CN101805526A - Synthesis and application of indole hemicyanine dye - Google Patents
Synthesis and application of indole hemicyanine dye Download PDFInfo
- Publication number
- CN101805526A CN101805526A CN201010144000A CN201010144000A CN101805526A CN 101805526 A CN101805526 A CN 101805526A CN 201010144000 A CN201010144000 A CN 201010144000A CN 201010144000 A CN201010144000 A CN 201010144000A CN 101805526 A CN101805526 A CN 101805526A
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- Prior art keywords
- compound
- fluorescence
- dye
- halogen
- indole
- Prior art date
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- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 title claims abstract description 30
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 title claims abstract description 15
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 title claims abstract description 15
- 230000015572 biosynthetic process Effects 0.000 title abstract description 5
- 238000003786 synthesis reaction Methods 0.000 title abstract 4
- 150000001875 compounds Chemical class 0.000 claims abstract description 103
- 239000000975 dye Substances 0.000 claims abstract description 48
- -1 Indole quaternary ammonium salts Chemical class 0.000 claims abstract description 26
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 20
- 229910052736 halogen Inorganic materials 0.000 claims description 28
- 150000002367 halogens Chemical class 0.000 claims description 28
- 239000007787 solid Substances 0.000 claims description 28
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 19
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 18
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims description 9
- 125000001814 trioxo-lambda(7)-chloranyloxy group Chemical group *OCl(=O)(=O)=O 0.000 claims description 8
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 7
- AMXLSXXWUVANCC-UHFFFAOYSA-N 1h-indole;naphthalene Chemical group C1=CC=C2NC=CC2=C1.C1=CC=CC2=CC=CC=C21 AMXLSXXWUVANCC-UHFFFAOYSA-N 0.000 claims description 6
- 125000000304 alkynyl group Chemical group 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- 229910020366 ClO 4 Inorganic materials 0.000 claims description 2
- 238000001215 fluorescent labelling Methods 0.000 claims description 2
- 125000000896 monocarboxylic acid group Chemical group 0.000 claims 9
- 210000004027 cell Anatomy 0.000 abstract description 44
- 238000010521 absorption reaction Methods 0.000 abstract description 21
- 239000000523 sample Substances 0.000 abstract description 21
- 238000003384 imaging method Methods 0.000 abstract description 12
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- 125000001424 substituent group Chemical group 0.000 abstract description 6
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- 230000008033 biological extinction Effects 0.000 abstract description 4
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- 239000002028 Biomass Substances 0.000 abstract description 2
- 238000010186 staining Methods 0.000 abstract description 2
- 150000001450 anions Chemical class 0.000 abstract 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 60
- 239000000243 solution Substances 0.000 description 35
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 28
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 27
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 24
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 16
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 15
- 239000002253 acid Substances 0.000 description 14
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- 210000001541 thymus gland Anatomy 0.000 description 14
- 244000309466 calf Species 0.000 description 13
- 239000000843 powder Substances 0.000 description 13
- PMUNIMVZCACZBB-UHFFFAOYSA-N 2-hydroxyethylazanium;chloride Chemical compound Cl.NCCO PMUNIMVZCACZBB-UHFFFAOYSA-N 0.000 description 12
- 125000000217 alkyl group Chemical group 0.000 description 12
- 238000001228 spectrum Methods 0.000 description 12
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 11
- 229940098773 bovine serum albumin Drugs 0.000 description 11
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- IPZJQDSFZGZEOY-UHFFFAOYSA-N dimethylmethylene Chemical compound C[C]C IPZJQDSFZGZEOY-UHFFFAOYSA-N 0.000 description 9
- 238000001914 filtration Methods 0.000 description 9
- 229960000890 hydrocortisone Drugs 0.000 description 9
- 238000000034 method Methods 0.000 description 9
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- 238000001514 detection method Methods 0.000 description 8
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- 238000010586 diagram Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 7
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000001476 alcoholic effect Effects 0.000 description 6
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 6
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- MZZINWWGSYUHGU-UHFFFAOYSA-J ToTo-1 Chemical compound [I-].[I-].[I-].[I-].C12=CC=CC=C2C(C=C2N(C3=CC=CC=C3S2)C)=CC=[N+]1CCC[N+](C)(C)CCC[N+](C)(C)CCC[N+](C1=CC=CC=C11)=CC=C1C=C1N(C)C2=CC=CC=C2S1 MZZINWWGSYUHGU-UHFFFAOYSA-J 0.000 description 5
- SMWDFEZZVXVKRB-UHFFFAOYSA-N anhydrous quinoline Natural products N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 5
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 150000002475 indoles Chemical class 0.000 description 5
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 4
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 4
- 229960005542 ethidium bromide Drugs 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 239000000463 material Substances 0.000 description 4
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- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical compound O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- NYGZLYXAPMMJTE-UHFFFAOYSA-M metanil yellow Chemical group [Na+].[O-]S(=O)(=O)C1=CC=CC(N=NC=2C=CC(NC=3C=CC=CC=3)=CC=2)=C1 NYGZLYXAPMMJTE-UHFFFAOYSA-M 0.000 description 3
- 210000002220 organoid Anatomy 0.000 description 3
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 description 2
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 2
- RGHHSNMVTDWUBI-UHFFFAOYSA-N 4-hydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1 RGHHSNMVTDWUBI-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- GRRMZXFOOGQMFA-UHFFFAOYSA-J YoYo-1 Chemical compound [I-].[I-].[I-].[I-].C12=CC=CC=C2C(C=C2N(C3=CC=CC=C3O2)C)=CC=[N+]1CCC[N+](C)(C)CCC[N+](C)(C)CCC[N+](C1=CC=CC=C11)=CC=C1C=C1N(C)C2=CC=CC=C2O1 GRRMZXFOOGQMFA-UHFFFAOYSA-J 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
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- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 2
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
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- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 1
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- 230000032258 transport Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Indole Compounds (AREA)
Abstract
The invention relates to synthesis and application of an indole hemicyanine dye, which belongs to the synthesis of a functional dye and the application of the functional dye as a fluorescent dye in the field of fine chemistry industry. Indole quaternary ammonium salts and the corresponding aldehyde are condensed to form compounds with the advantages of large molar extinction coefficient, good light stability, easy synthesis and the like. The molecules have pH probe property, solid-state fluorescent property and binding property with biomacromolecules according to different substituents and anions. Being used as a pH probe, the compound can adjust the pKa of the probe according to the different substituents, and can be used for two-color living cell imaging; being used as a solid-state emission dye, the compound can adjust the solid-state emission wavelength according to different substituents and anions; being used as a biological molecule probe, the compound has good very response to the biological molecule, and living cell membrane permeability ability, can be used for cell staining, can prevent the interference of biomass autofluorescence because of longer absorption and emission wavelengths, and can also reduce the damage of exciting light to cells or biological tissues.
Description
Technical field
The present invention relates to the synthetic of a class indole hemicyanine dye and use, belong to the synthetic of a class functional dye in the field of fine chemical and as the application of fluorescence dye.
Background technology
The cell inner proton is all played the part of an important role in many cell processes, as: the growth of cell, the adjusting of calcium, endocytosis, cytoadherence etc.The kinetics of research internal pH is vital to the regulation mechanism of understanding physiological function in many cells.The method that detects internal pH is a lot, and as microelectrode, nucleus magnetic resonance etc., and fluorescent microscope is sensitiveer, more convenient than these methods, so the fluorescent pH probe was widely used in monitoring the variation of internal pH in recent years.In general, the pH scope that has two broads in the cell: one is about 6.8-7.4, as tenuigenin; Another is about 4.5-6.0, and promptly so-called acid organoid is as lysosome.With various fluorophores is that the pH probe of parent is in the news, and some has been used to tissue or viable cell imaging, and some is commercialization also.But the pH probe with two emissions is seldom reported.Particularly the acid pH probe nearly all is based on photoinduction transfer transport (PET) mechanism, does not therefore have wavelength change.Though commercialization probe DND160 is acid pH probe (the Diwu Z with two emissions, Chen CS, Zhang C Chem.Biol.1999,6 (7): 411-418), but its shortcoming is obvious: the absorption of probe and emission wavelength are all very short, pair cell causes damage easily, but also can produce the interference of biomass autofluorescence.Two emissions of therefore synthetic longer wavelength, the probe of low pKa will have the better application prospect.
The styrenic indole cyanine dyes has good light stability, and suitable is water-soluble, good permeability of cell membrane and in cell, have stronger fluorescence, and also this class dyestuff is synthetic very simple, and yield is also very high, but insensitive to pH usually.Hydroxyl is an important pH probe functional group, many pH probes that contain hydroxyl be in the news (Tang B, Liu X, Xu KH, Huang H, YangGW, An LG.Chem.Comm.2007,3726-3728).Need have lower pKa and be applied to the pH probe that cell acid soaks picture, in dye molecule, introduce the pKa that electron-withdrawing group can reduce molecule.
In recent years, the organic compound with solid fluorescence had been subjected to extensive concern, because they not only can be used for carrying out fundamental research, but also may be applied to photoelectric field, as Organic Light Emitting Diode, and photo-translating system.Luminous behavior and the solid-state performance of understanding them are very important, because in actual applications, these materials generally are to use with the form of film.Yet the organic compound with solid-state emission seldom.This mainly is because in crystal habit or unbodied solid phase (for example film), piles up very closely between the fluorophore, even have the brightest emitted fluorescence group, also can cause serious cancellation.In addition, solid-state exciting light and emission light have very strong optical extinction coefficient, and this is very deleterious (Ozdemir T, Atilgan S to solid fluorescence, Kutuk I, Yildirim, LT, Tulek A, Bayindir M, Akkaya E U.Org.Lett.2009,11 (10): 2105-2107), especially when the Stokes shift of fluorophore is very little.Therefore, the exploitation of solid state fluorescence molecule efficiently is a problem with challenge.Recent years, a lot of documents (Ooyama Y, Mamura T, Yoshida K.E.J.Org.Chem.2007,5010-5019 are arranged; Shimizu M, Takeda Y, Higashi M.Angew.Chem.Int.Ed.2009,48 (20): 3653-3656; ShimizuM, Takeda Y, Higashi M, Hiyama T.Angew.Chem.Int.Ed.2009,48 (20): 1-5) report solid emitted fluorescence unity structure, set forth its luminescence mechanism, they all are the conjugated systems that has the larger side chain, can suppress intermolecular gathering like this; And most molecules are confession-acceptor forms, and stronger intramolecularly prototropy (ICT) effect is arranged, and can increase the Stokes shift of fluorophore, reduce the fluorescence self-quenching.
Above-mentioned a lot of research has been set forth the relation of solid fluorescence character and packing of molecules structure on the basis of X-ray single crystal diffraction.Studies show that: the successive intramolecular hydrogen bond is the principal element that causes the solid state fluorescence cancellation between the fluorophore of intermolecular strong π-π effect and vicinity.Therefore, the novel strong attitude emitted fluorescence of design is rolled into a ball and at first will be reduced the intermolecular interaction that causes fluorescent quenching.The intermolecular accumulative method of current elimination mainly is that introducing large volume substituting group or introducing have sterically hindered substituting group structure nonplanar structure, solves the problem of assembling cancellation fluorescence with this.Styryl dye has stronger ICT effect, also can make it have stronger solid state fluorescence by introducing bigger substituting group, and the good light stability of this class dyestuff is easy to synthetic and purifying, is suitable for the research of photoelectric field.
Fluorescence dye is used widely in every field of science and technology as functional pigmented, and especially the research at aspects such as life science, clinical treatment diagnosis, immunoassay detections gets most of the attention in the whole world.At present, phenanthridines class (EB, PI), acridine (AO), imidazoles (Hoechst, DAPI) and flower cyanines man same clan commercialization fluorescence dyes such as (Cy, TOTO, SYTO) all play an important role in fields such as genomics technology, nucleic acid quantification detection, blood cell analysis.Yet these dyestuffs all exist application limit separately.One shows that mainly most fluorescence dye is subject to the fixed cell sample.For example TOPRO, TOTO family dyestuff, ethidium bromide (EB), propidium iodide (PI) etc. need could carry out effective fluorescent mark to biological sample by the permeability or the similar method of film disintegration that makes that increase cytolemma.Yet this fixing means often observation of pair cell and biological tissue's true form has negative impact [Kozubek S, Lukasova E, Amrichova J, Kozubek M, Liskova A, SlotovaJ.Anal Biochem 2000; 282:29-38].Simultaneously, acridines such as ethidium bromide, phenanthridines class dyestuff has very big toxicity and carinogenicity.They are two years old, there is the exciting light of quite a few fluorescence dye to be in ultraviolet region, as fluorescence dye 4 ' that can single-minded identification thymus nucleic acid (DNA), 6-diamino-2-phenylindone quinoline compound (DAPI), Hoechst33258, Hoechst34580 etc., after DNA combines, put down the generation blue-fluorescence at ultraviolet excitation.Because components such as the nucleic acid in the UV-light pair cell, albumen can cause serious damage, so this class fluorescence use in fluorescence microscopy is subjected to restriction [Davis SK, the Bardeen CJ.Photochem Photobiol 2003 of optical excitation time; 77:675-679].Therefore, research and develop out and have the good fluorescence spectrum property, toxicity is little, and the novel fluorescence dyestuff of viable cell permeability remains the key and the core of development such as the fields that promote fluorescence analysis and life science.
In the fluorescence dye of numerous kinds, cyanine fluorochrome is wide with its wavelength region, advantages such as molar extinction coefficient is big, and fluorescence quantum yield is moderate are used widely as biomolecules fluorescent probe, CD and VCD recording materials, sensitive materials photosensitizers, photoelectric conversion material etc.Most typical in the asymmetric cyanine compounds is TOTO and analogue (YOYO) and derivatives class (TOPRPO).TOTO (thiazole orange dimer), YOYO (oxazole yellow dimer) have many positive charges asymmetric cyanine fluorochrome of high affinity by a class of Glazer study group exploitation to nucleic acid, and the structure of the length by changing the polymethylene chain and the fragrant parent nucleus (thiazole, oxazole, quinoline, pyridine and indoline) at two ends can obtain different heterodimer analogue and derivative.This class dyestuff does not almost have fluorescence in solution, reduced the fluorescence background in the testing process and disturbed, and combines back fluorescence with nucleic acid and strengthens.Some kind commercialization in this type of dyestuff, as: SYTOX Blue, TOTO, POPO, BOBO, YO-PRO etc.But the most of molecule of these commercial dyestuffs is bigger, and complex structure belongs to the non-permeability of viable cell, can only be applied to the in vitro identification and the detection of nucleic acid.
In recent years, some styrenic hemicyanine dyes had been used to the test of DNA gradually, comprising pyridines, benzothiazoles, quinoline and indoles etc.Though occurred two dyestuffs of being connected by flexible chain (electrically charged or neutral) in first three class hemicyanine dye, the two hemicyanine dyes of indoles that are used for biomolecule detection are considerably less, and the indoles pair hemicyanine dyes that are used for Protein Detection do not appear in the newspapers.It should be noted that, the rarely seen report of structure that has the viable cell permeability in above-mentioned all two cyanine dyes, may because more than two cyanine dyes of report with the carbochain that contains positive charge as linking group, increased the water-soluble of dyestuff, reduced the penetrating ability of cytolemma, hindered these dyestuffs in the application of viable cell organoid aspect visual with high-performance.Suitable lipotropy can make probe pass cytolemma and can be at film surface sediment (J Colston, RW Horobin, F Rashid-Doubell, J Pediani, KK Johal.Biotechnic; Histochemistry 2003,78 (6): 323-332); Simultaneously, the indoles cyanine dyes that does not contain water-soluble group has good membrane permeability.On the other hand, long wavelength's dyestuff not only can be avoided absorption and the fluorescence of organism self, and can also reduce the damage of laser pair cell when using.
Therefore, be fluorophore with the indole hemicyanine dye, by the connection of different connecting arms, reduce its background fluorescence, make it have the better application prospect aspect biomolecules identification.
Summary of the invention
The purpose of this invention is to provide that a class formation is simple, good light stability, be easy to the new compound of synthetic.According to substituent difference, this compounds can be used for the function fluorescence dye of different field.
The technical solution used in the present invention is: a class indole hemicyanine dye molecule has general structure I:
Wherein: R
1=H, halogen, nitro, sulfonic group, R
2=H; Or R
1R
2Form the naphthalene indole ring
R
3And R
4Independently be selected from H, hydroxyl, halogen, nitro, C separately
1-4Alkyl;
R
5Be H, hydroxyl, N ((CH
2)
mR
7)
2, wherein: R
7=H, OH, OAc, COOH, COOCH
3, phenyl;
R
6Be (CH
2)
pR
8, wherein: R
8=H, OH, C=C, alkynyl, OAc, COOH, COOCH
3, COOC
2H
5, p-(C
6H
4) R
9, wherein: R
9=H, CH
3, halogen, NO
2, CN, COOH, L-A;
Y
-=halide-ions, ClO
4 -, PF
6 -Or TsO
-
L=-C
1-18Alkyl-, p-(CH
2)
q(C
6H
4) (CH
2)
q-;
M=1～18, n=1 or 2, p=0～18, q=1-6.
As n=1, R
5During for hydroxyl, have structural formula II:
Wherein: R1=H, halogen, nitro, sulfonic group, R2=H; Or R1R2 forms the naphthalene indole ring
R3 and R4 independently are selected from H, hydroxyl, halogen, nitro, C1-4 alkyl separately;
R6 is (CH2) pR8, wherein: and R8=H, OH, C=C, alkynyl, OAc, COOH, COOCH3, COOC2H5, p-(C6H4) R9, wherein: R9=H, CH3, halogen, NO2, CN, COOH;
Y-=halide-ions, ClO4-, PF6-or TsO-, wherein: P=1～18.
When R2=H, n=1, form structural formula II I:
Wherein: R1=H, halogen, nitro, sulfonic group.
R3 and R4 independently are selected from H, hydroxyl, halogen, nitro, C1-4 alkyl separately;
R5 is H, hydroxyl, N ((CH2) mR7) 2, wherein: R7=H, OH, OAc, COOH, COOCH3, phenyl;
R6 is (CH2) pR8, wherein: and R8=H, OH, OAc, COOH, COOCH3, COOC2H5, p-(C6H4) R9, wherein: R9=H, CH3, halogen, NO2, CN, COOH;
Y-=halide-ions, ClO4-, PF6-or TsO-;
m＝1～18；P＝0～18。
When R6=L-A, have general structure IV:
Wherein: R1=H, halogen, nitro, sulfonic group, R2=H; Or R1R2 forms the naphthalene indole ring
R3 and R4 independently are selected from H, hydroxyl, halogen, nitro, C1-4 alkyl separately;
R5 is H, N ((CH2) mR7) 2, wherein: R7=H, OH, OAc, COOH, COOCH3, phenyl;
Y-=halide-ions, ClO4-, PF6-or TsO-;
The L=-C1-18 alkyl-or (CH2) q-of p-(CH2) q (C6H4);
m＝1～18，n＝1、2，p＝1～18，q＝1-6。
Described have the compound of structural formula II as the pH fluorescent probe.
Described have the compound of structural formula II I as the solid fluorescence compound.
Described have the compound of structural formula IV as biological fluorescent labeling.
Owing to adopted above technical scheme, the beneficial effect that possesses when making new compound among the present invention as probe biomolecule is:
(1) new pair of dye molecule of the present invention forms the intramolecularly gathering, reduces the dyestuff autofluorescence, and the binding ability of dyestuff and biomolecules is strengthened, and improved detection sensitivity.And within the specific limits can be to the biomolecules detection by quantitative.
(2) part reactive monoazo dyestuffs compound of the present invention has increased a conjugation methine chain, makes maximum absorption wavelength can increase about 100nm, and emission wavelength ranges is wide, can reach the near-infrared region of 650nm～900nm, can avoid the fluorescence background of biology self to disturb.The molar extinction coefficient of dyestuff is big, and is highly sensitive, and good light stability can be applicable to fields such as biomolecules identification, clinical treatment diagnosis, immunoassay detection.
(3) the two dye molecules of part of the present invention have the living cell membrane permeability, can be used for the viable cell fluorescence imaging.
(4) reactive monoazo dyestuffs product side effect of the present invention is little, and raw material is easy to get, and is simple in structure, generally can synthesize target molecule by the reaction of 2 to 4 steps, is easy to industrialization.
Description of drawings
Fig. 1 is the compd A of embodiment 2 when changing with pH, the changing trend diagram of absorption spectrum.X-coordinate is wavelength (nm), and ordinate zou is relative absorption intensity.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; PH meter, model: LEICI.The concentration of compd A is 2 μ M.
Fig. 2 is the compd A of embodiment 2 when changing with pH, the changing trend diagram of fluorescence spectrum.X-coordinate is wavelength (nm), and ordinate zou is a relative intensity of fluorescence.Used instrument is a spectrophotofluorometer, model: FP-6500; PH meter, model: LEICI.The concentration of compd A is 2 μ M.
Fig. 3 is the acid base equilibrium mechanism of compd A and the resonance structure of existence.
Fig. 4 is the compd B of embodiment 3 when changing with pH, the changing trend diagram of absorption spectrum.X-coordinate is wavelength (nm), and ordinate zou is relative absorption intensity.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; PH meter, model: LEICI.The concentration of compd B is 2 μ M.
Fig. 5 is the compd B of embodiment 3 when changing with pH, the changing trend diagram of fluorescence spectrum.X-coordinate is wavelength (nm), and ordinate zou is a relative intensity of fluorescence.Used instrument is a spectrophotofluorometer, model: FP-6500; PH meter, model: LEICI.The concentration of compd B is 2 μ M.
Fig. 6 is that compd B can be estimated the pKa of compd B in the matched curve of 570nm place fluorescence intensity and pH among the embodiment 3.
Fig. 7 be the compd A of embodiment 2 to the burnt micro-imaging photo of the painted copolymerization of viable cell MCF-7 (human breast cancer cell), from left to right be followed successively by green channel imaging picture, red channel imaging picture, two channels stack picture and bright field picture.The concentration of compd A is 1.5 μ M.Used instrument is a confocal laser scanning microscope, CLSM, model: TCS-SP2.Exciting light passage: green glow: 458nm, ruddiness: 543nm.
Fig. 8 is the solid-state emmission spectrum of Compound C, D, E and known compound S 1 among the embodiment 4,5,6.Used instrument is a spectrophotofluorometer, model: LS 55.
Fig. 9 is powder color and the solid fluorescence photo of Compound C and known compound S1 among the embodiment 4.
Figure 10 be the compound G of embodiment 8 and known compound S1 in three (methylol) aminomethane hydrochloride damping fluid of pH 7.4, concentration 10mM, combine with calf thymus DNA that front and back absorb, emmission spectrum changes comparison diagram.X-coordinate is wavelength (nm), and ordinate zou is relative absorption, fluorescence intensity.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500.The concentration of compound G and S1 is 2 μ M, and the concentration of calf thymus DNA is 100 μ M.
Figure 11 be the compound G of embodiment 8 in three (methylol) aminomethane hydrochloride damping fluid of pH 7.4, concentration 10mM, combine back emmission spectrum changing trend diagram with the calf thymus DNA of different concns.X-coordinate is wavelength (nm), and ordinate zou is a relative intensity of fluorescence.Used instrument is a spectrophotofluorometer, model: FP-6500.The concentration of compound G is 2 μ M, and the concentration of calf thymus DNA is 0-50 μ M.
Figure 12 is the fluorescence intensity of the compound G among the embodiment 8 and the variation tendency (illustration) and the linear relationship of DNA concentration.
Figure 13 is that compound H and known compound S2 combine absorption, the change in fluorescence comparison diagram of front and back respectively in three (methylol) aminomethane hydrochloride damping fluid of pH 7.0, concentration 10mM among the embodiment 9 with bovine serum albumin BSA.X-coordinate is wavelength (nm), and ordinate zou is relative absorption, fluorescence intensity.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500.The concentration of compound H and S2 is 2 μ M, and the concentration of bovine serum albumin BSA is 50 μ M.
Figure 14 be among the embodiment 9 compound H in three (methylol) aminomethane hydrochloride damping fluid of pH 7.0, concentration 10mM, with bovine serum albumin BSA concentration rising change in fluorescence trend map.X-coordinate is wavelength (nm), and ordinate zou is a relative intensity of fluorescence.Used instrument is a spectrophotofluorometer, model: FP-6500.The concentration of compound H is 2 μ M, and the concentration of bovine serum albumin BSA is 0-10 μ M.
Figure 15 is the fluorescence intensity of compound H and the variation tendency (illustration) and the linear relationship of BSA concentration.
Figure 16 is the compound F 17-hydroxy-corticosterone of embodiment 7 and the Compound I among the embodiment 10 imaging photo to viable cell PC12.From left to right be followed successively by compound F 17-hydroxy-corticosterone to the bright-field imagery photo of viable cell PC12, compound F 17-hydroxy-corticosterone fluorescence imaging photo to viable cell PC12; Compound I is to the bright-field imagery photo of viable cell PC12, the Compound I fluorescence imaging photo to viable cell PC12.Dye strength 3 μ M, incubation time 30min.Exciting light is WB510-570nm, and instrument is Nikon eclipase TE 2000-5.
Embodiment
Unless otherwise indicated, term used herein has following implication.
Term used herein " alkyl " comprises straight chained alkyl and branched-chain alkyl.As mention single alkyl as " propyl group ", and then only refer in particular to straight chained alkyl, as mention single branched-chain alkyl as " sec.-propyl ", then only refer in particular to branched-chain alkyl.For example, " C1-6 alkyl " comprises C1-4 alkyl, C1-3 alkyl, methyl, ethyl, n-propyl, sec.-propyl and the tertiary butyl.Similarly rule also is applicable to other group that uses in this specification sheets.
Term used herein " halogen " comprises fluorine, chlorine, bromine and iodine.
Term used herein " benzyl " is meant-the CH2-Ph group.When with " the optional replacement " modification benzyl, refer to that this benzyl can unsubstituted form exist, and perhaps can be replaced in any suitable position by suitable substituents.Suitable substituents includes but not limited to H, C1-18 alkyl, CN, COOH, NH2, NO2, OH, SH, C1-6 alkoxyl group, C1-6 alkylamino, C1-6 amido, halogen or C1-6 haloalkyl etc., as long as the final compound that forms has the character of the present invention's expectation.Preferred benzyl is by COOH, NH2, OH, C1-6 alkoxyl group, the optional replacement of halogen.
Use Y-to represent negative ion herein, it can be any suitable negative ion, includes but not limited to inorganic negative ion or organic negative ion, for example halide-ions, ClO4-, PF6-, BF4-, CH3COO-or OTs-.
In compound of Formula I of the present invention, preferred R1 and R2 independently are selected from R1 separately, and R2 is H, halogen, nitro, sulfonic group; Or
Most preferably R1 and R2 are H.
Preferred R3 and R4 independently are selected from H or hydroxyl, methoxyl group, halogen, nitro, C1-4 alkyl separately; More preferably R3 and R4 independently are selected from H or C1-2 alkyl separately; Most preferably R3 and R4 are H.
Preferred R5 is H, hydroxyl, N ((CH2) mR7) 2; Most preferably R5 is hydroxyl, N (CH3) 2.
Preferred R6 is (CH2) pR8, R8=H, OH, OAc, COOH, COOCH3, COOC2H5, p-(C6H4) R9, A, R9=H, CH3, halogen, NO2, CN, COOH; Most preferably R6 is CH3, C=C, alkynyl, carboxyl substituted phenyl ring, A;
Preferred L is (CH2) q of C1-18 alkyl or p-(CH2) q (C6H4), the q=C1-6 alkyl; More preferably L is (CH2) q of C1-14 alkyl or p-(CH2) q (C6H4), the q=C1-4 alkyl; More preferably L is (CH2) q of C1-12 alkyl or p-(CH2) q (C6H4) again, the q=C1-2 alkyl; Most preferably L is C1-10 alkyl or p-(CH2) (C6H4) (CH2).
Preferred Y-is halide-ions, ClO4-, PF6-, BF4-, CH3COO-or OTs-, and most preferably Y-is Cl-, Br-.
The invention provides the pH probe character and the biologic applications thereof of above-mentioned general formula I I compound.
The present invention also provides above-mentioned compound of formula III solid state fluorescence character.
The present invention also provides a kind of utilize above-mentioned general formula I V compound, its conjugate or the application of its composition aspect biological stain.
The beneficial effect that compound of the present invention possesses when being used as fluorescence dye is:
-part of compounds has two emission characteristics, can be used for double-colored cell imaging.
Form intramolecularly in the-new compound molecule and assemble, dyestuff is significantly reduced with the preceding fluorescence of combining of nucleic acid, increase, improved detection sensitivity in conjunction with back fluorescence intensity and fluorescence quantum yield.
-new compound molecule has the good cell membrane permeability, and range of application increases.
-part reactive monoazo dyestuffs compound can reach more than the 650nm fluorescent emission wavelength by increasing the methine chain, avoids the fluorescence background of biology self to disturb.
-new compound can use that cheapness, volume are little, the red laser diode of stable performance is as light source, reduces use cost greatly.
-new compound product side effect is little, and raw material is easy to get, and is simple in structure, generally can synthesize target molecule by the reaction of 4 to 5 steps, easily industrialization.
These feature and advantage of the present invention and other feature and advantage will become apparent with reference to the following drawings with after the specific embodiment of the present invention.
The compounds of this invention can be directly used in the dyeing of biological sample with described salt form herein.In addition, the derivative of The compounds of this invention also can be used for the dyeing of biological sample, and described derivative includes but not limited to conjugate.
Typically, conjugate uses in fluorescence-activated cell sorter (FACS)." conjugate " used herein is meant that fluorescence dye of the present invention is connected the compound that forms by covalent linkage with other molecule.Can be with the molecule that fluorescence dye of the present invention is puted together and cell or cellular constituent specificity bonded molecule, include but not limited to antibody, antigen, acceptor, part, enzyme, substrate, coenzyme etc.Usually, specimen and fluorescence conjugate incubation for some time, make this fluorescence conjugate combine with some cell or cellular constituent specificity in the specimen, this fluorescence conjugate can be called as dyeing with combining also of cell or cellular constituent.This staining procedure can carry out repeatedly successively, or carries out multiple dyeing simultaneously with multiple conjugates.After dyeing was finished, sample was analyzed in fluorescence-activated cell sorter, the fluorescence dye of the present invention in the excitation light source excites conjugate wherein, and determinator is measured the emission light that is produced by the excited fluorescent dyestuff.
The present invention also provides the composition that comprises formula II compound or its conjugate, and described composition is used for the dyeing of biological sample.
Composition of the present invention also can comprise needed other component of biological sample dyeing, for example solvent, osmotic pressure regulator, pH regulator agent, tensio-active agent etc. except that comprising formula II compound or its conjugate.These components all are known in the industry.
Composition of the present invention can exist with aqueous solution form, perhaps can exist to face other suitable form that is formulated as solution with preceding water.
Aspect another, the present invention also provides and uses above-mentioned formula II compound or its conjugate or comprise the method for formula II compound compositions with stained biological samples, and this method comprises to be made above-mentioned formula II compound or its conjugate or comprise the step that formula II compound compositions contacts with biological sample.Term used herein " contact " can be included in solution or the solid phase and contact.
In order to illustrate that the optimization of compound of the present invention on application performance improves, known compound S1, S2 compare explanation as object of reference among the embodiment 14,15,17.Wherein compound S 1 and S2 structure are as follows:
Embodiment 1
Dyestuff intermediate 1-methyl-5-nitro-2,3,3-trimethylammonium-3H indoles quaternary ammonium salt synthetic:
With 20mmol 5-nitro-2,3,3-trimethylammonium-3H indoles and 40mmol methyl iodide join 100ml and contain in the round-bottomed flask of 20ml toluene argon shield.Stop behind the reaction reflux sustained reaction 20h.Mixture cooled and filtered precipitation is also used the ether washing leaching cake.Obtain flaxen pressed powder after the drying, thick yield 85%.
Embodiment 2
The preparation of compd A:
With 5mmol 1-methyl-5-nitro-2,3,3-trimethylammonium-3H indoles quaternary ammonium salt and 6mmol p-Hydroxybenzaldehyde join 25ml and contain in the 10ml alcoholic acid round-bottomed flask, and nitrogen protection behind the stirring at normal temperature 24h, places reaction solution under-20 ℃ and spends the night.Collect the solid of separating out by filtering, and use ethyl acetate drip washing, vacuum-drying then obtains the orange/yellow solid powder, yield 85%.
1H?NMR(400MHz，DMSO-d6，ppm)：1.87(s，6H，C(CH3)2)，4.12(s，3H，CH3-N+)，7.00(d，2H，J＝8.4，Ar-H)，7.52(d，1H，J＝16，CH＝CH)，8.06(d，1H，J＝8.8，Ar-H)，8.23(d，2H，J＝8.4，Ar-H)，8.50(d，1H，J＝8.8，Ar-H)，8.56(d，1H，J＝16，CH＝CH)，8.83(s，1H，Ar-H)，11.09(s，1H，OH).13C?NMR(100MHz，DMSO-d6，ppm)：25.45，34.44，52.02，109.29，115.36，116.67，118.63，125.14，126.09，134.65，144.20，146.59，147.00，156.89，154.39，184.64.HRMS(TOF?MS?ES+)calcd.for?C19H19N203[M-I-]+323.1390，found?323.1394.
Embodiment 3
The preparation of compd B:
With 5mmol 1-propargyl-2,3,3-trimethylammonium-3H indoles quaternary ammonium salt and 5mmol 3-Br-4-hydroxy benzaldehyde join 25ml and contain in the round-bottomed flask of 10ml methyl alcohol, and nitrogen protection behind the reflux 4h, places reaction solution under-20 ℃ and spends the night.Collect the solid of separating out by filtering, and use ethyl acetate drip washing, vacuum-drying then obtains the orange solids powder, yield 88%.
1H?NMR(400MHz，CDCl3，ppm)：1.73(s，6H，C(CH3)2)，3.31(t，1H，J＝2.38，Alkyne-H)5.41(s，2H，CH2-N+)，6.82(d，2H，J＝8.8，Ar-H)，7.19(d，1H，J＝15.6，CH＝CH)，7.46(t，1H，J＝7.2，Ar-H)，7.54(t，1H，J＝7.6，Ar-H)，7.67(d，1H，J＝8.0，Ar-H)，7.75(d，1H，J＝7.2，Ar-H)，7.99(d，1H，J＝8.8，Ar-H)，8.19(d，1H，J＝15.6，CH＝CH)，8.45(s，1H，Ar-H).13C?NMR(100MHz，DMSO-d6，ppm)：26.09，33.92，51.16，72.24，81.35，113.8，114.68，119.45，123.05，125.24，127.79，129.14，133.73，137.06，142.66，142.92，151.49，165.83，179.00.HRMS(TOF?MS?EI+)calcd.for?C19H18BrNO[M-Br-+H+](100％)380.0645，found?380.0650；[M-Br-+H++2](100％)：382.0630，found：382.0636
Embodiment 4
The preparation of Compound C:
With 5mmol 1-(4-carboxylic benzyl)-2,3,3-trimethylammonium-5-bromo-3H benzindole quaternary ammonium salt and 5.5mmol 4-N; the N dimethylamine benzaldehyde joins 25ml and contains in the 10ml alcoholic acid round-bottomed flask; nitrogen protection behind the reflux 2h, places reaction solution under-20 ℃ and spends the night.Collect the solid of separating out by filtering, and use ethyl acetate drip washing, vacuum-drying then obtains the yellow-green colour pressed powder, yield 92%.
1H?NMR(400MHz，DMSO-d6，ppm)：1.83(s，6H，C(CH3)2)，3.18(s，6H，N(CH3)2)，5.91(s，2H，CH2-N+)，6.89(d，2H，J＝8.8，Ar-H)，7.35(d，1H，J＝15.2，CH＝CH)，7.43-7.45(m，3H，Ar-H)，7.56(d，2H，J＝6.8，Ar-H)，7.81(d，1H，J＝6.4，Ar-H)，7.95(d，2H，J＝8.0，Ar-H)，8.06(d，2H，J＝8.0，Ar-H)，8.43(d，1H，J＝15.2，CH＝CH).HRMS(TOFMS?EI+)calcd.for?C28H27BrN2O2[M-Cl--H+]502.2151，found?502.2162.
Embodiment 5
The preparation of Compound D:
With 5mmol 1-ethyl-2,3,3-trimethylammonium-3H indoles quaternary ammonium salt and 6mmol 4-N, N-diethanolamine benzaldehyde join 25ml and contain in the 10ml alcoholic acid round-bottomed flask, and nitrogen protection behind the reflux 2h, places reaction solution under-20 ℃ and spends the night.Collect the solid of separating out by filtering, and use ethyl acetate drip washing, vacuum-drying then obtains the green solid powder, yield 95%.
1H?NMR(400MHz，DMSO-d6，ppm)：1.40(t，3H，J＝7.2，CH3CH2)，1.77(s，6H，C(CH3)2)，3.39(s，4H，NCH2CH2OH)，3.67(s，4H，NCH2CH2OH)，4.56(q，2H，J＝7.2，CH2-N+)，4.91(s，2H，NCH2CH2OH)，6.96(d，2H，J＝8.8，Ar-H)，7.26(d，1H，J＝15.6，CH＝CH)，7.48(s，1H，Ar-H)，7.56(d，1H，J＝7.2，Ar-H)，7.80(d，1H，J＝6.8，Ar-H)，8.10(d，2H，J＝8.8，Ar-H)，8.34(d，1H，J＝15.6，CH＝CH).MS：[M-I-]+＝379.2；HRMS(TOF?MS?EI+)cal?cd.for?C24H30N2O2[M-I--H+]378.2307，found?378.2299.
Embodiment 6
The preparation of compd E:
With 5mmol 1-carboxylic amyl group-2,3,3-trimethylammonium-5-methoxyl group-3H indoles quaternary ammonium salt and 5.5mmol4-N; N-dibutylamine benzaldehyde joins 25ml and contains in the round-bottomed flask of 10ml ethylene glycol monomethyl ether; nitrogen protection behind the reflux 1.5h, places reaction solution under-20 ℃ and spends the night.Collect the solid of separating out by filtering, and use ethyl acetate drip washing, vacuum-drying then obtains blackish green pressed powder, yield 92%.
1H?NMR(400MHz，DMSO-d6，ppm)：0.9(t，6H，J＝7.2，CH3)，1.32-1.54(m，13H，CH2)，1.57(m，2H，CH2)，1.76(s，6H，C(CH3)2)，1.80(m，2H，CH2)，2.20(t，2H，J＝7.2，CH2COOH)，3.28(s，4H，NCH2)，4.52(t，2H，J＝7.2，CH2-N+)，6.90(d，2H，J＝9.2，Ar-H)，7.26(d，1H，J＝15.6，CH＝CH)，7.48(d，1H，J＝7.2，Ar-H)，7.55(d，1H，J＝7.2，Ar-H)，7.72(d，1H，J＝8.0，Ar-H)，7.79(d，1H，J＝7.2，Ar-H)，8.10(s，1H，Ar-H)，8.35(d，1H，J＝15.6，CH＝CH)，12.00(s，1H，COOH).HRMS(TOF?MS?EI+)calcd.for?C33H47N2O3[M-Br-]+519.3581，found519.3590.
Embodiment 7
The preparation of compound F 17-hydroxy-corticosterone:
With 5mmol 1,1 '-(1,4-fourth two bases)-two 2; 3,3-trimethylammonium-3H indoles quaternary ammonium salt and 11mmol 4-N, N dimethylamine benzaldehyde join 25ml and contain in the 10ml alcoholic acid round-bottomed flask; nitrogen protection behind the reflux 2h, places reaction solution under-20 ℃ and spends the night.Collect the solid of separating out by filtering, and use ethyl acetate drip washing, vacuum-drying then obtains the purple pressed powder, yield 90%.
1H?NMR(400MHz，DMSO-d6，ppm)：1.68(s，12H，C(CH3)2)，2.01(s，4H，CH2CH2N+)，3.17(s，12H，N(CH3)2)，4.65(s，4H，CH2CH2-N+)，6.79(d，4H，J＝9.2，Ar-H)，7.30(d，2H，J＝15.6，CH＝CH)，7.46(t，2H，J＝7.6，Ar-H)，7.52(t，2H，J＝7.6，Ar-H)，7.77(d，2H，J＝7.2，Ar-H)，7.81(d，2H，J＝7.2，Ar-H)，8.12(d，4H，J＝8.8，Ar-H)，8.32(d，2H，J＝15.6，CH＝CH).13C?NMR(100MHz，DMSO-d6，ppm)：24.91，26.45，44.40，50.81，104.63，112.11，113.58，122.34，122.81，127.46，128.72，134.45，141.05，142.56，154.56，154.72，179.38.HRMS(TOFMS?ES+)calcd.for?C44H52N42+[M-2Br-]2+636.4181，found?636.4168.
Embodiment 8
The preparation of compound G:
With 5mmol 1,1 '-(1, the 6-dihexyl)-two 2; 3,3-trimethylammonium-5-bromo-3H indoles quaternary ammonium salt and 12mmol 4-N, N dimethylamine benzaldehyde join 25ml and contain in the 10ml alcoholic acid round-bottomed flask; nitrogen protection behind the reflux 2h, places reaction solution under-20 ℃ and spends the night.Collect the solid of separating out by filtering, and use ethyl acetate drip washing, vacuum-drying then obtains the pink solid powder, yield 83%.
1H?NMR(400MHz，DMSO-d6，ppm)：1.51(m，4H，CH2CH2CH2N+)，1.75(s，12H，C(CH3)2)，1.77(m，4H，CH2CH2CH2N+)，3.17(s，12H，N(CH3)2)，4.54(t，4H，J＝6.6，CH2CH2CH2-N+)，6.87(d，4H，J＝8.8，Ar-H)，7.27(d，2H，J＝15.6，CH＝CH)，7.49(m，4H，Ar-H)，7.70(t，2H，J＝6.8，Ar-H)，7.78(d，2H，J＝6.8，Ar-H)，8.12(s，2H，Ar-H)，8.34(d，2H，J＝15.2，CH＝CH).13C?NMR(100MHz，DMSO-d6，ppm)：25.68，26.58，27.68，45.04，50.91，104.57，111.08，112.24，113.57，122.28，122.86，127.52，128.79，131.58，141.07，142.72，154.59，179.34.HRMS(TOF?MS?ES+)cal?cd.for?C46H54Br2N42+[M-2Br-]2+820.2704，found820.2706.
Embodiment 9
The preparation of compound H:
With 5mmol 1,1 '-(1,4-benzene second two bases)-two 2; 3,3-trimethylammonium-3H indoles quaternary ammonium salt and 12mmol 4-N, N dimethylamine base cinnamic aldehydes joins 25ml and contains in the 10ml alcoholic acid round-bottomed flask; nitrogen protection behind the reflux 4h, places reaction solution under-20 ℃ and spends the night.Collect the solid of separating out by filtering, and use ethyl acetate drip washing, vacuum-drying then obtains the blue solid powder, yield 75%.
1H?NMR(400MHz，DMSO-d6，ppm)：1.77(s，12H，C(CH3)2)，2.84(t，4H，J＝7.2，CH2CH2N+)，3.09(s，12H，N(CH3)2)，5.10(t，4H，J＝7.2，CH2-N+)，6.81(d，4H，J＝8.0，Ar-H)，7.06(d，2H，J＝14.4，CH＝CH)，7.25(t，2H，J＝12.8，CH＝CH)，7.35-7.84(m，18H，Ar-H+CH＝CH)，8.48(t，2H，J＝12.8，CH＝CH).13C?NMR(100MHz，DMSO-d6，ppm)：26.47，30.85，47.68，48.56，50.96，110.16，112.44，113.68，122.943，123.77，127.31，127.66，128.86，132.14，133.82，141.12，142.53，153.27，153.9，157.83，179.66.HRMS(TOF?MS?ES+)calcd.for?C54H60N42+[M-2I-]2+764.4807，found?764.4801.
Embodiment 10
The preparation of Compound I:
With 5mmol 1; 1 '-(1; 10-base in the last of the ten Heavenly stems two)-two 2; 3; 3-trimethylammonium-3H benzindole quaternary ammonium salt and 13mmcl 4-N, N dimethylamine base vinylbenzene formaldehyde join 25ml and contain in the round-bottomed flask of 10ml ethylene glycol monomethyl ether nitrogen protection; behind the reflux 3h, reaction solution is placed under-20 ℃ and spend the night.Collect the solid of separating out by filtering, and use ethyl acetate drip washing, vacuum-drying then obtains the mazarine pressed powder, yield 72%.
1H?NMR(400MHz，DMSO-d6，ppm)：1.39(m，8H，CH2)，1.54(m，4H，CH2CH2CH2N+)，1.71(s，12H，C(CH3)2)，1.80(m，4H，CH2CH2CH2N+)，3.08(s，12H，N(CH3)2)，4.42(t，4H，J＝7.2，CH2CH2CH2-N+)，6.82(d，4H，J＝8.0，Ar-H)，7.15(d，2H，J＝14.8，CH＝CH)，7.41(d，2H，J＝12.8，CH＝CH)，7.60(d，4H，J＝8.4，Ar-H)，7.71(t，2H，J＝8.0，Ar-H)，7.78(d，2H，J＝12.8，CH＝CH)，7.82(t，2H，J＝8.0，Ar-H)，7.93(d，2H，J＝8.8，Ar-H)，7.97(d，4H，J＝8.4，Ar-H)，8.22(d，2H，J＝8.4，Ar-H)，8.35(t，2H，J＝12.8，CH＝CH).13C?NMR(100MHz，DMSO-d6，ppm)：25.26，26.01，26.74，27.91，29.74，45.69，51.31，53.34，110.95，112.69，114.07，123.28，123.34，124.21，126.65，128.34，129.40，132.31，133.50，137.43，141.33，143.14，145.72，153.00，153.41，157.19，179.46.HRMS(TOF?MS?ES+)calcd.forC62H72N42+[M-2Cl-]2+872.5746，found?872.5754.
Embodiment 11
Under the different pH, the absorption of compd A and the mensuration of emmission spectrum:
Configuration concentration is the compd A of 1mM, the DMSO of B (dimethyl sulfoxide (DMSO)) solution respectively, get 40 μ L respectively, be diluted to 20mL, place the twoport flask, with dense hydrochloric acid soln solution is transferred to acidity, use the pH of rare sodium hydroxide solution regulator solution then, simultaneously absorption and the emmission spectrum of recording solution under this pH.Along with the reduction of pH, compound raises at the absorption peak at 465nm place, and the absorption peak at 366nm and 560nm place descends, and has two isobestic points, and showing has two kinds of acid base equilibriums to exist.And be that the emission peak of excitation wavelength raises simultaneously with 465nm and 560nm.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500; PH meter, model: LEICI.
Embodiment 12
Under the different pH, the estimation of the absorption of compd B and the mensuration of emmission spectrum and pKa:
Configuration concentration is DMSO (dimethyl sulfoxide (DMSO)) solution of the compd B of 1mM respectively, get 40 μ L respectively, be diluted to 20mL, place the twoport flask, with dense hydrochloric acid soln solution is transferred to acidity, use the pH of rare sodium hydroxide solution regulator solution then, simultaneously absorption and the emmission spectrum of recording solution under this pH.Along with the reduction of pH, compound raises at the absorption peak at 430nm place, and the absorption peak at 550nm place descends, and there is an isobestic point in the 475nm place, shows to have a kind of acid base equilibrium.And be that the emission peak of excitation wavelength raises along with the rising of its corresponding emission wavelength intensity with 430nm and 550nm.Fluorescence intensity and pH to compound do the sigmoidal match, and the pH of 1/2 maximum strength correspondence is the pKa of compound on the curve.The pKa that obtains compd B is 5.2, shows that compound is applicable to the acid organoid imaging of viable cell.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500; PH meter, model: LEICI.
Embodiment 13
Confocal laser scanning microscope, CLSM is observed the dyeing of compd A to viable cell MCF-7 down:
Add be furnished with compd A, PBS damping fluid 12 μ L that concentration is 2 μ M in six orifice plates of having cultivated the MCF-7 cell, in 37 ℃, the cell culture incubator of 5%CO2, hatch 30min.Then, PBS concussion rinsing 5min * 3 add cell culture medium, confocal laser scanning microscope, CLSM (TCS-SP2, Germany) observation of cell form again.Choose representative area, green (458nm), red (543) two channels excite, and observe triplicate with oily mirror (1000 *).Fig. 7 from left to right is followed successively by green channel imaging picture, red channel imaging picture, two channels stack picture and bright field picture.Can be observed compd A to the intracytoplasmic acidic region specific stain of MCF-7 as figure, and can double-colored imaging.Used instrument is a confocal laser scanning microscope, CLSM, model: TCS-SP2.
Embodiment 14
The mensuration of the solid state spectrum of Compound C, D, E:
Measuring the compounds solid state emmission spectrum is to be undertaken by the principle of emission.Directly powder is encased in the test trough during test, regulates sample angle, the light of reflection is entered on the receptor just, the record emmission spectrum.Used instrument is a spectrophotofluorometer, model: LS 55.
For more intuitive comparison, taken under the daylight of Compound C and known compound S1 solid fluorescence photo under the powder color and ultraviolet lamp.
Embodiment 15
Compound G and known compound S1 combine the mensuration of front and back absorption, emmission spectrum and fluorescence intensification factor with calf thymus DNA:
Configuration concentration is DMSO (dimethyl sulfoxide (DMSO)) solution of compound G, the S1 of 1mM, gets 6 μ L respectively, and three (methylol) aminomethane hydrochloride damping fluid that adds pH 7.4,10mM is diluted to 3mL, places cuvette, measures its fluorescence intensity.Dispose the aqueous solution of certain density calf thymus DNA, by the absorbance at its 260nm place of uv-absorbing spectrophotometric determination, demarcating its concentration is 1.5mM.DMSO (dimethyl sulfoxide (DMSO)) the solution 6 μ L of compound G, S1 that get in addition concentration respectively and be 1mM are in cuvette, again respectively to wherein adding the calf thymus DNA solution 200 μ L that concentration is 1.5mM, three (methylol) aminomethane hydrochloride damping fluid that adds pH 7.4,10mM at last is diluted to 3mL, measures its fluorescence intensity.After the effect, it is big that the absorption intensity of S1 becomes, and wavelength does not change; And the absorption spectrum alteration of form of G.Under the same terms (same substrate concentration and DNA concentration), known dye S1, after calf thymus DNA combined, relative intensity of fluorescence increased by 5.9 (I/I0=190/32=5.9) doubly; And compound G is with after calf thymus DNA combines, and relative intensity of fluorescence can increase by 91 (I/I0=600/6.6=91) doubly.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500.
Embodiment 16
Compound G combines the mensuration of back fluorescence emission spectrum with the calf thymus DNA of different concns:
Configuration concentration is DMSO (dimethyl sulfoxide (DMSO)) solution of the compound G of 1mM, gets 6 μ L, and three (methylol) aminomethane hydrochloride damping fluid that adds pH7.4,10mM is diluted to 3mL, places cuvette, measures its fluorescence intensity.Dispose the aqueous solution of certain density calf thymus DNA, by the absorbance at its 260nm place of uv-absorbing spectrophotometric determination, demarcating its concentration is 1.5mM.DMSO (dimethyl sulfoxide (DMSO)) the solution 6 μ L of compound G that get in addition concentration respectively and be 1mM are in cuvette, again respectively to wherein add different volumes, concentration is the calf thymus DNA solution of 1.5mM, three (methylol) aminomethane hydrochloride damping fluid that adds pH 7.4,10mM at last is diluted to 3mL, measures its fluorescence intensity.Along with the increase of DNA concentration, the fluorescence intensity of compound G constantly raises.When DNA concentration surpassed 30 μ M, fluorescence enhancing trend slowed down.In this concentration range, compound G fluorescence intensity and DNA concentration are carried out linear fit, find that there is good linear relationship in both by this part.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500.
Embodiment 17
Compound H and known compound S2 combine the mensuration of front and back absorption, emmission spectrum and fluorescence intensification factor with bovine serum albumin BSA:
Configuration concentration is the compound H of 1mM, the DMSO of S2 (dimethyl sulfoxide (DMSO)) solution, gets 6 μ L respectively, and three (methylol) aminomethane hydrochloride damping fluid that adds pH 7.4,10mM is diluted to 3mL, places cuvette, measures its fluorescence intensity.The aqueous solution of the bovine serum albumin BSA of configuration 5mM.Getting concentration respectively in addition is that the compound H of 1mM, the DMSO of S2 (dimethyl sulfoxide (DMSO)) solution 6 μ L are in cuvette, again respectively to wherein adding the calf thymus DNA solution 30 μ L that concentration is 5mM, three (methylol) aminomethane hydrochloride damping fluid that adds pH 7.0,10mM at last is diluted to 3mL, measures its fluorescence intensity.After the effect, the absorption intensity of S2 diminishes, and wavelength does not change; And the absorption spectrum alteration of form of G and with red shift of wavelength.Under the same terms (same substrate concentration and BSA concentration), known dye S2, after bovine serum albumin BSA combined, relative intensity of fluorescence increased by 1.5 (I/I0=40.2/26.8=1.5) doubly; And compound H is with after bovine serum albumin BSA combines, and relative intensity of fluorescence can increase by 19 (I/I0=159.6/8.3=19) doubly.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500.
Embodiment 18
Compound H combines the mensuration of back fluorescence emission spectrum with the bovine serum albumin BSA of different concns:
Configuration concentration is DMSO (dimethyl sulfoxide (DMSO)) solution of the compound H of 1mM, gets 6 μ L, and three (methylol) aminomethane hydrochloride damping fluid that adds pH7.0,10mM is diluted to 3mL, places cuvette, measures its fluorescence intensity.The aqueous solution of the bovine serum albumin BSA of configuration 5mM.Get the DMSO that concentration is the compound H of 1mM (dimethyl sulfoxide (DMSO)) solution 6 μ L respectively in cuvette in addition, again respectively to wherein add different volumes, concentration is the bovine serum albumin BSA solution of 5mM, three (methylol) aminomethane hydrochloride damping fluid that adds pH 7.0,10mM at last is diluted to 3mL, measures its fluorescence intensity.Along with the increase of BSA concentration, the fluorescence intensity of compound H constantly raises.When BSA concentration surpassed 8.5 μ M, fluorescence enhancing trend slowed down.In this concentration range, compound H fluorescence intensity and BSA concentration are carried out linear fit, find that there is good linear relationship in both by this part.Used instrument is a ultraviolet-visible pectrophotometer, model: Hp8453; Spectrophotofluorometer, model: FP-6500.
Embodiment 19
The viable cell permeability test of compound F 17-hydroxy-corticosterone, I:
Add be furnished with compound F 17-hydroxy-corticosterone, I, concentration is in dried six orifice plates of having cultivated the PC12 cell of the PBS damping fluid 12 μ L of 1.5 μ M, hatches 30min in 37 ℃, the cell culture incubator of 5%CO2.Then, PBS concussion rinsing 5min * 3 add cell culture medium, confocal laser scanning microscope, CLSM (Nikoneclipase TE 2000-5) observation of cell form again.Choose representative area, 510-570nm excites, imaging three times.Figure 16 from left to right is followed successively by compound F 17-hydroxy-corticosterone to the bright-field imagery photo of viable cell PC12, the compound F 17-hydroxy-corticosterone fluorescence imaging photo to viable cell PC12; Compound I is to the bright-field imagery photo of viable cell PC12, the Compound I fluorescence imaging photo to viable cell PC12.As figure can be observed compound F 17-hydroxy-corticosterone, I has the good cell membrane permeability.Used instrument is Nikon eclipase TE 2000-5.
Above content be in conjunction with concrete preferred implementation to further describing that the present invention did, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.As fluorescence dye is a kind of purposes of new compound of the present invention; can not assert that compound of the present invention only is used for fluorescence dye; for the general technical staff of the technical field of the invention; under based on the consideration of The compounds of this invention as the same function mechanism of fluorescence dye; can also make some simple inferences; draw other application purpose of compound of the present invention, all should be considered as belonging to protection scope of the present invention.
Claims (7)
1. a class indole hemicyanine dye, it is characterized in that: described dye molecule has general structure I:
Wherein: R
1=H, halogen, nitro, sulfonic group, R
2=H; Or R
1R
2Form the naphthalene indole ring
R
3And R
4Independently be selected from H, hydroxyl, halogen, nitro, C separately
1-4Alkyl;
R
5Be H, hydroxyl, N ((CH
2)
mR
7)
2, wherein: R
7=H, OH, OAc, COOH, COOCH
3, phenyl;
R
6Be (CH
2)
pR
8, wherein: R
8=H, OH, C=C, alkynyl, OAc, COOH, COOCH
3, COOC
2H
5, p-(C
6H
4) R
9, wherein: R
9=H, CH
3, halogen, NO
2, CN, COOH, L-A;
Y
-=halide-ions, ClO
4 -, PF
6 -Or TsO
-
L=-C
1-18Alkyl-, p-(CH
2)
q(C
6H
4) (CH
2)
q-;
M=1～18, n=1 or 2, p=0～18, q=1-6.
2. according to the described class indole hemicyanine dye of claim 1, it is characterized in that: as n=1, R
5During for hydroxyl, have structural formula II:
Wherein: R1=H, halogen, nitro, sulfonic group, R2=H; Or R1R2 forms the naphthalene indole ring
R3 and R4 independently are selected from H, hydroxyl, halogen, nitro, C1-4 alkyl separately;
R6 is (CH2) pR8, wherein: and R8=H, OH, C=C, alkynyl, OAc, COOH, COOCH3, COOC2H5, p-(C6H4) R9, wherein: R9=H, CH3, halogen, NO2, CN, COOH;
Y-=halide-ions, ClO4-, PF6-or TsO-;
p＝1～18。
3. according to the described class indole hemicyanine dye of claim 1, it is characterized in that: when R2=H, n=1, form structural formula II I:
Wherein: R1=H, halogen, nitro, sulfonic group.
R3 and R4 independently are selected from H, hydroxyl, halogen, nitro, C1-4 alkyl separately;
R5 is H, hydroxyl, N ((CH2) mR7) 2, wherein: R7=H, OH, OAc, COOH, COOCH3, phenyl;
R6 is (CH2) pR8, wherein: and R8=H, OH, OAc, COOH, COOCH3, COOC2H5, p-(C6H4) R9, wherein: R9=H, CH3, halogen, NO2, CN, COOH;
Y-=halide-ions, ClO4-, PF6-or TsO-;
m＝1～18；p＝0～18。
4. according to the described class indole hemicyanine dye of claim 1, it is characterized in that: when R6=L-A, have general structure IV:
Wherein: R1=H, halogen, nitro, sulfonic group, R2=H; Or R1R2 forms the naphthalene indole ring
R3 and R4 independently are selected from H, hydroxyl, halogen, nitro, C1-4 alkyl separately;
R5 is H, N ((CH2) mR7) 2, wherein: R7=H, OH, OAc, COOH, COOCH3, phenyl;
Y-=halide-ions, ClO4-, PF6-or TsO-;
The L=-Cl-18 alkyl-or (CH2) q-of p-(CH2) q (C6H4);
m＝1～18，n＝1、2，p＝1～18，q＝1-6。
5. according to the described class indole hemicyanine dye of claim 2, it is characterized in that: described have the compound of structural formula II as the pH fluorescent probe.
6. according to the described class indole hemicyanine dye of claim 3, it is characterized in that: described have the compound of structural formula II I as the solid fluorescence compound.
7. according to the described class indole hemicyanine dye of claim 4, it is characterized in that: described have the compound of structural formula IV as biological fluorescent labeling.
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| CN103113284A (en) * | 2013-01-18 | 2013-05-22 | 大连理工大学 | Half cyanine dye compound, preparation method and application of half cyanine dye compound |
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| CN103113283A (en) * | 2013-01-22 | 2013-05-22 | 天津理工大学 | Method for preparing hemicyanine plastocyanin temperature-sensitive molecular optical probe containing electron-donating group |
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