 1 to 6 carbon atoms, hydroxyl group, carboxy group, sulfonyl group, alkoxycarbonyl group, halogen atom, or amino group.
- the monovalent substituent in R 2a and R 2b is an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom.
- one of R 2a and R 2b is hydrogen and the other is an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom.
- both R 2a and R 2b are alkyl groups having 1 to 6 carbon atoms.
- the monovalent substituent represented by R 2a and R 2b may be a functional group capable of introducing a label site or a target accumulation site.
- functional groups capable of introducing a label site or a target accumulation site are carboxyl group, alkyl group having carboxyl group, ester group, alkyl ester group, amino group, amide group, alkylamino group, isothiocyanate group, Examples include a sulfonyl chloride group, a haloalkyl group, a haloacetamide group, an azide group, an alkynyl group, and a carboxyl group, an alkyl group having a carboxyl group, an amino group, and an amide group are particularly preferable.
- one of R 2a and R 2b is a monovalent substituent (preferably an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom).
- the other is a functional group capable of introducing a label site or a target accumulation site.
- at least one of R 1 may be a functional group capable of introducing a label site or a target accumulation site, all R 1 may be hydrogen, or at least one of R 1 One may be a monovalent substituent such as an alkyl group having 1 to 6 carbon atoms, and the remaining R 1 may be hydrogen.
- R 3 and R 4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
- the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups,
- the alkyl group represented by R 3 or R 4 may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or the like.
- R 3 and R 4 are each independently preferably a hydrogen atom or a halogen atom. When R 3 and R 4 are both hydrogen atoms, or R 3 and R 4 are both fluorine atoms or chlorine atoms. More preferred.
- R 5 and R 6 each independently represent an alkyl group or aryl group having 1 to 6 carbon atoms, but R 5 and R 6 are each independently An alkyl group having 1 to 3 carbon atoms is preferred, and R 5 and R 6 are both preferably methyl groups.
- the alkyl group represented by R 5 and R 6 may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, and the like, for example, R 5 or R 6 represents The alkyl group may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or the like.
- the aryl group may be either a monocyclic aromatic group or a condensed aromatic group, and the aryl ring has one or more ring-constituting heteroatoms.
- a nitrogen atom, an oxygen atom, or a sulfur atom may be contained.
- the aryl group is preferably a phenyl group.
- One or more substituents may be present on the aryl ring. As the substituent, for example, one or two or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups and the like may be present.
- R 5 and R 6 are absent.
- X is a phosphorus atom
- one of —R 5 and —R 6 may be ⁇ O.
- one of —R 5 and —R 6 is ⁇ O, and the other represents an alkyl group or aryl group having 1 to 6 carbon atoms.
- R 7 and R 8 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom, and are the same as those described for R 3 and R 4. is there. It is preferable that R 7 and R 8 are both hydrogen atoms, both chlorine atoms, or both fluorine atoms.
- R 9 and R 10 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
- R 9 or R 10 may be combined with R 3 or R 7 to form a 5- to 7-membered heterocyclyl or heteroaryl containing a nitrogen atom to which R 9 or R 10 is bonded.
- it may contain 1 to 3 further heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom as a ring member, and the heterocyclyl or heteroaryl has 1 to 6 carbon atoms.
- Alkyl alkenyl having 2 to 6 carbon atoms, or alkynyl having 2 to 6 carbon atoms, aralkyl group having 6 to 10 carbon atoms (benzyl group, phenethyl group, etc.), alkyl substitution having 6 to 10 carbon atoms It may be substituted with an alkenyl group.
- heterocyclyl or heteroaryl thus formed include, but are not limited to, pyrrolidine, piperidine, hexamethyleneimine, pyrrole, imidazole, pyrazole, oxazole, thiazole and the like.
- Y represents a substituted or unsubstituted aryl group or heteroaryl group.
- the pKa of the piperazine ring amino group can be changed by introducing an aryl group or heteroaryl group, which is an electron-withdrawing group, onto the piperazine ring amino group, which facilitates the pKa of the pH probe. It becomes possible to adjust to.
- the aryl group include a phenyl group and a naphthyl group, and a phenyl group is preferable.
- heteroaryl group examples include pyridyl group, pyrazyl group, pyrimidyl group, pyridazyl group, indolyl group, benzofuranyl group, benzothienyl group, benzothiazolyl group, pyrrolyl group, furanyl group, thienyl group, imidazolyl group, and thiazolyl group.
- a pyridyl group is preferred.
- the pKa is further reduced or increased by further introducing an electron withdrawing group or electron donating group into the aryl group or heteroaryl group introduced onto the piperazine ring amino group. It is possible to make adjustments.
- the substituent introduced into the aryl group or heteroaryl group includes, as an electron withdrawing group, a nitro group, a sulfonyl group, a carbonyl group, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom or an iodine atom), a carbon number of 1 to 6 alkoxy groups and the like, and a fluorine atom and a sulfonyl group are preferable.
- the aryl group or heteroaryl group can have two or more of the electron withdrawing groups described above, and these electron withdrawing groups may be the same or different.
- Examples of the electron donating group include an amino group, a methoxy group, and an alkyl group having 1 to 6 carbon atoms, including a t-butyl group, a sec-butyl group, an n-butyl group, an iso-propyl group, an n-propyl group, An ethyl group, a methyl group, an amino group, and a methoxy group are preferable.
- the aryl group or heteroaryl group can have two or more of the above electron donating groups, and these electron donating groups may be the same or different.
- m is an integer of 0-6.
- m is preferably 0 or 1 in order to obtain an effective electron withdrawing effect of the aryl group or heteroaryl group introduced as Y.
- One preferred embodiment of the present invention is when Y is a substituted or unsubstituted phenyl group and m is 1, that is, a substituted or unsubstituted benzyl group is introduced into the piperazine ring amino group.
- One preferred aspect of the present invention is a compound or a salt thereof in which Y is a phenyl group substituted with a fluorine atom in the general formula (I), and m is 1.
- One preferable aspect of the present invention is a compound or a salt thereof in which Y is a phenyl group substituted with a sulfonyl group in the general formula (I), and m is 1.
- One preferable aspect of the present invention is a compound or a salt thereof in which Y is a phenyl group substituted with two sulfonyl groups in the general formula (I), and m is 1.
- X represents a silicon atom, oxygen atom, carbon atom, phosphorus atom or germanium atom, preferably a silicon atom or germanium atom, and particularly preferably a silicon atom.
- One aspect of embodiment 1 of the present invention is the following general formula (II): Or a salt thereof.
- R 1 to R 2b , R 4 to R 8 , R 10 , X, Y and m are as described above for the general formula (I).
- R 11 to R 14 each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. In one preferred embodiment of the present invention, R 11 to R 14 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms (preferably a methyl group or an ethyl group). In one preferred embodiment of the present invention, R 11 to R 14 are all hydrogen atoms.
- Non-limiting examples of compounds of general formula (I) or (II) of the present invention include the following compounds:
- the compounds represented by the general formulas (I) and (II) of the present invention can exist as acid addition salts or base addition salts.
- the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, or organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate.
- the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, organic amine salts such as ammonium salt, and triethylamine salt.
- a salt with an amino acid such as glycine may be formed.
- the compounds of the present invention or salts thereof may exist as hydrates or solvates, and these substances are also within the scope of the present invention.
- the compounds represented by the general formulas (I) and (II) of the present invention may have one or two or more asymmetric carbons depending on the type of substituent, but one or two or more asymmetric carbons may be present.
- stereoisomers such as optically active substances based on asymmetric carbons and diastereoisomers based on two or more asymmetric carbons, any mixture of stereoisomers, racemates, etc. are all included in the scope of the present invention. Is done.
- At least one of R 1 in the general formula (I) or (II) is a functional group capable of introducing a label site or a target accumulation site, and the functional group is A compound or a salt thereof bonded to a crosslinking group (hereinafter also referred to as “embodiment 2”).
- a functional group capable of introducing a label site or a target accumulation site a carbonyl group, an alkylcarbonyl group, an ester group, an alkyl ester group, an amino group, an alkylamino group, an amide group, an isothiocyanate group, a chloride group
- a sulfonyl group, a haloalkyl group, a haloacetamido group, an azide group, an alkynyl group and the like can be mentioned, and a carbonyl group and an alkylcarbonyl group are particularly preferable.
- the crosslinking group may be any crosslinking group as long as it functions as a spacer for linking the functional group capable of introducing the label site or target accumulation site and the label site or target accumulation site.
- substituted or unsubstituted hydrocarbon groups alkanes, alkenes, alkynes, cycloalkanes, aromatic hydrocarbons, etc.
- ethylene glycol groups diethylene glycol groups, triethylene glycol groups, polyethylene glycol groups, alkyl cysteates and heterocyclic rings
- a group for example, a piperidinyl group and the like, but are not limited thereto.
- the crosslinking group has a functional group capable of introducing a label site or a target accumulation site, a functional group capable of binding to the label site or the target accumulation site at one or both ends thereof. Also good.
- a functional group include an amino group, a carbonyl group, a carboxyl group, an amide group, a propargyl group, and the like.
- Embodiment 2 is a compound represented by the following general formula (Ia) or a salt thereof.
- R 3 to R 10 , X, Y, and m are as defined in the general formula (I).
- X ′ is a structure after a functional group capable of introducing the above-described label site or target accumulation site is bonded to T, and T is the above-described bridging group.
- the bridging group may have a functional group capable of introducing a label site or a target accumulation site, a functional group capable of binding to the label site or the target accumulation site at one or both ends thereof.
- Examples of such a functional group include an amino group, a carbonyl group, a carboxyl group, an amide group, a propargyl group, and the like.
- R 1 ′ is hydrogen or the same or different monovalent substituent defined as R 1 in the general formula (I), and the details thereof are as described for the compound of the general formula (I).
- R 1 ′ is preferably hydrogen.
- R 2a and R 2b each independently represent hydrogen or a monovalent substituent, provided that both R 2a and R 2b are not hydrogen. Also, one of R 2a and R 2b may be the X'-T, in this case, the other of R 2a and R 2b is a monovalent substituent (preferably, alkyl having 1-6 carbon Group, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom).
- n is an integer of 0 to 2
- one of R 2a and R 2b is X′-T, and the other is a monovalent substituent (preferably an alkyl group having 1 to 6 carbon atoms, 1 to 6 alkoxy groups or halogen atoms).
- — (X′-T) can be introduced at any of the 2-6 positions of the benzene ring.
- n is 1 and p is 2, in which case R 1 ′ may be the same or different.
- one of R 2a and R 2b may be X′-T, in which case the other of R 2a and R 2b is a monovalent substituent (preferably having 1 to 6 carbon atoms).
- Embodiment 2 is a compound represented by the following general formula (IIa) or a salt thereof.
- R 4 to R 8 , R 10 , X, Y and m are as defined in the general formula (I), and R 11 to R 14 are each independently a hydrogen atom, carbon X ′, T, R 1 ′, R 2a to R 2b , n and p are as defined in the general formula (Ia).
- n is 1 and p is 2, in which case R 1 ′ may be the same or different.
- one of R 2a and R 2b may be X′-T, in which case the other of R 2a and R 2b is a monovalent substituent (preferably having 1 to 6 carbon atoms).
- -X'-T in general formula (Ia) or (IIa) is selected from:
- At least one of R 1 in the general formula (I) or (II) is a functional group capable of introducing a label site or a target accumulation site, and the functional group is It is a compound or a salt thereof which is bonded to a crosslinking group, and in which the crosslinking group is further bonded to a label site or a target accumulation site.
- at least one R 1 in the general formula (I) or (II) is a functional group capable of introducing a label site or a target accumulation site, The group is a compound or a salt thereof bound to a label site or a target accumulation site without using a bridging group.
- the two types of embodiments are collectively referred to as “embodiment 3” hereinafter.
- the functional group and the crosslinking group capable of introducing the label site or the target accumulation site are as described in the second embodiment.
- label sites or target accumulation sites include N-hydroxysuccinimide ester, Halo tag ligand (eg, 2- (2-((6-chlorohexyl) oxy) ethoxy) ethaneamino group), weakly basic amine, maleimide, Examples include a thiocyanate group, a sulfonyl chloride group, a haloalkyl group, a haloacetamide group, an azide group, an alkynyl group, a benzylguanine derivative, or a benzylcytosine derivative.
- Halo tag ligand eg, 2- (2-((6-chlorohexyl) oxy) ethoxy) ethaneamino group
- weakly basic amine maleimide
- Examples include a thiocyanate group, a sulfonyl chloride group, a haloalkyl group, a haloacetamide group, an azide group,
- the label site or target accumulation site also includes a polyethylene glycol group that may have a modifying group at one or both ends, and examples of the modifying group include an amino group, a carbonyl group, and a carboxyl group.
- a polyethylene glycol group having a modifying group is 3- (2- (2- (2-aminoethoxy) ethoxy) ethoxy) propanoic acid.
- Embodiment 3 Since the compound included in Embodiment 3 or a salt thereof becomes strongly fluorescent for the first time in an acidic environment, a specific protein or the like can be labeled, and it can be localized in acidic organelle cells. Various phenomena involving intracellular acidic vesicles can be visualized in real time.
- a compound in which a label site or a target accumulation site is introduced into a part of a functional group capable of introducing a label site or a target accumulation site that is, a functional group capable of introducing a label site or a target accumulation site
- a compound having both a label site or a substituent introduced with a target accumulation site are also included in Embodiment 3.
- Embodiment 3 is a compound represented by the following general formula (Ib) or a salt thereof.
- R 3 to R 10 , X, Y and m are as defined in the general formula (I).
- X ′ is a structure after a functional group capable of introducing the above-described label site or target accumulation site is bonded to T, and T ′, if present, is a cross-link described above This is the structure after the group is bonded to S, where S is the label site or target accumulation site.
- R 1 ′ is hydrogen or the same or different monovalent substituent defined as R 1 in the general formula (I), and the details thereof are as described for the compound of the general formula (I).
- R 1 ′ is preferably hydrogen.
- R 2a and R 2b each independently represent hydrogen or a monovalent substituent, provided that both R 2a and R 2b are not hydrogen.
- One of R 2a and R 2b can be X ′-(T ′) — S, and in this case, the other of R 2a and R 2b is a monovalent substituent (preferably having a carbon number of 1 To 6 alkyl groups, alkoxy groups having 1 to 6 carbon atoms or halogen atoms).
- n is an integer of 0 to 2
- one of R 2a and R 2b is X ′-(T ′) — S, and the other is a monovalent substituent (preferably having 1 to 6 carbon atoms).
- n 1 and p is 2.
- R 1 ′ may be the same or different.
- one of R 2a and R 2b may be X ′-(T ′) — S, in which case the other of R 2a and R 2b is a monovalent substituent (preferably An alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom).
- T ′ may or may not be present (if not present, S is directly bonded to X ′).
- the crosslinking group may have a functional group capable of introducing a label site or a target accumulation site, a functional group capable of binding to the label site or the target accumulation site at one or both ends thereof.
- examples of such a functional group include an amino group, a carbonyl group, a carboxyl group, an amide group, and a propargyl group.
- Embodiment 3 is a compound represented by the following general formula (IIb) or a salt thereof.
- R 4 to R 8 , R 10 , X, Y and m are as defined in the general formula (I), and R 11 to R 14 are each independently a hydrogen atom, carbon Represents an alkyl group or a halogen atom having a number of 1 to 6, and X ′, T ′, R 1 ′, S, n and p are as defined in formula (Ib).
- n is 1 and p is 2, in which case R 1 ′ may be the same or different.
- one of R 2a and R 2b may be X ′-(T ′) — S, in which case the other of R 2a and R 2b is a monovalent substituent (preferably An alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom).
- —S in general formula (Ib) or (IIb) is selected from:
- the compounds of general formulas (Ib) and (IIb) can be labeled with a specific protein or the like and can be localized in acidic organelle cells, It becomes possible to visualize various phenomena related to the vesicle in real time.
- N-hydroxysuccinimide ester or polyethylene glycol having a terminal modified with a carboxyl group or the like is introduced into the molecule as a label site, it is labeled with dextran, which is a sugar polymer, It becomes possible to localize the fluorescent probe of the present invention in intracellular acidic vesicles.
- the fluorescent probe of the present invention can be accumulated in acidic vesicles.
- a Halo tag ligand is introduced as a label site, a specific label to the Halo tag becomes possible.
- a protein in which a Halo tag is fused to VAMP2 which is a synaptic vesicle marker (VAMP2-Halo tag) is expressed in nerve cells, and the fluorescent probe of the present invention into which a Halo tag ligand is introduced is added thereto. This allows specific labeling of fluorescent probes to synaptic vesicles.
- the compounds of Embodiments 2 and 3 of the present invention can exist as acid addition salts or base addition salts.
- the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, or organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate.
- the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, organic amine salts such as ammonium salt, and triethylamine salt.
- a salt with an amino acid such as glycine may be formed.
- the compounds of the present invention or salts thereof may exist as hydrates or solvates, and these substances are also within the scope of the present invention.
- the compounds of Embodiments 2 and 3 of the present invention may have one or two or more asymmetric carbons depending on the type of substituent, but they are optically active substances based on one or two or more asymmetric carbons.
- stereoisomers such as diastereoisomers based on two or more asymmetric carbons, any mixture of stereoisomers, racemates, and the like are included in the scope of the present invention.
- Another aspect of the present invention is a fluorescent probe comprising a compound of the general formula (I), (II), (Ia), (IIa), (Ib) or (IIb) or a salt thereof.
- Another aspect of the present invention is a method for measuring an acidic region in a cell, wherein (a) the general formula (I), (II), (Ia), (IIa), (Ib) or (IIb) And a salt thereof, and (b) a step of measuring fluorescence emitted from the compound or salt thereof in the cell.
- One aspect of the present invention is a method for measuring an acidic region in which intracellular acidic organelles exist.
- a cell is contacted with a compound of the general formula (I), (II), (Ia), (IIa), (Ib) or (IIb) or a salt thereof.
- Forming a cell (b) incubating the contacted cell for the compound to enter the cell to form a labeled cell, and (c) forming the labeled cell. Irradiating with an appropriate wavelength at which fluorescence is measured, thereby monitoring the pH inside the cell, thereby monitoring the pH inside the living cell.
- the cells include normal cells, cancer cells, nerve cells and the like. The method according to item.
- a further aspect of the present invention provides a carrier comprising: (a) a carrier molecule bound to a compound of general formula (I), (II), (Ia), (IIa), (Ib) or (IIb) or a salt thereof; Forming a conjugate; (b) contacting the carrier conjugate with a cell to form a contacted cell; and (c) incubating the contacted cell to form an incubated solution.
- a carrier in the solution comprising: (d) irradiating the incubated solution to form an irradiated solution; and (e) detecting fluorescence emission from the irradiated solution.
- Carrier molecules include amino acids, peptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, haptens, psoralens, drugs, hormones, lipids, lipid aggregates, synthetic polymers, polymer microparticles, biological cells, viruses And combinations thereof.
- the carrier molecule is an amino acid (including amino acids that are protected or substituted by phosphates, carbohydrates, or C1-C22 carboxylic acids), or polymers of amino acids (such as peptides or proteins). is there.
- the carrier molecule also comprises at least 5 amino acids, more preferably 5 to 36 amino acids.
- peptides include, but are not limited to, neuropeptides, cytokines, toxins, protease substrates, and protein kinase substrates.
- Other exemplary peptides are organelle localization peptides, i.e., peptides that serve to target bound compounds for localization within specific subcellular structures by cellular transport mechanisms. May function.
- Preferred protein carrier molecules include enzymes, antibodies, lectins, glycoproteins, histones, albumins, lipoproteins, transferrin, avidin, streptavidin, protein A, protein G, phycobiliproteins and other fluorescent proteins, hormones, toxins and Growth factors.
- the protein carrier molecule is an antibody, antibody fragment, avidin, streptavidin, toxin, lectin, growth factor, bacterial particle or cell receptor binding partner.
- nucleic acid polymer carrier molecules are single-stranded or multi-stranded, natural or synthetic DNA or RNA oligonucleotides, or DNA / RNA hybrids, or unusual linkers (morpholine derivatized phosphate (AntiVirals, Inc., Oregon) ), Or peptide nucleic acids (such as N- (2-aminoethyl) glycine units) that contain less than 50 nucleotides, more typically less than 25 nucleotides. .
- the carrier molecule is typically a dextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, polysaccharide such as starch, agarose and cellulose, or poly (ethylene glycol) Contains a carbohydrate or polyol that is a polymer.
- the polysaccharide carrier molecule includes dextran, agarose, or FICOLL.
- the carrier molecule comprises lipids (typically comprising 6-25 carbons) including glycolipids, phospholipids, and sphingolipids.
- the carrier molecule comprises a lipid vesicle (such as a liposome) or it is a lipoprotein (see below). Some lipophilic substituents are useful to facilitate transport of bound dye to cells or organelles.
- Step (a) N-Boc-piperazine (2.41 g, 12.9 mmol) and 2-bromo-4-fluorobenzaldehyde (2.19 g, 10.8 mmol) were dissolved in DMF (30 mL) and K 2 CO 3 (2.24 g, 16.2 mmol) was added and stirred at 100 ° C. for 18 hours. After the solvent was distilled off under reduced pressure, water was added to the residue, and the mixture was extracted with dichloromethane and washed with brine. The organic layer was dried over Na 2 SO 4 and the solvent was distilled off under reduced pressure.
- Step (a) 2,6-diMe PiperaIndoSiR (3.5 mg, 6.03 ⁇ mol) was dissolved in methanol (2 mL), 3-fluorobenzaldehyde (1.3 ⁇ L, 12.06 ⁇ mol) and acetic acid (20 ⁇ L) were added, and the mixture was stirred at room temperature for 10 minutes. .
- Sodium cyanoborohydride (1.5 mg, 24 ⁇ mol) was added to the reaction solution, and the mixture was stirred at room temperature for 16 hours. Water was added to the mixture and the mixture was extracted with dichloromethane. The organic layer was dried over Na 2 SO 4 and the solvent was distilled off under reduced pressure.
- 2,6-diMe PiperaIndoSiR (2.4 mg, 4.16 ⁇ mol) was dissolved in methanol (2 mL) and sodium 2-formylbenzene-1-sulfonate (1.7 mg, 8.32 ⁇ mol) and acetic acid (2.4 ⁇ L, 41.6 ⁇ mol) was added and the mixture was stirred at room temperature for 10 minutes.
- Sodium cyanoborohydride (0.5 mg, 8.32 ⁇ mol) was added to the reaction solution, and the mixture was stirred at room temperature for 13 hours. Water was added to the mixture and the mixture was extracted with dichloromethane. The organic layer was dried over Na 2 SO 4 and the solvent was distilled off under reduced pressure.
- Step (a) Dehydrated tetrahydrofuran (10 mL) was added to tert-butyl 4-bromo-3,5-dimethylbenzoate (182 mg, 0.638 mmol) in a flask that had been dried and purged with argon, and the reaction solution was cooled to ⁇ 78 ° C. 1M sec-BuLi (0.64 mL, 0.64 mL) was added to the reaction solution, and the mixture was stirred at -78 degrees for 30 minutes. BocPiperaIndoSiXanthone (61 mg, 0.128 mmol) was added to the reaction solution, and the mixture was heated to reflux for 2.5 hours.
- Step (b) 4-COOH-2,6-diMe PiperaIndoSiR (12.8 mg, 20.5 ⁇ mol) was dissolved in methanol (5 mL), and disodium 4-formylbenzene-1,3-disulfonate (19.1 mg, 61.5 ⁇ mol). Then, acetic acid (250 ⁇ L) was added and stirred at room temperature for 30 minutes. To the reaction solution, 2-picoline borane (4.4 mg, 41 ⁇ mol) was added and stirred at room temperature for 16 hours.
- Example 5 The spectral characteristics of Compound 1 (2,6-diMe BnPiperaIndoSiR) of the present invention were evaluated. Absorption spectrum, fluorescence spectrum and excitation spectrum were measured using Shimadzu UV-1650PC absorptiometer and Hitachi F-4500 fluorometer. The results are shown in Figures 2a-e.
- FIG. 2a represents the assumed chemical equilibrium equation for 2,6-diMe BnPiperaIndoSiR.
- FIGS. 2b, 2c, and 2d show absorption, fluorescence, and excitation spectra of 2,6-diMe BnPiperaIndoSiR in 100 mM sodium phosphate buffer (containing 1% DMSO) with different pHs, respectively.
- the absorbance at 580 nm decreases and the absorbance at 663 nm increases as the pH of the solvent changes from acidic to basic, indicating that 2,6-diMe BnPiperaIndoSiR functions as a ratio type pH probe.
- the pK a decreased 0.9 by comparing the aliphatic amino group of the piperazine with 2-Me MePiperaIndoSiR by benzylated (compound obtained by introducing a N-methyl piperazine ring). This is thought to be due to the electron withdrawing effect of the benzyl group.
- Example 6 The spectral properties of Compound 2 (SiRpH3) of the present invention were evaluated. Absorption spectrum, fluorescence spectrum and excitation spectrum were measured using Shimadzu UV-1650PC absorptiometer and Hitachi F-4500 fluorometer. The results are shown in FIGS. 3a-c.
- FIG. 3a, 3b, and 3c show absorption, fluorescence, and excitation spectra in 100 mM sodium phosphate buffer (containing 1% DMSO) having different pH values of SiRpH3, respectively.
- FIG. 3d shows a plot of fluorescence intensity ratio (ratio) versus pH divided by 700 nm fluorescence intensity upon excitation at 580 nm divided by fluorescence intensity upon excitation at 663 nm.
- the absorption wavelength of Compound 2 of the present invention was increased from about 580 nm to about 660 nm to about 80 nm as the solution pH was changed from acidic to basic.
- the fluorescence wavelength did not change greatly even when the pH of the solution changed. Therefore, the wavelength of the excitation spectrum also increased greatly as the pH of the solution changed from acidic to basic. Based on this characteristic, it was possible to calculate the pH of the solution in the cuvette by measuring the ratio in the cuvette.
- SiRpH3 measured in 100 mM sodium phosphate buffer are shown in Table 2.
- SiRpH3 substituted with a fluorine atom had absorption maximums at about 580 nm and about 660 nm depending on the pH, similar to 2,6-diMe BnPiperaIndoSiR, and showed a sufficient fluorescence quantum yield as a fluorescent probe.
- Example 7 The spectral characteristics of the compound 3 (SiRpH4) of the present invention were evaluated. Absorption spectrum, fluorescence spectrum and excitation spectrum were measured using Shimadzu UV-1650PC absorptiometer and Hitachi F-4500 fluorometer. The results of the absorption spectrum and excitation spectrum are shown in FIGS. 4a-b.
- FIG. 4a and 4b show absorption and excitation spectra in a 100 mM sodium phosphate buffer (containing 1% DMSO) having different pH values of SiRpH4, respectively.
- FIG. 4c shows a plot of fluorescence intensity ratio (ratio) versus pH divided by 700 nm fluorescence intensity upon excitation at 580 nm divided by fluorescence intensity upon excitation at 663 nm.
- SiRpH4 showed the same optical characteristics as SiRpH3, but due to the ortho-position sulfone group, the pKa of the ratio value change shifted to the basic side compared to SiRpH3.
- Example 8 The spectral characteristics of the compound 4 (SiR pH 5) of the present invention were evaluated. Absorption spectrum, fluorescence spectrum and excitation spectrum were measured using Shimadzu UV-1650PC absorptiometer and Hitachi F-4500 fluorometer. The results of the absorption spectrum and the excitation spectrum are shown in FIGS. 5b to 5c.
- FIG. 5a represents the assumed chemical equilibrium equation for SiRpH5.
- FIGS. 5b and 5c show absorption and excitation spectra in 100 mM sodium phosphate buffer (containing 1% DMSO) having different pH values of SiR pH5, respectively.
- SiRpH5 showed the same optical characteristics as SiRpH4, but the pKa of the ratio value change was shifted to the acidic side compared to SiRpH4 due to the para-position sulfone group, and showed a value suitable for the measurement of acidic organelle.
- Table 3 shows the optical properties of SiRpH5 measured in 100 mM sodium phosphate buffer. SiRpH5 had absorption maximums at about 590 nm and about 670 nm, and showed a sufficient fluorescence quantum yield as a fluorescent probe.
- FIG. 6 shows a plot of ratio value versus pH for each compound.
- each organelle in the cell has a unique pH
- by developing and using pH probe having a pK a suitable pH measurement for each by using the pH probe nucleus organelle more precisely the It is thought that the pH of the organelle can be measured.
- SiRpH5-PEG 6 -SE (Compound 6) having N-hydroxysuccinimidyl ester via a PEG linker and capable of forming a covalent bond with an amino group of a biopolymer was synthesized according to Scheme 6 below.
- Steps (a) and (b) To the flask were added SiRpH5 (15 mg, 17.2 ⁇ mol), N-hydroxysuccinimide (2.2 mg, 18.9 ⁇ mol), EDC hydrochloride (3.6 mg, 18.9 mmol), DMF (2 mL) followed by DIPEA (6. 6 ⁇ L, 37.8 ⁇ mol) was added, and the mixture was stirred at room temperature for 20 hours.
- SiRpH5-PEG 6 -BG (Compound 7) having a benzylguanine structure via a PEG linker and capable of forming a covalent bond with a SNAP-Tag protein was synthesized according to the following scheme 7.
- Step (a) To the flask was added SiRpH5-PEG 6 -SE (1.8 mg, 1.38 ⁇ mol), BG-NH 2 (1.1 mg, 4.14 ⁇ mol), DMF (1 mL), and then DIPEA (1.4 ⁇ L, 8.28 ⁇ mol). Was added and stirred at room temperature for 4 hours.
- Example 11 PH measurement of intracellular organelles using SiRpH5 (A) pH measurement of lysosomes After adding dextran, a polysaccharide, to the extracellular fluid and incorporating it into the cells, the dextran is selectively lysosomally incubated for several hours. It is known to be accumulated in In order to measure the pH of lysosomes using this probe, SiRpH5-Dex was prepared by labeling SiRpH5 with 10 kDa aminodextran.
- Tfn recycling endosome Transferrin
- TfnR transferrin receptor
- SiRpH5-Tfn was prepared by labeling SiRpH5 on holotransferrin, which is an iron ion-transferrin complex.
- Fluorescence derived from SiRpH5-Tfn was observed from the site overlapping with the fusion protein of Rab11 which is a marker protein of recycling endosome and green fluorescent protein GFP.
- Rab11 which is a marker protein of recycling endosome and green fluorescent protein GFP.
- the pH was calculated from the ratio value of the recycling endosome surrounded by the dotted line using the calibration curve, it was calculated to be 5.8. This value was comparable to the reported value for recycling endosomes. From this, it is considered that the pH of various intracellular organelles can be measured by changing the protein that labels the probe.
- the fluorophore Si rhodamine used as the mother nucleus of the pH probe is a fluorescent dye exhibiting high fluorescence quantum yield and light fading resistance. Therefore, the present invention can achieve high-accuracy imaging of target molecules labeled with a small amount of molecules and time-lapse imaging that requires long-time excitation light irradiation, which was difficult with conventional pH probes with low fluorescence quantum yield. It becomes. Moreover, since environmental sensitivity is low, it is expected that highly reliable pH measurement will be possible in various organelles in cells.
- the pH probe of the present invention is expected to be a useful tool for detailed analysis of various life phenomena.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
Off/On型pHプローブは励起、蛍光検出をそれぞれ1波長のみで行うため、比較的単純な光学系でも利用可能という利点がある。しかしながら、細胞の収縮やプローブの細胞外への漏出等のプローブ濃度の増減を蛍光強度の増減すなわちpHの変化として観測してしまうため、Off/On型のpHプローブを用いて細胞内のpHを定量的に測定することは難しい。
[1]以下の一般式(I):
(式中、
R1は、水素原子を示すか、又はベンゼン環上に存在する1ないし3個の同一又は異なる一価の置換基を示し;
R2a及びR2bは、それぞれ独立に、水素又は一価の置換基を示し、但し、R2a及びR2bの両方が水素ではなく;
R3及びR4は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
R5及びR6は、存在する場合は、それぞれ独立に、炭素数1~6個のアルキル基又はアリール基を示し、
ここで、Xが酸素原子の場合は、R5及びR6は存在せず、
Xがリン原子の場合は、-R5及び-R6の一方は、=Oであってもよい;
R7及びR8は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
R9及びR10は、それぞれ独立に、水素原子又は炭素数1~6個のアルキル基を示し、
R9又はR10は、R3又はR7と一緒になって、R9又はR10が結合している窒素原子を含む5~7員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される1~3個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数1~6個のアルキル、炭素数2~6個のアルケニル、又は炭素数2~6個のアルキニル、炭素数6~10個のアラルキル基、炭素数6~10個のアルキル置換アルケニル基で置換されていてもよく;
Yは、置換又は無置換のアリール基又はヘテロアリール基を示し;
Xは、珪素原子、酸素原子、炭素原子、リン原子又はゲルマニウム原子を示し;
mは、0~6の整数である。)
で表される化合物又はその塩。
[2]Yが置換又は無置換のフェニル基である、[1]に記載の化合物又はその塩。
[3]Yがフェニル基、フッ素原子で置換されたフェニル基、又はスルホニル基で置換されたフェニル基である、[2]に記載の化合物又はその塩。
[4]mが0又は1である、[1]~[3]のいずれか1項に記載の化合物又はその塩。
[5]R1の少なくとも1つは、カルボキシ基、カルボキシ基を有するアルキル基、エステル基、アルキルエステル基、アミノ基、アミド基、アルキルアミノ基、イソチオシアネート基、塩化スルホニル基、ハロアルキル基、ハロアセトアミド基、アジド基、又はアルキニル基から選ばれる、[1]~[4]のいずれか1項に記載の化合物又はその塩。
[6]R1の少なくとも1つは、カルボキシ基、カルボキシル基を有するアルキル基、アミノ基、アミド基である、[5]に記載の化合物又はその塩。
[7]R2a及びR2bにおける一価の置換基が、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である、[1]~[6]のいずれか1項に記載の化合物又はその塩。
[8]R2a及びR2bの一方は水素であり、他方は炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である、[7]に記載の化合物又はその塩。
[9]R2a及びR2bの両方が炭素数1~6個のアルキル基である、[7]に記載の化合物又はその塩。
[10]以下の一般式(II):
(式中、
R1~R2b、R4~R8、R10、X、Y及びmは、一般式(I)で定義したとおりであり、R11~R14は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示す。)
で表される、[1]に記載の化合物又はその塩。
[11]Yが置換又は無置換のフェニル基である、[10]に記載の化合物又はその塩。
[12]Yがフェニル基、フッ素原子で置換されたフェニル基、又はスルホニル基で置換されたフェニル基である、[11]に記載の化合物又はその塩。
[13]mが0又は1である、[11]又は[12]に記載の化合物又はその塩。
[14]R1の少なくとも1つは、カルボキシ基、カルボキシ基を有するアルキル基、エステル基、アルキルエステル基、アミノ基、アミド基、アルキルアミノ基、イソチオシアネート基、塩化スルホニル基、ハロアルキル基、ハロアセトアミド基、アジド基、又はアルキニル基から選ばれる、[10]~[13]のいずれか1項に記載の化合物又はその塩。
[15]R1の少なくとも1つは、カルボキシ基、カルボキシル基を有するアルキル基、アミノ基、アミド基である、[14]に記載の化合物又はその塩。
[16]R2a及びR2bにおける一価の置換基が、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である、[10]~[15]のいずれか1項に記載の化合物又はその塩。
[17]R2a及びR2bの一方は水素であり、他方は炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である、[16]に記載の化合物又はその塩。
[18]R2a及びR2bの両方が炭素数1~6個のアルキル基である、[16]に記載の化合物又はその塩。
[19]以下の一般式(Ia):
(式中、
R3及びR4は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基、又はハロゲン原子を示し;
R5及びR6は、存在する場合は、それぞれ独立に、炭素数1~6個のアルキル基又はアリール基を示し、
ここで、Xが酸素原子の場合は、R5及びR6は存在せず、
Xがリン原子の場合は、-R5及び-R6の一方は、=Oであってもよい;
R7及びR8は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
R9及びR10は、それぞれ独立に、水素原子又は炭素数1~6個のアルキル基を示し、
R9又はR10は、R3又はR7と一緒になって、R9又はR10が結合している窒素原子を含む5~7員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される1~3個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数1~6個のアルキル、炭素数2~6個のアルケニル、又は炭素数2~6個のアルキニル、炭素数6~10個のアラルキル基、炭素数6~10個のアルキル置換アルケニル基で置換されていてもよく;
Yは、置換又は無置換のアリール基又はヘテロアリール基を示し;
Xは、珪素原子、酸素原子、炭素原子、リン原子又はゲルマニウム原子を示し;
mは、0~6の整数であり;
X’は、ラベル部位又は標的集積部位を導入することが可能な官能基がTと結合した後の構造を示し;
Tは、架橋基を示し、該架橋基は、その片方または両方の端部に、ラベル部位又は標的集積部位を導入することが可能な官能基、ラベル部位又は標的集積部位と結合することができる官能基を有してもよい;
R1’は、水素、又は同一又は異なる一価の置換基であり;
(i)R2a及びR2bは、それぞれ独立に、水素又は一価の置換基を示し、但し、R2a及びR2bの両方が水素ではなく、又は
(ii)R2a及びR2bの一方は、X’-Tであり、R2a及びR2bの他方は、一価の置換基であり;
nは0~2の整数であり、pは1~3の整数であり、但し、n+p=3であり、ここで、nが0の場合は、R2a及びR2bの一方は、X’-Tであり、他方は、一価の置換基である。)
で表される、化合物又はその塩。
[20]以下の一般式(IIa):
(式中、
R4~R8、R10、X、Y及びmは、一般式(Ia)で定義した通りであり;
R11~R14は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
X’、T、R1’、R2a~R2b、n及びpは一般式(Ia)で定義した通りである。)
[19]に記載の化合物又はその塩。
[21]-X’-Tは、以下から選択される、[19]又は[20]に記載の化合物又はその塩。
[22]以下の一般式(Ib):
(式中、
R3及びR4は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基、又はハロゲン原子を示し;
R5及びR6は、存在する場合は、それぞれ独立に、炭素数1~6個のアルキル基又はアリール基を示し、
ここで、Xが酸素原子の場合は、R5及びR6は存在せず、
Xがリン原子の場合は、-R5及び-R6の一方は、=Oであってもよい;
R7及びR8は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
R9及びR10は、それぞれ独立に、水素原子又は炭素数1~6個のアルキル基を示し、
R9又はR10は、R3又はR7と一緒になって、R9又はR10が結合している窒素原子を含む5~7員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される1~3個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数1~6個のアルキル、炭素数2~6個のアルケニル、又は炭素数2~6個のアルキニル、炭素数6~10個のアラルキル基、炭素数6~10個のアルキル置換アルケニル基で置換されていてもよく;
Yは、置換又は無置換のアリール基又はヘテロアリール基を示し;
Xは、珪素原子、酸素原子、炭素原子、リン原子又はゲルマニウム原子を示し;
mは、0~6の整数であり;
X’は、生体高分子標識部位を導入することが可能な官能基がTと結合した後の構造を示し;
T’は、存在する場合は、架橋基がSと結合した後の構造であり;
Sは。ラベル部位又は標的集積部位を示し;
R1’は、水素、又は同一又は異なる一価の置換基であり;
R2a及びR2bは、
(i)それぞれ独立に、水素又は一価の置換基を示し、但し、R2a及びR2bの両方が水素ではなく、又は
(ii)R2a及びR2bの一方は、X’-(T’)-Sであり、R2a及びR2bの他方は、一価の置換基であり;
nは0~2の整数であり、pは1~3の整数であり、但し、n+p=3であり、ここで、nが0の場合は、R2a及びR2bの一方は、X’-(T’)-Sであり、他方は、一価の置換基である。)
で表される化合物又はその塩。
[23]以下の一般式(IIb):
(式中、
R4~R8、R10、X、Y及びmは、一般式(Ib)で定義した通りであり;
R11~R14は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
X’、T’、R1’、S、R2a~R2b、n及びpは一般式(Ib)で定義した通りである。)
で表される、[23]に記載の化合物又はその塩。
[24]-Sは、以下から選択される、[22]又は[23]に記載の化合物又はその塩。
[25][1]~[24]のいずれか1項に記載の化合物又はその塩を含む蛍光プローブ。
[26]細胞内の酸性領域の測定方法であって、
(a)[1]~[24]のいずれか1項に記載の化合物又はその塩を細胞内に導入する工程、及び
(b)当該化合物又はその塩が細胞内で発する蛍光を測定する工程
を含む方法。
[27]細胞内酸性小器官の存在する酸性領域を測定する[26]に記載の方法。
を、提供するものである。
R1の少なくとも1つは、ラベル部位又は標的集積部位を導入することが可能な官能基である場合においては、その他のR1は水素であっても、上記の一価の置換基(炭素数1~6個のアルキル基、炭素数1~6個のアルケニル基、炭素数1~6個のアルキニル基、炭素数1~6個のアルコキシ基、水酸基、カルボキシ基、スルホニル基、アルコキシカルボニル基、ハロゲン原子、又はアミノ基)であってもよい。
本発明の1つの好ましい態様においては、R2a及びR2bにおける一価の置換基は、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である。
また、本発明の1つの好ましい態様においては、R2a及びR2bの一方は水素であり、他方は炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である。
また、本発明の1つの更に好ましい態様においては、R2a及びR2bの両方が炭素数1~6個のアルキル基である。理論に拘束されることを意図するものではないが、R2a及びR2bの両方が炭素数1~6個のアルキル基であると、プローブの溶液中における安定性を向上させることができるためである。
また、R9又はR10は、R3又はR7と一緒になって、R9又はR10が結合している窒素原子を含む5~7員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される1~3個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数1~6個のアルキル、炭素数2~6個のアルケニル、又は炭素数2~6個のアルキニル、炭素数6~10個のアラルキル基(ベンジル基、フェネチル基等)、炭素数6~10個のアルキル置換アルケニル基で置換されていてもよい。このようにして形成されるヘテロシクリル又はヘテロアリールとしては、例えば、ピロリジン、ピペリジン、ヘキサメチレンイミン、ピロール、イミダゾール、ピラゾール、オキサゾール、チアゾールなどが挙げられるが、これらに限定されない。
アリール基としては、フェニル基、ナフチル基が挙げられるが、フェニル基が好ましい。ヘテロアリール基としては、ピリジル基、ピラジル基、ピリミジル基、ピリダジル基、インドリル基、ベンゾフラニル基、ベンゾチエニル基、ベンゾチアゾリル基、ピロリル基、フラニル基、チエニル基、イミダゾリル基、チアゾリル基が挙げられるが、ピリジル基が好ましい。
また、本発明においては、ピペラジン環アミノ基上に導入したアリール基又はヘテロアリール基に更に電子求引性基や、電子供与性基を導入することにより、さらにpKaを低下させたり、あるいは上昇させたりといった調整をすることが可能である。
アリール基又はヘテロアリール基に導入する置換基は、電子求引性基としては、ニトロ基、スルホニル基、カルボニル基、ハロゲン原子(フッ素原子、塩素原子、臭素原子又はヨウ素原子)、炭素数1~6のアルコキシ基等が挙げられるが、フッ素原子、スルホニル基が好ましい。アリール基又はヘテロアリール基は、上記の電子求引性基を2以上有することができ、これら電子求引性基は同一又は異なっていてもよい。
電子供与基としては、アミノ基、メトキシ基、炭素数1~6のアルキル基が挙げられるが、t-ブチル基、sec-ブチル基、n-ブチル基、iso-プロピル基、n-プロピル基、エチル基、メチル基、アミノ基、メトキシ基が好ましい。アリール基又はヘテロアリール基は、上記の電子供与基を2以上有することができ、これら電子供与基は同一又は異なっていてもよい。
本発明の1つの好ましい態様において、R11~R14は、それぞれ独立に、水素原子又は炭素数1~6個のアルキル基(好ましくは、メチル基、エチル基)である。
本発明の1つの好ましい態様において、R11~R14はいずれも水素原子である。
ここで、ラベル部位又は標的集積部位を導入することが可能な官能基としては、カルボニル基、アルキルカルボニル基、エステル基、アルキルエステル基、アミノ基、アルキルアミノ基、アミド基、イソチオシアネート基、塩化スルホニル基、ハロアルキル基、ハロアセトアミド基、アジド基、アルキニル基などが挙げられ、特にカルボニル基、アルキルカルボニル基が好ましい。
該架橋基は、その片方または両方の端部に、ラベル部位又は標的集積部位を導入することが可能な官能基、ラベル部位又は標的集積部位と結合することができる官能基を有してもよい。このような官能基として、例えば、アミノ基、カルボニル基、カルボキシル基、アミド基、プロパギル基などが挙げられる。
また、R1’は、水素、又は一般式(I)のR1として定義した同一又は異なる一価の置換基であり、その詳細は一般式(I)の化合物について説明した通りである。
R1’は、好ましくは、水素である。
また、R2a及びR2bの一方は、X’-Tであることができ、この場合、R2a及びR2bの他方は、一価の置換基(好ましくは、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子)である。
ここで、nが0の場合は、R2a及びR2bの一方は、X’-Tであり、他方は、一価の置換基(好ましくは、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子)である。
一般式(Ia)で表される好ましい側面において、nは1であり、pは2であり、この場合、R1’は同一であっても異なっていてもよい。この場合、R2a及びR2bの一方は、X’-Tであってもよく、その場合には、R2a及びR2bの他方は、一価の置換基(好ましくは、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子)である。
一般式(IIa)で表される好ましい側面において、nは1であり、pは2であり、この場合、R1’は同一であっても異なっていてもよい。この場合、R2a及びR2bの一方は、X’-Tであってもよく、その場合には、R2a及びR2bの他方は、一価の置換基(好ましくは、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子)である。
また、本発明のもう一つの実施態様は、一般式(I)又は(II)におけるR1の少なくとも1つが、ラベル部位又は標的集積部位を導入することが可能な官能基であって、当該官能基は、架橋基を介さずに、ラベル部位又は標的集積部位と結合した化合物又はその塩である。
上記2種類の実施態様を総称して以下「実施態様3」とも言う。
ラベル部位又は標的集積部位を導入することが可能な官能基、架橋基については、実施態様2で説明した通りである。
また、R1’は、水素、又は一般式(I)のR1として定義した同一又は異なる一価の置換基であり、その詳細は一般式(I)の化合物について説明した通りである。
R1’は、好ましくは、水素である。
また、R2a及びR2bの一方は、X’-(T’)-Sであることができ、この場合、R2a及びR2bの他方は、一価の置換基(好ましくは、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子)である。
ここで、nが0の場合は、R2a及びR2bの一方は、X’-(T’)-Sであり、他方は、一価の置換基(好ましくは、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子)である。
一般式(Ib)で表される好ましい側面において、nは1であり、pは2であり、この場合、R1’は同一であっても異なっていてもよい。この場合、R2a及びR2bの一方は、X’-(T’)-Sであってもよく、その場合には、R2a及びR2bの他方は、一価の置換基(好ましくは、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子)である。
また、架橋基は、その片方または両方の端部に、ラベル部位又は標的集積部位を導入することが可能な官能基、ラベル部位又は標的集積部位と結合することができる官能基を有してもよく、このような官能基として、例えば、アミノ基、カルボニル基、カルボキシル基、アミド基、プロパギル基などが挙げられる。
一般式(IIb)で表される化合物の好ましい側面において、nは1であり、pは2であり、この場合、R1’は同一であっても異なっていてもよい。この場合、R2a及びR2bの一方は、X’-(T’)-Sであってもよく、その場合には、R2a及びR2bの他方は、一価の置換基(好ましくは、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子)である。
また、本発明のもう1つの態様は、細胞内の酸性領域の測定方法であって、(a)一般式(I)、(II)、(Ia)、(IIa)、(Ib)又は(IIb)の化合物又はその塩を細胞内に導入する工程、及び(b)当該化合物又はその塩が細胞内で発する蛍光を測定する工程を含む方法である。
また、本発明の1つの側面は、細胞内酸性小器官の存在する酸性領域を測定する方法である。
ここで、細胞としては、正常細胞、癌細胞、神経細胞等が挙げられる。
項に記載の方法。
(1)合成中間体1の合成
以下のスキーム1により合成中間体1(tert-ブチル4-(1,10,10-トリメチル-5-オキソ-2,3,5,10-テトラヒドロ-1H-ベンゾ[5,6]シリノ[3,2-f]インドール-8-イル)ピペラジン-1-カルボキシレート(BocPiperaIndoSiXanthone)を合成した。
N-Boc-ピペラジン(2.41g、12.9mmol)および2-ブロモ-4-フルオロベンズアルデヒド(2.19g、10.8mmol)をDMF(30mL)に溶解させ、K2CO3(2.24g、16.2mmol)を加えて100℃で18時間攪拌した。溶媒を減圧留去した後に残渣に水を加え、この混合物をジクロロメタンで抽出し、食塩水で洗浄した。有機層をNa2SO4で乾燥して溶媒を減圧留去した後、メタノール(30mL)、水素化ホウ素ナトリウム(494mg、13.0mmol)を加えて室温で3時間攪拌した。溶媒を減圧留去した後、残差に水を加え、この混合物をジクロロメタンで抽出した。有機層をNa2SO4で乾燥し、溶媒を減圧留去した後、カラムクロマトグラフィー(シリカゲル、酢酸エチル/n-ヘキサン)で精製して2-ブロモ-4-(1-Bocピペラジル)-ベンジルアルコール(3.46g、9.33mmol、収率86%)を得た。
1H NMR (300 MHz, CDCl3): δ 1.48 (s, 9H), 2.02 (t, J = 6.6 Hz, 1H), 3.13 (t, J = 5.1 Hz, 4H), 3.56 (t, J = 5.1 Hz, 4H), 4.66 (d, J = 6.6 Hz, 2H), 6.85 (dd, J = 8.1, 2.2 Hz, 1H), 7.08 (d, J = 2.1 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 28.40, 48.89, 64.77, 80.05, 115.35, 120.09, 123.85, 130.03, 130.87, 151.76, 154.64; HRMS (ESI+) Calcd for [M+H]+, 371.0970, Found, 371.0922 (-4.8 mmu).
6-ブロモ-1-メチルインドリン(23mg、0.108mmol)、2-ブロモ-4-(1-Bocピペラジル)-ベンジルアルコール(41mg、0.110mmol)をジクロロメタン(5mL)に溶解させ、BF3OEt2(28μL、0.220mmol)を加えて室温で6時間攪拌した。反応溶液に水を加え、この混合物をジクロロメタンで抽出した。有機層をNa2SO4で乾燥して溶媒を減圧留去した後、カラムクロマトグラフィー(シリカゲル、酢酸エチル/n-ヘキサン)で精製し、4-(3-ブロモ-4-((6-ブロモ-1-メチルインドリン-5-イル)メチル)フェニル)Bocピペラジン(53mg、0.0937mmol、収率87%)を得た。
1H NMR (300 MHz, CDCl3): δ 1.48 (s, 9H), 2.73 (s, 3H), 2.82 (t, J = 8.1 Hz, 2H,), 3.10 (t, J = 5.1 Hz, 4H,), 3.30 (t, J = 8.1 Hz, 2H), 3.56 (t, J = 5.1 Hz, 4H), 4.00 (s, 2H), 6.65 (s, 1H), 6.70 (s, 1H), 6.77 (dd, J = 8.7, 2.1 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 7.13 (d, J = 2.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 28.31, 28.40, 35.96, 40.38, 49.13, 56.22, 79.95, 110.81, 115.64, 120.25, 123.13, 125.36, 126.09, 127.47, 130.20, 130.77, 131.20, 150.52, 153.13, 154.65; HRMS (ESI+) Calcd for [M+H]+, 566.0841, Found, 566.0850 (+0.9 mmu).
乾燥させアルゴン置換したフラスコに4-(3-ブロモ-4-((6-ブロモ-1-メチルインドリン-5-イル)メチル)フェニル)Bocピペラジン(1150mg、2.03mmol)と脱水テトラヒドロフラン(30mL)を加えた。-78℃に冷却後、1Msec-ブチルリチウム(4.26mL、4.26mmol)を加え、20分間攪拌した。そのままの温度でジクロロジメチルシラン(257μL、2.23mmol)を脱水テトラヒドロフラン10mLに溶解してゆっくりと加え、室温に戻して13時間攪拌した。2N塩酸で反応を停止してNaHCO3で中和した。この混合物をジクロロメタンで抽出して食塩水で洗浄し、有機層をNa2SO4で乾燥させた後に溶媒を減圧留去した後、カラムクロマトグラフィー(シリカゲル、酢酸エチル/n-ヘキサン)で一部の副生成物を除いた。残渣をアセトン(30mL)に溶解し、0℃に冷却してKMnO4(237mg、1.50mmol)を少量ずつ1時間かけて加え、さらに同じ温度で1時間攪拌した。ジクロロメタンを加え、ろ紙を用いて吸引ろ過した後、水を加え、この混合物をジクロロメタンで抽出し、食塩水で洗浄した。有機層をNa2SO4で乾燥して溶媒を減圧留去した後、カラムクロマトグラフィー(シリカゲル、酢酸エチル/n-ヘキサン)で精製し、tert-ブチル4-(1,10,10-トリメチル-5-オキソ-2,3,5,10-テトラヒドロ-1H-ベンゾ[5,6]シリノ[3,2-f]インドール-8-イル)ピペラジン-1-カルボキシレート(52mg、0.109mmol、収率5%)を得た。
1H NMR (400 MHz, CDCl3): δ 0.45 (s, 6H), 1.49 (s, 9H), 2.91 (s, 3H), 3.06 (t, J = 9.0 Hz, 2H), 3.35 (t, J = 5.0 Hz, 4H), 3.49 (t, J = 9.0 Hz, 2H), 3.62 (t, J = 5.0 Hz, 4H), 6.49 (s, 1H), 7.01-7.04 (m, 2H), 8.20 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ -1.19, 27.88, 28.33, 34.36, 47.56, 54.63, 79.97, 107.74, 116.22, 117.61, 126.02, 130.95, 131.37, 132.20, 132.56, 140.21, 140.29, 151.98, 154.48, 154.91, 184.99; HRMS (ESI+) Calcd for [M+H]+, 478.2526, Found, 478.2483 (-4.3 mmu).
上記で得た合成中間体1から以下のスキーム2により本発明の化合物1(2,6-diMe BnPiperaIndoSiR)を得た。
乾燥させアルゴン置換したフラスコにtert-ブチル4-(1,10,10-トリメチル-5-オキソ-2,3,5,10-テトラヒドロ-1H-ベンゾ[5,6]シリノ[3,2-f]インドール-8-イル)ピペラジン-1-カルボキシレート(42mg、0.0879mmol)、脱水テトラヒドロフラン(10mL)、2,6-ジメチルフェニルマグネシウムブロミド(2.64mL、2.64mmol)を加え3.5時間加熱還流した。反応液を室温に戻した後に2N塩酸水溶液で反応を停止して飽和炭酸水素ナトリウム水溶液を加えて中和した後、混合物をジクロロメタンで抽出し、有機層をNa2SO4で乾燥させ、溶媒を減圧留去した。残渣にトリフルオロ酢酸(5mL)、ジクロロメタン(5mL)を加えて室温で3時間攪拌した。残渣をヘキサンで洗った後にHPLC(溶離液、27%アセトニトリル/0.1%トリフルオロ酢酸/水(0分)から72%アセトニトリル/0.1%TFA/水(20分);流速=25.0mL/min)で精製し、2,6-diMe PiperaIndoSiR(3mg、5.17μmol、収率6%)を得た。
HRMS (ESI+) Calcd for [M]+, 466.2679, Found, 466.2653 (-2.6 mmu).
2,6-diMe PiperaIndoSiR(3mg、5.17μmol)をジクロロメタン(5mL)、メタノール(1mL)に溶解させ、ベンズアルデヒド(1μL、9.91μmol)および酢酸(20μL)を加え室温で10分攪拌した。反応液にシアノ水素化ホウ素ナトリウム(1.26mg、20μmol)を加えて室温で23時間攪拌した。混合物に水を加えてジクロロメタンで抽出し、有機層をNa2SO4で乾燥させ、溶媒を減圧留去した。残渣にジクロロメタン(10mL)、p-クロラニル(2mg、8.13μmol)を添加して室温で1時間攪拌した後に溶媒を減圧留去した。残渣をHPLC(溶離液、27%アセトニトリル/0.1%トリフルオロ酢酸/水(0分)から64%アセトニトリル/0.1%TFA/水(20分);流速=5.0mL/minで精製し、2,6-diMe BnPiperaIndoSiR(1.5mg、2.24μmol、収率43%)を得た。
1H NMR (400 MHz, CD3OD): δ 0.49 (s, 6H), 0.60 (s, 3H), 1.90 (s, 6H), 2.51 (t, J = 5.0 Hz, 4H), 2.91 (t, J = 7.0 Hz, 2H), 3.25 (s, 3H), 3.49 (s, 2H), 3.54 (t, J = 5.0 Hz, 4H), 3.85 (t, J = 7.0 Hz, 4H), 6.58 (dd, J = 8.6, 2.8 Hz, 1H), 6.72 (s, 1H), 6.89 (d, J = 8.6 Hz, 1H), 6.97 (s, 1H), 7.10-7.12 (m,3H), 7.18-7.28 (m, 6H); HRMS (ESI+) Calcd for [M]+, 556.3148, Found, 556.3134 (-1.4 mmu).
(1)化合物2の合成
以下のスキーム3により化合物2(SiRpH3)を合成した。
2,6-diMe PiperaIndoSiR(3.5mg、6.03μmol)をメタノール(2mL)に溶解させ、3-フルオロベンズアルデヒド(1.3μL、12.06μmol)および酢酸(20μL)を加え室温で10分攪拌した。反応液にシアノ水素化ホウ素ナトリウム(1.5mg、24μmol)を加えて室温で16時間攪拌した。混合物に水を加えてジクロロメタンで抽出し、有機層をNa2SO4で乾燥させ、溶媒を減圧留去した。残渣にジクロロメタン(5mL)、p-クロラニル(3mg、12.2μmol)を添加して室温で3時間攪拌した後に溶媒を減圧留去した。残渣をHPLC(溶離液、27%アセトニトリル/0.1%トリフルオロ酢酸/水(0分)から64%アセトニトリル/0.1%TFA/水(20分);流速=25.0mL/minで精製し、SiRpH3(1.5mg、1.87μmol、収率31%)を得た。
1H NMR (400 MHz, CD3OD): δ 0.60 (s, 6H, a), 1.97 (s, 6H, b), 3.01 (t, J = 6.5 Hz, 2H, c), 3.38 (brs, 4H, d), 3.44 (s, 3H, e), 3.84 (brs, 4H, f), 4.03 (t, J = 6.5 Hz, 2H, g), 4.38 (s, 2H, h), 6.82 (s, 1H, i), 6.90 (dd, J = 9.4, 3.0 Hz, 1H, j), 6.99 (d, J = 9.4 Hz, 1H, k), 7.23-7.38 (m,6H, l), 7.45-7.56 (m, 3H, m); HRMS (ESI+) Calcd for [M]+, 574.3054, Found, 574.3032 (-2.2 mmu)
(1)化合物3の合成
以下のスキーム4により化合物3(SiRpH4)を合成した。
(1)化合物4の合成
以下のスキーム5により化合物4(SiRpH5)を合成した。
乾燥させアルゴン置換したフラスコにtert-ブチル4-ブロモ-3,5-ジメチルベンゾエート(182mg、0.638mmol)に脱水テトラヒドロフラン(10mL)を加えて反応液を-78℃に冷却した。反応液に1M sec-BuLi(0.64mL、0.64mL)を加え、-78度で30分間攪拌した。反応液にBocPiperaIndoSiXanthone(61mg、0.128mmol)を加えて2.5時間加熱還流した。反応液を室温に戻した後に2N塩酸水溶液で反応を停止した後、混合物をジクロロメタンで抽出し、有機層をNa2SO4で乾燥させ、溶媒を減圧留去した。残渣にトリフルオロ酢酸(5mL)を加えて室温で30分間攪拌した。残渣をヘキサンで洗った後にHPLC(溶離液、24%アセトニトリル/0.1%トリフルオロ酢酸/水(0分)から64%アセトニトリル/0.1%TFA/水(20分);流速=25.0mL/min)で精製し、4-COOH-2,6-diMe PiperaIndoSiR(50mg、80.2μmol、収率63%)を得た。
1H NMR (300 MHz, CD3CN): δ 0.56 (s, 6H, a), 2.00 (s, 6H, b), 2.91 (t, J = 6.1 Hz, 2H, c), 3.30 (t, J = 4.5 Hz, 4H, d), 3.33 (s, 3H, e), 3.81 (t, J =4.5 Hz, 4H, f), 3.94 (t, J = 6.1 Hz, 4H, g), 6.73-6.77 (m, 2H, h), 6.87 (d, J = 9.6 Hz, 1H, i), 7.29 (s, 1H, j), 7.36 (d, J = 2.4 Hz, 1H, k), 7.86 (s, 2H, l); 13C NMR (100 MHz, CD3OD) δ -1.39, 19.84, 26.39, 35.08, 43.68, 44.41, 56.88, 116.00, 119.30, 121.79, 129.64, 129.99, 132.04, 133.46, 137.28, 137.43, 137.60, 144.22, 144.64, 152.62, 156.24, 160.24, 164.39, 168.28; HRMS (ESI+) Calcd for [M]+, 510.2577, Found, 510.2557 (-2.0 mmu)
4-COOH-2,6-diMe PiperaIndoSiR(12.8mg、20.5μmol)をメタノール(5mL)に溶解させ、4-ホルミルベンゼン-1,3-ジスルホン酸二ナトリウム(19.1mg、61.5μmol)および酢酸(250μL)を加え室温で30分攪拌した。反応液に2-ピコリンボラン(4.4mg、41μmol)を加えて室温で16時間攪拌した。混合物の溶媒を減圧留去し、残渣をHPLC(溶離液、28%アセトニトリル/0.1%トリフルオロ酢酸/水(0分)から60%アセトニトリル/0.1%TFA/水(20分);流速=25.0mL/minで精製し、SiRpH5(11.2mg、12.8μmol、収率63%)を得た。
1H NMR (400 MHz, 20 mM pD 9.7 phosphate buffered D2O): δ 0.48 (s, 6H, a), 1.87 (s, 6H, b), 2.63 (brs, 4H, c), 2.81 (t, J = 7.0 Hz, 2H, d), 3.31 (s, 3H, e), 3.50 (brs, 4H, f), 3.90 (t, J = 7.0 Hz, 4H, g), 4.00 (s, 2H, h), 6.51 (d, J = 9.4 Hz, 1H, i), 6.75 (s, 1H, j), 6.84 (d, J = 9.4 Hz, 1H, k), 7.28 (s, 1H, l), 7.38 (s, 1H, m), 7.65 (s, 2H, n), 7.72 (d, J = 8.2 Hz, 1H, o), 7.88 (d, J = 8.2 Hz, 1H, p), 8.30 (s, 1H, q); 13C NMR (100 MHz, CD3OD:D2O = 1:3) δ -1.11, 20.10, 26.73, 35.45, 45.53, 52.91, 57.57, 59.77, 117.07, 119.58, 120.19, 122.40, 126.87, 129.41, 130.21, 130.34, 130.81, 131.79, 133.57, 135.96, 137.56, 138.12, 138.47, 144.92, 145.32, 146.11, 147.92, 152.72, 157.61, 160.90, 163.91, 170.85; HRMS (ESI+) Calcd for [M]+, 760.2183, Found, 760.2137 (-4.6 mmu)
本発明の化合物1(2,6-diMe BnPiperaIndoSiR)のスペクトル特性を評価した。吸収スペクトル、蛍光スペクトル及び励起スペクトルを島津UV-1650PC吸光光度計、日立F-4500蛍光光度計を用いて測定した。結果を図2a~eに示す。
本発明の化合物2(SiRpH3)のスペクトル特性を評価した。吸収スペクトル、蛍光スペクトル及び励起スペクトルを島津UV-1650PC吸光光度計、日立F-4500蛍光光度計を用いて測定した。結果を図3a~cに示す。
フッ素原子を置換したSiRpH3は、2,6-diMe BnPiperaIndoSiRと同様にpHに応じて約580nmと約660nmに吸収極大を有し、蛍光プローブとして十分な蛍光量子収率を示した。
本発明の化合物3(SiRpH4)のスペクトル特性を評価した。吸収スペクトル、蛍光スペクトル及び励起スペクトルを島津UV-1650PC吸光光度計、日立F-4500蛍光光度計を用いて測定した。吸収スペクトルと励起スペクトルの結果を図4a~bに示す。
本発明の化合物4(SiRpH5)のスペクトル特性を評価した。吸収スペクトル、蛍光スペクトル及び励起スペクトルを島津UV-1650PC吸光光度計、日立F-4500蛍光光度計を用いて測定した。吸収スペクトルと励起スペクトルの結果を図5b~cに示す。
SiRpH5は約590nmと約670nmに吸収極大を有し、蛍光プローブとして十分な蛍光量子収率を示した。
[実施例9]
PEGリンカーを介してN-ヒドロキシスクシニルイミジルエステルを有し、生体高分子のアミノ基と共有結合を形成できるSiRpH5-PEG6-SE(化合物6)を以下のスキーム6により合成した。
フラスコにSiRpH5(15mg、17.2μmol)、N-ヒドロキシスクシンイミド(2.2mg、18.9μmol)、EDC塩酸塩(3.6mg、18.9mmol)、DMF(2mL)を加えた後にDIPEA(6.6μL、37.8μmol)を添加し、室温で20時間攪拌した。反応液をHPLC(溶離液、28%アセトニトリル/0.1%トリフルオロ酢酸/水(0分)から60%アセトニトリル/0.1%TFA/水(20分);流速=25.0mL/min)で精製し、溶媒を減圧留去した。残渣にNH2-PEG6-OH(4.9mg、13.7μmol)、DIPEA(25μL、0.14mmol)、水(50μL)を加えて室温で15時間攪拌した。反応液をHPLC(溶離液、28%アセトニトリル/0.1%トリフルオロ酢酸/水(0分)から60%アセトニトリル/0.1%TFA/水(20分);流速=25.0mL/min)で精製し、溶媒を減圧留去した(SiRpH5-PEG6-OH)。残渣にN-ヒドロキシスクシンイミド(4mg、35μmol)、EDC塩酸塩(6.7mg、0.11mmol)、DMF(3mL)を加えた後にDIPEA(20μL、37.8μmol)を添加し、室温で17時間攪拌した。反応液をHPLC(溶離液、24%アセトニトリル/0.1%トリフルオロ酢酸/水(0分)から44%アセトニトリル/0.1%TFA/水(20分);流速=25.0mL/min)で精製し、SiRpH5-PEG6-SE(4.1mg、3.44μmol、収率27%)を得た。
PEGリンカーを介してベンジルグアニン構造を有し、SNAP-Tagタンパクと共有結合を形成できるSiRpH5-PEG6-BG(化合物7)を以下のスキーム7により合成した。
フラスコにSiRpH5-PEG6-SE(1.8mg、1.38μmol)、BG-NH2(1.1mg、4.14μmol)、DMF(1mL)を加えた後にDIPEA(1.4μL、8.28μmol)を添加し、室温で4時間攪拌した。反応液をHPLC(溶離液、16%アセトニトリル/0.1%トリフルオロ酢酸/水(0分)から48%アセトニトリル/0.1%TFA/水(20分);流速=25.0mL/min)で精製し、SiRpH5-PEG6-BG(1.7mg、1.16μmol、収率84%)を得た。
SiRpH5を用いた細胞内オルガネラのpH測定
(A)リソソームのpH測定
多糖類であるデキストランを細胞外液に添加して細胞内に取り込ませた後に数時間インキュベーションを行うことで、デキストランをリソソーム選択的に集積させられると知られている。本プローブを用いてリソソームのpH測定を行うため、SiRpH5を10kDaアミノデキストランに標識したSiRpH5-Dexを作成した。
反応容器に10kDaアミノデキストラン(1.3mg、0.13μmol)、0.1M炭酸水素ナトリウム水溶液(460μL)を加えた。その後、SiRpH5-PEG6-SE(0.64μg、0.49μmol)を160μLのDMSOに溶解させた溶液を添加し、室温で4時間攪拌した。反応液をPD-10ゲル濾過カラム(GE-Healthcare)を用いて精製し、凍結乾燥させてSiRpH5-Dexを得た。10mg/mLのSiRpH5-Dex水溶液を調整し、吸光度を測定することでプローブの平均ラベル化率は2.6nmolプローブ/1nmol Dextranと決定した。
(a)実験のイメージ図(図7a参照):SiRpH5-Dexをリソソームに集積させ、その後リソソーム塩基性試薬である塩化アンモニウム水溶液を添加することでリソソームのpHを塩基性化させる。
(b)SiRpH5-Dexをロードした細胞を10%ホルマリンで固定化し、細胞外液をpH4.5-7.4のリン酸ナトリウム緩衝液に交換し、撮像を行い作成した細胞内pHの検量線を図7bに示す。
(c)200μg/mLのSiRpH5-Dexを含む培地でMEF細胞を2時間インキュベーションし、その後プローブを含まない培地と交換し、3時間インキュベーションを行った。共焦点顕微鏡で観察を行うと、リソソームマーカータンパク質であるVamp7と黄色蛍光タンパク質Venusの融合タンパクの近傍からSiRpH5-Dex由来の蛍光が観察された(図7c)。点線で囲った部位のレシオ値から検量線を用いてpHを計算すると、4.7と算出された。その後、最終濃度33mMの塩化アンモニウム水溶液を添加したところ、リソソームのpHは6.2まで塩基性化したことが分かった。これらの値は報告されているリソソームのpHや塩基性化試薬添加後のリソソームのpHと同程度であることから、本発明を用いて生細胞内のpH測定が行えることが分かった。
鉄輸送タンパク質であるトランスフェリン(Tfn)はトランスフェリン受容体(TfnR)を介してエンドサイトーシスされて初期エンドソームへ輸送され、その後リサイクリングエンドソームへ輸送されることが知られている。本プローブを用いてリサイクリングエンドソームのpH測定を行うため、鉄イオン-トランスフェリン複合体であるホロトランスフェリンにSiRpH5を標識したSiRpH5-Tfnを作成した。
反応容器にHolo-Transferrin(9.04mg、0.113μmol)、1000μLの0.1Mホウ酸バッファー(pH8.0)を加えた。その後、SiRpH5-PEG6-SE(1.18mg、0.91μmol)を200μLのDMSOと800μLの0.1Mホウ酸バッファー(pH8.0)に溶解させた溶液を反応容器に添加し、室温で1時間攪拌した。反応液をPD-10ゲル濾過カラム(GE-Healthcare)を用いて精製し、凍結乾燥させてSiRpH5-Tfnを得た。5mg/mLのSiRpH5-Tfn水溶液を調整し、吸光度を測定することでプローブの平均ラベル化率は7.3nmolプローブ/1nmolTfnと決定した。
(a)実験のイメージ図(図8a参照):SiRpH5-Tfnをリサイクリングエンドソーム(RE)に集積させ、pH測定を行う。
(b)SiRpH5-Tfnをロードした細胞を4%ホルムアルデヒドで固定化し、細胞外液をpH4.0-7.4のHEPSバッファーに交換し、撮像を行うことで作成した細胞内pHの検量線を図8bに示す。
(c)25μg/mLのSiRpH5-Tfnを含むバッファー中でCOS-1細胞を50分間インキュベーションし、その後プローブを含まないバッファーと交換し、15分間インキュベーションを行い、共焦点顕微鏡で撮像した(図8c)。リサイクリングエンドソームのマーカータンパク質であるRab11と緑色蛍光タンパク質GFPの融合タンパクと重なる部位からSiRpH5-Tfn由来の蛍光が観察された。点線で囲ったリサイクリングエンドソームのレシオ値から検量線を用いてpHを計算すると、5.8と算出された。この値は報告されているリサイクリングエンドソームの値と同程度であった。
このことから、プローブを標識するタンパク質を変えることで、様々な細胞内小器官のpH測定が行えると考えられる。
以上のように、本発明のpHプローブは、様々な生命現象の詳細な解析に向けて有用なツールとなることが期待される。
Claims (27)
- 以下の一般式(I):
(式中、
R1は、水素原子を示すか、又はベンゼン環上に存在する1ないし3個の同一又は異なる一価の置換基を示し;
R2a及びR2bは、それぞれ独立に、水素又は一価の置換基を示し、但し、R2a及びR2bの両方が水素ではなく;
R3及びR4は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
R5及びR6は、存在する場合は、それぞれ独立に、炭素数1~6個のアルキル基又はアリール基を示し、
ここで、Xが酸素原子の場合は、R5及びR6は存在せず、
Xがリン原子の場合は、-R5及び-R6の一方は、=Oであってもよい;
R7及びR8は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
R9及びR10は、それぞれ独立に、水素原子又は炭素数1~6個のアルキル基を示し、
R9又はR10は、R3又はR7と一緒になって、R9又はR10が結合している窒素原子を含む5~7員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される1~3個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数1~6個のアルキル、炭素数2~6個のアルケニル、又は炭素数2~6個のアルキニル、炭素数6~10個のアラルキル基、炭素数6~10個のアルキル置換アルケニル基で置換されていてもよく;
Yは、置換又は無置換のアリール基又はヘテロアリール基を示し;
Xは、珪素原子、酸素原子、炭素原子、リン原子又はゲルマニウム原子を示し;
mは、0~6の整数である。)
で表される化合物又はその塩。 - Yが置換又は無置換のフェニル基である、請求項1に記載の化合物又はその塩。
- Yがフェニル基、フッ素原子で置換されたフェニル基、又はスルホニル基で置換されたフェニル基である、請求項2に記載の化合物又はその塩。
- mが0又は1である、請求項1~3のいずれか1項に記載の化合物又はその塩。
- R1の少なくとも1つは、カルボキシ基、カルボキシ基を有するアルキル基、エステル基、アルキルエステル基、アミノ基、アミド基、アルキルアミノ基、イソチオシアネート基、塩化スルホニル基、ハロアルキル基、ハロアセトアミド基、アジド基、又はアルキニル基から選ばれる請求項1~4のいずれか1項に記載の化合物又はその塩。
- R1の少なくとも1つは、カルボキシ基、カルボキシル基を有するアルキル基、アミノ基、アミド基である、請求項5に記載の化合物又はその塩。
- R2a及びR2bにおける一価の置換基が、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である、請求項1~6のいずれか1項に記載の化合物又はその塩。
- R2a及びR2bの一方は水素であり、他方は炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である、請求項7に記載の化合物又はその塩。
- R2a及びR2bの両方が炭素数1~6個のアルキル基である、請求項7に記載の化合物又はその塩。
- Yが置換又は無置換のフェニル基である、請求項10に記載の化合物又はその塩。
- Yがフェニル基、フッ素原子で置換されたフェニル基、又はスルホニル基で置換されたフェニル基である、請求項11に記載の化合物又はその塩。
- mが0又は1である、請求項11又は12に記載の化合物又はその塩。
- R1の少なくとも1つは、カルボキシ基、カルボキシ基を有するアルキル基、エステル基、アルキルエステル基、アミノ基、アミド基、アルキルアミノ基、イソチオシアネート基、塩化スルホニル基、ハロアルキル基、ハロアセトアミド基、アジド基、又はアルキニル基から選ばれる請求項10~13のいずれか1項に記載の化合物又はその塩。
- R1の少なくとも1つは、カルボキシ基、カルボキシル基を有するアルキル基、アミノ基、アミド基である、請求項14に記載の化合物又はその塩。
- R2a及びR2bにおける一価の置換基が、炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である、請求項10~15のいずれか1項に記載の化合物又はその塩。
- R2a及びR2bの一方は水素であり、他方は炭素数1~6個のアルキル基、炭素数1~6個のアルコキシ基又はハロゲン原子である、請求項16に記載の化合物又はその塩。
- R2a及びR2bの両方が炭素数1~6個のアルキル基である、請求項16に記載の化合物又はその塩。
- 以下の一般式(Ia):
(式中、
R3及びR4は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基、又はハロゲン原子を示し;
R5及びR6は、存在する場合は、それぞれ独立に、炭素数1~6個のアルキル基又はアリール基を示し、
ここで、Xが酸素原子の場合は、R5及びR6は存在せず、
Xがリン原子の場合は、-R5及び-R6の一方は、=Oであってもよい;
R7及びR8は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
R9及びR10は、それぞれ独立に、水素原子又は炭素数1~6個のアルキル基を示し、
R9又はR10は、R3又はR7と一緒になって、R9又はR10が結合している窒素原子を含む5~7員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される1~3個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数1~6個のアルキル、炭素数2~6個のアルケニル、又は炭素数2~6個のアルキニル、炭素数6~10個のアラルキル基、炭素数6~10個のアルキル置換アルケニル基で置換されていてもよく;
Yは、置換又は無置換のアリール基又はヘテロアリール基を示し;
Xは、珪素原子、酸素原子、炭素原子、リン原子又はゲルマニウム原子を示し;
mは、0~6の整数であり;
X’は、ラベル部位又は標的集積部位を導入することが可能な官能基がTと結合した後の構造を示し;
Tは、架橋基を示し、該架橋基は、その片方または両方の端部に、ラベル部位又は標的集積部位を導入することが可能な官能基、ラベル部位又は標的集積部位と結合することができる官能基を有してもよい;
R1’は、水素、又は同一又は異なる一価の置換基であり;
(i)R2a及びR2bは、それぞれ独立に、水素又は一価の置換基を示し、但し、R2a及びR2bの両方が水素ではなく、又は
(ii)R2a及びR2bの一方は、X’-Tであり、R2a及びR2bの他方は、一価の置換基であり;
nは0~2の整数であり、pは1~3の整数であり、但し、n+p=3であり、ここで、nが0の場合は、R2a及びR2bの一方は、X’-Tであり、他方は、一価の置換基である。)
で表される、化合物又はその塩。 - 以下の一般式(Ib):
(式中、
R3及びR4は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基、又はハロゲン原子を示し;
R5及びR6は、存在する場合は、それぞれ独立に、炭素数1~6個のアルキル基又はアリール基を示し、
ここで、Xが酸素原子の場合は、R5及びR6は存在せず、
Xがリン原子の場合は、-R5及び-R6の一方は、=Oであってもよい;
R7及びR8は、それぞれ独立に、水素原子、炭素数1~6個のアルキル基又はハロゲン原子を示し;
R9及びR10は、それぞれ独立に、水素原子又は炭素数1~6個のアルキル基を示し、
R9又はR10は、R3又はR7と一緒になって、R9又はR10が結合している窒素原子を含む5~7員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される1~3個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数1~6個のアルキル、炭素数2~6個のアルケニル、又は炭素数2~6個のアルキニル、炭素数6~10個のアラルキル基、炭素数6~10個のアルキル置換アルケニル基で置換されていてもよく;
Yは、置換又は無置換のアリール基又はヘテロアリール基を示し;
Xは、珪素原子、酸素原子、炭素原子、リン原子又はゲルマニウム原子を示し;
mは、0~6の整数であり;
X’は、生体高分子標識部位を導入することが可能な官能基がTと結合した後の構造を示し;
T’は、存在する場合は、架橋基がSと結合した後の構造であり;
Sは。ラベル部位又は標的集積部位を示し;
R1’は、水素、又は同一又は異なる一価の置換基であり;
R2a及びR2bは、
(i)それぞれ独立に、水素又は一価の置換基を示し、但し、R2a及びR2bの両方が水素ではなく、又は
(ii)R2a及びR2bの一方は、X’-(T’)-Sであり、R2a及びR2bの他方は、一価の置換基であり;
nは0~2の整数であり、pは1~3の整数であり、但し、n+p=3であり、ここで、nが0の場合は、R2a及びR2bの一方は、X’-(T’)-Sであり、他方は、一価の置換基である。)
で表される化合物又はその塩。 - 請求項1~24のいずれか1項に記載の化合物又はその塩を含む蛍光プローブ。
- 細胞内の酸性領域の測定方法であって、
(a)請求項1~24のいずれか1項に記載の化合物又はその塩を細胞内に導入する工程、及び
(b)当該化合物又はその塩が細胞内で発する蛍光を測定する工程
を含む方法。 - 細胞内酸性小器官の存在する酸性領域を測定する請求項26に記載の方法。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2017509316A JP6742576B2 (ja) | 2015-03-27 | 2016-01-15 | pH感受性蛍光プローブ |
US15/561,444 US10815379B2 (en) | 2015-03-27 | 2016-01-15 | pH sensitive fluorescent probe |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015065543 | 2015-03-27 | ||
JP2015-065543 | 2015-03-27 | ||
US201562261438P | 2015-12-01 | 2015-12-01 | |
US62/261,438 | 2015-12-01 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016157937A1 true WO2016157937A1 (ja) | 2016-10-06 |
Family
ID=57005553
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2016/051119 WO2016157937A1 (ja) | 2015-03-27 | 2016-01-15 | pH感受性蛍光プローブ |
Country Status (3)
Country | Link |
---|---|
US (1) | US10815379B2 (ja) |
JP (1) | JP6742576B2 (ja) |
WO (1) | WO2016157937A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2018090536A (ja) * | 2016-12-02 | 2018-06-14 | 国立大学法人 東京大学 | 近赤外蛍光レシオ型プローブ |
EP3461815A1 (de) * | 2017-09-29 | 2019-04-03 | ATTO-TEC GmbH | Neue poly-sulfonierte fluoreszenz-farbstoffe |
JP2022510369A (ja) * | 2018-12-02 | 2022-01-26 | ザ・ユニバーシティ・オブ・シカゴ | 試料中のpHおよびカルシウムまたは塩化物イオン濃度を決定する方法 |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP7100363B2 (ja) * | 2016-12-01 | 2022-07-13 | 国立大学法人 東京大学 | 深赤色蛍光プローブ |
CN110183482B (zh) * | 2019-06-27 | 2022-03-01 | 河南师范大学 | 一种监测溶酶体pH的近红外荧光探针及其制备方法和应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102659744A (zh) * | 2012-05-09 | 2012-09-12 | 大连理工大学 | 一类对pH变化敏感的罗丹明荧光探针、合成方法及应用 |
WO2013035767A1 (ja) * | 2011-09-07 | 2013-03-14 | 国立大学法人 東京大学 | 酸性環境検出蛍光プローブ |
WO2013134686A1 (en) * | 2012-03-09 | 2013-09-12 | Promega Corporation | pH SENSORS |
-
2016
- 2016-01-15 WO PCT/JP2016/051119 patent/WO2016157937A1/ja active Application Filing
- 2016-01-15 US US15/561,444 patent/US10815379B2/en active Active
- 2016-01-15 JP JP2017509316A patent/JP6742576B2/ja active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013035767A1 (ja) * | 2011-09-07 | 2013-03-14 | 国立大学法人 東京大学 | 酸性環境検出蛍光プローブ |
WO2013134686A1 (en) * | 2012-03-09 | 2013-09-12 | Promega Corporation | pH SENSORS |
CN102659744A (zh) * | 2012-05-09 | 2012-09-12 | 大连理工大学 | 一类对pH变化敏感的罗丹明荧光探针、合成方法及应用 |
Non-Patent Citations (5)
Title |
---|
DAISUKE ASANUMA ET AL.: "Acidic-pH-Activatable Fluorescence Probes for Visualizing Exocytosis Dynamics", ANGEWANDTE CHEMIE, vol. 53, no. 24, 2014, pages 6085 - 6089, XP055201374 * |
DANIEL AIGNER ET AL.: "New fluorescent pH sensors based on covalently linkable PET rhodamines", TALANTA, vol. 99, 2012, pages 194 - 201, XP028936871 * |
MASAYUKI ISA ET AL.: "High-Throughput Screening System To Identify Small Molecules That Induce Internalization and Degradation of HER2", ACS CHEMICAL BIOLOGY, vol. 9, no. 10, 2014, pages 2237 - 2241, XP055201369 * |
XIAOYU GUAN ET AL.: "Development of a new rhodamine-based FRET platform and its application as a Cu2+ probe", ORGANIC & BIOMOLECULAR CHEMISTRY, vol. 12, no. 23, 2014, pages 3944 - 3949, XP055201096 * |
YAN-RU ZHANG ET AL.: "A ratiometric fluorescent probe for sensing HOCl based on a coumarin-rhodamine dyad", CHEMICAL COMMUNICATIONS, vol. 50, no. 91, 2014, pages 14241 - 14244, XP055201087 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2018090536A (ja) * | 2016-12-02 | 2018-06-14 | 国立大学法人 東京大学 | 近赤外蛍光レシオ型プローブ |
EP3461815A1 (de) * | 2017-09-29 | 2019-04-03 | ATTO-TEC GmbH | Neue poly-sulfonierte fluoreszenz-farbstoffe |
EP3461814A1 (de) * | 2017-09-29 | 2019-04-03 | ATTO-TEC GmbH | Neue poly-sulfonierte fluoreszenz-farbstoffe |
US11180657B2 (en) | 2017-09-29 | 2021-11-23 | Atto-Tec Gmbh | Polysulfonated fluorescence dyes |
JP2022510369A (ja) * | 2018-12-02 | 2022-01-26 | ザ・ユニバーシティ・オブ・シカゴ | 試料中のpHおよびカルシウムまたは塩化物イオン濃度を決定する方法 |
JP7307797B2 (ja) | 2018-12-02 | 2023-07-12 | ザ・ユニバーシティ・オブ・シカゴ | 試料中のpHおよびカルシウムまたは塩化物イオン濃度を決定する方法 |
Also Published As
Publication number | Publication date |
---|---|
US20180118943A1 (en) | 2018-05-03 |
JP6742576B2 (ja) | 2020-08-19 |
JPWO2016157937A1 (ja) | 2018-02-08 |
US10815379B2 (en) | 2020-10-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2016157937A1 (ja) | pH感受性蛍光プローブ | |
JP6351511B2 (ja) | 非対称Siローダミン及びロドールの合成 | |
US20050227299A1 (en) | Fluorescent dyes (AIDA) for solid phase and solution phase screening | |
Xu et al. | A novel highly selective chemosensor based on curcumin for detection of Cu2+ and its application for bioimaging | |
US20100015725A1 (en) | Luminescent 1-hydroxy-2-pyridinone chelates of lanthanides | |
US20070134737A1 (en) | Fluorophore compounds and their use in biological systems | |
US10837909B2 (en) | Super-resolution fluorescent imaging probe | |
WO2006025887A2 (en) | Long wavelength thiol-reactive fluorophores | |
US9688857B2 (en) | Fluorescent probe | |
WO2013035767A1 (ja) | 酸性環境検出蛍光プローブ | |
WO2014136781A1 (ja) | 蛍光プローブ | |
Jin et al. | Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH monitoring | |
Zatsikha et al. | Functionalized bispyridoneannelated BODIPY–Bright long-wavelength fluorophores | |
JP5887011B2 (ja) | 蛍光プローブ | |
Chen et al. | Detection of thiophenol in buffer, in serum, on filter paper strip, and in living cells using a red-emitting amino phenothiazine boranil based fluorescent probe with a large Stokes shift | |
Bříza et al. | Pentamethinium fluorescent probes: The impact of molecular structure on photophysical properties and subcellular localization | |
US20040054195A1 (en) | Xanthene derivatives | |
WO2002055512A1 (en) | Xanthene derivatives | |
US7541467B2 (en) | Fluorescent zinc ion sensor | |
JP6456592B2 (ja) | 蛍光寿命イメージングプローブ | |
CN113416196B (zh) | 一种苯并噻二唑-tb类化合物及其合成方法和应用 | |
Cao et al. | Self-assemble nanostructured ensembles for detection of guanosine triphosphate based on receptor structure modulated sensitivity and selectivity | |
Guan et al. | Norcyanine dyes with benzo [c, d] indolium moiety: Spectral sensitivity with pH change for fluorescence pH imaging in living cells | |
Yu et al. | Two–photon excitable red fluorophores for imaging living cells | |
US9133220B2 (en) | Boron-containing 5-arylidene-3,5-dihydro-4H-imidazol-4-ones |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16771823 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2017509316 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15561444 Country of ref document: US |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 16771823 Country of ref document: EP Kind code of ref document: A1 |