CN103687857A - Cytosolic fluorescent ion indicators - Google Patents
Cytosolic fluorescent ion indicators Download PDFInfo
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- CN103687857A CN103687857A CN201180055664.2A CN201180055664A CN103687857A CN 103687857 A CN103687857 A CN 103687857A CN 201180055664 A CN201180055664 A CN 201180055664A CN 103687857 A CN103687857 A CN 103687857A
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- Prior art keywords
- crown ether
- dibenzo
- diaza
- sequestrant
- fluorophore
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- 0 CC(C(c1ccc(*)c(C)c1)c(ccc(C)c1)c1O1)(C=C2)C1=CC2=* Chemical compound CC(C(c1ccc(*)c(C)c1)c(ccc(C)c1)c1O1)(C=C2)C1=CC2=* 0.000 description 6
- IQUPABOKLQSFBK-UHFFFAOYSA-N [O-][N+](c(cccc1)c1O)=O Chemical compound [O-][N+](c(cccc1)c1O)=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- PNGXCJQDDFDAPR-UHFFFAOYSA-N [O-][N+](c(cccc1)c1OCCOCCOCl)=O Chemical compound [O-][N+](c(cccc1)c1OCCOCCOCl)=O PNGXCJQDDFDAPR-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
- C07D311/90—Xanthenes with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
Abstract
Novel fluorophores for fluorescent ion indicators incorporate water-solubilizing functional groups. These fluorophores enable visible-wavelength analysis of ion concentrations with chelators that are insoluble or poorly soluble in water. A green version of the fluorophore comprises a putative fluorescein with carboxylate appendages. An orange version of the fluorophore comprises a putative rhodamine, also with carboxylate appendages. Attaching the fluorophore to a 12-crown-4, a 15-crown-5, a 18-crown-6, a 21-crown-7, or a 24-crown-8 produces lithium, sodium, potassium, rubidium, and cesium indicator families, respectively. Attaching the fluorophore to diaza [1,1,1], [1,1,2], [1,2,2], [2,2,2], [2,2,3], [2,3,3], or [3,3,3] cryptands produces more selective lithium, sodium, potassium, rubidium, and cesium indicator families, respectively. Attaching the fluorophore to BAPTA, APTRA, or half- BAPTA or their analogs produces new calcium, magnesium, and other metal ions indicator families that strongly resist leakage from the cell.
Description
In author: A Kuaxi (AKWASI MINTA) and P. Roger, ACunico plum difficult to understand is drawn (P.ROGELIO ESCAMILLA)
Proxy: A Sangte research company (ASANTE RESEARCH, LLC)
Submission date: on September 20th, 2011
Related application
The application is involved in the U.S. Provisional Patent Application submitted on September 20th, 2010 number 61/384,695, and requires the right of priority from this application.The application is involved in the U.S. Provisional Patent Application submitted on March 17th, 2011 number 61/453,664, and it is combined in to this by reference.
Invention field
The present invention relates to the purposes that a kind of fluorophore and this fluorophore and chelating moiety combine to provide new fluorescence ion indicator.
Background of invention
Fluorescence ion indicator typically comprises that is connected to the chelating moiety on fluorophore, make to excite and/or the variation of emission wavelength, or excite and/or the variation of emissive porwer, or the two occurs when in conjunction with this ion.For the ionic concn in water sensing medium, this fluorescence ion indicator should be water-soluble.For intracellular measurement, this indicator should be not disturbing the speed of this analysis to leak out from this cell.For convenience's sake, this indicator should be loaded in Non-Invasive mode, for example, as a kind of acetoxy-methyl ester.
Many sequestrants, as crown ether and cave ether, shortcoming is very poor water solubility, has limited thus the measuring ion to organic solvent.Yet if these sequestrants are connected on a water miscible fluorophore, they can produce a kind of fluorescence ion indicator for water medium.
For sequestrant soluble in water, as BAPTA, this water miscible fluorophore can strengthen water-soluble and, for intracellular measurement, the cell that can strengthen fluorescence ion indicator retains.
Two kinds of these type of water miscible fluorophores, hydroxyl xanthone variant and rosamine variant, be that Asante Green(is green)
tM(I) and Asante Orange(orange)
tM(II).
Summary of the invention
The present invention discloses new for measuring the fluorescence ion indicator of the concentration of metal ion.
This fluorescence ion indicator comprise a kind of combination water solubilising functional group, wavelength visible fluorophore, the chelating moiety of this indicator is not needed in conjunction with identical group.Therefore a kind of like this fluorophore makes to have water insoluble or to be insoluble in the wavelength analysis of ionic concn of sequestrant of water visible.
Because an application of these indicator comprises the measurement of the kytoplasm concentration of ion, this water solubilizing group, should be can shelter as the hydrophobic part that can see through this lipid cytolemma.Such hydrophobic part is typically easy to by cellular component, be cut so that this indicator is got back to its water-soluble form.The most universal hydrophobicity mask is acetoxy-methyl ester, and it can cut by the nonspecific esterase in cytosol.
For the application of identical cell, this fluorescence ion indicator should retain the sufficiently long time its leakage can not had a negative impact to experimental result in this cell.Water solubilising carboxylate salt contributes to this indicator to be retained in cell.
For calcium ion, the BAPTA chelating moiety of this indicator comprises carboxylate salt four kinds of water solubilisings and cell-retention.For alkaline-earth metal, this chelating moiety typically comprises crown ether or the cave ether without carboxylate salt functional group.In this case, this fluorophore provides necessary carboxylate salt functional group.
In the situation that sequestrant is if do not comprised the BAPTA of part water solubilising and cell-retention, this fluorophore only increases wetting ability and strengthens the reservation of this indicator.
The UV fluorescence excitation group of some prior aries has acetoxy-methyl ester-functional group that can shelter, water solubilising; As those in Fura-2, Indo-1, SBFI, PBFI or SBFO.Seldom there is the visible fluorescence excitation group to have these characteristics, and even rarer covalently bound to sequestrant.
The green version of this fluorophore comprises a kind of fluorescein of supposition, this fluorescein be halo to guarantee in the pH insensitivity at physiology pH place and to have shown two kinds of carboxylate salt official energy at physiology pH place.
The orange version of this fluorophore comprises the basic rhodamine having through a kind of supposition of the alkylating amino/imino-of carboxylate salt (immino) sense.
Thereby this fluorophore is attached to and on 12-crown ether-4,15-crown ether-5, hexaoxacyclooctadecane-6-6, heptaoxacycloheneicosane-7-7 or octaoxacyclotetracosane-8-8, produces respectively lithium, sodium, potassium, rubidium, He Se indicator family.
This fluorophore is attached to diaza [1,1,1], [1,1,2], [1,2,2], [2,2,2], [2,2,3], [2,3,3] or [3,3,3] thus on the ether of cave, produce and have more optionally lithium, sodium, potassium, rubidium, He Se indicator family respectively.
Thereby this fluorophore is attached to and on BAPTA or its analogue, produces a kind of new, calconcarboxylic acid family of the anti-leakage from this cell consumingly, and this gives the credit to the other carboxylate salt on this fluorophore.These calconcarboxylic acids show larger brightness and the dynamicrange of indicator (for example Fluo-4) than prior art.
Thereby this fluorophore is attached to and on APTRA or its analogue, produces a kind of new, magnesium indicator family of the anti-leakage from this cell consumingly, and this gives the credit to the other carboxylate salt on this fluorophore.
Thereby this fluorophore is attached to and on half-BAPTA or its analogue, produces a kind of new, indicator family of the anti-leakage from this cell consumingly, and this gives the credit to the other carboxylate salt on this fluorophore.
Brief Description Of Drawings
Fig. 1 shows in an acellular water droplet is fixed, the response of sodium fluorescent indicator ANG-1 to ever-increasing na concn.
Fig. 2 shows in an acellular water droplet is fixed, the response of sodium fluorescent indicator ANG-2 to ever-increasing na concn.
Fig. 3 shows in an acellular water droplet is fixed, the response of sodium fluorescent indicator ANG-3 to ever-increasing na concn.
Fig. 4 shows in an acellular water droplet is fixed, the response of potassium fluorescent indicator APG-1 to ever-increasing potassium concn.
Fig. 5 shows in an acellular water droplet is fixed, the response of potassium fluorescent indicator APG-2 to ever-increasing potassium concn.
Fig. 6 shows in an acellular water droplet is fixed, the response of calcium fluorescent indicator ACG-1 to ever-increasing calcium concn.
Fig. 7 shows in HEK293 cell, and due to the capsaicine agonism (capsaicin agonization) of TRPV1 passage, sodium fluorescent indicator ANG-1 is to Na in ever-increasing cell
+the response of concentration.
Fig. 8 shows in REF52 cell, due to Na
+the interior stream of ionophore linear gramicidins-promotion, sodium fluorescent indicator ANG-1 is to Na in ever-increasing cell
+the response of concentration.
Fig. 9 shows in stellate cell, and due to the crow bar glycosides inhibition of sodium pump, sodium fluorescent indicator ANG-1 is to Na in ever-increasing cell
+the response of concentration.
Figure 10 shows in REF52 inoblast, due to Na
+the interior stream that ionophore SQI-Pr-promotes, sodium fluorescent indicator ANG-2 is to Na in ever-increasing cell
+the response of concentration.
Figure 11 shows in REF52 inoblast, and sodium fluorescent indicator ANG-2 is to Na in cell
+amphotericin-B consumption, and then to Na in ever-increasing cell
+the response of concentration.
Figure 12 shows in REF52 inoblast, and sodium fluorescent indicator APG-1 is to K in cell
+amphotericin-B consumption, and then to K in ever-increasing cell
+the response of concentration.
Figure 13 has compared Ca
2+the quantum yield of-saturated ACG-1 to fluorescein.
Figure 14 shows in the vagal Sensory neurone of rat, the response of calcium fluorescent indicator ACG-1 to the depolarize step from 1-1000msec scope.
Fig. 1 shows in an acellular water droplet is fixed, sodium fluorescent indicator (diaza-15-crown ether-5 sequestrant; Z
1=O
-+tMA; Z
2=O; R
1=Cl; R
2=(CH
2)
ncOO
-+tMA, n=2; R
3=R
6=R
7=R
8=R
9=H) response to ever-increasing na concn.
Fig. 1
6show in an acellular water droplet is fixed sodium fluorescent indicator (diaza-15-crown ether-5 sequestrant; Z
1=O
-+tMA; Z
2=O; R
1=H; R
2=(CH
2)
ncOO
-+tMA, n=2; R
3=R
7=R
8=R
9=H, R
6=OMe) response to ever-increasing na concn.
The explanation of embodiment
Asante Green(is green)
tMwith Asante Orange(orange)
tMthereby the covalently bound fluorescence ion indicator to producing water-soluble and cell-retention on some sequestrant, comprises for those of lithium, sodium, potassium, rubidium, caesium, magnesium, calcium and thallium.
I.Asante Green(is green) II.Asante Orange(orange)
X=F、Cl、Br R
1=(CH
2)
nCOO
-，n=0、1、2、3
R
1=(CH
2)
ncOO
-, n=0,1,2,3 R
2=H or (CH
2)
ncOO
-, n=0,1,2,3
In this embodiment, these ion indicator comprise via as shown in a connector (as figure LINKER(connector)) the Asante Green(that is connected on a kind of sequestrant is green) or Asante Orange(orange) as shown in fluorophore (as figure FLUOROPHORE(fluorophore)).When this sequestrant comprises a kind of diaza crown ether, this fluorophore can be via as shown in a non-annularity connector (as figure NON-ANNULATED LINKER(non-annularity connector)) azepine or the amine official that are connected to this crown ether can go up, and other azepines of this crown ether or amine official can be with as shown in aromatic senses that can comprise a plurality of parts (as figure NON-ANNULATED CAP(non-annularity cap)) add cap, thereby increase or reduce sequestrant affinity, strengthen the reservation of cell, this indicator is positioned to cytolemma or other cellular compartment places, or activating reaction reactive site is for being attached to synthetic or natural polymkeric substance.When this sequestrant comprises a kind of ring-type diaza crown ether or cave ether, this fluorophore can be via as shown in a loop connecting thing (as figure ANNULATED LINKER(loop connecting thing)) an azepine official being connected to this crown ether can go up, yet other azepines official of this crown ether can be with as shown in an aromatic sense of ring-type that can comprise a plurality of parts (as figure ANNULATED CAP(non-annularity cap)) add cap, thereby increase or reduce sequestrant affinity, strengthen the reservation of cell, this indicator is positioned to cytolemma or other cellular compartment places, or activating reaction reactive site is for being attached to synthetic or natural polymkeric substance.
FLUOROPHORE(fluorophore)
Z
1=OR
4or NR
5ar
5b
Z
2=O or NR
5ar
5b
R
1=H, F, Cl, Br or (CH
2)
ncO
2r
4, n=0,1 or 2
R
2=H, F, Cl, Br or (CH
2)
ncO
2r
4, n=0,1 or 2
R
3=H、F、Cl、Br
R
4=H, salt (that is, TMA
+, K
+) or AM(CH
2oCOCH
3) or any other pharmacy acceptable salt and ester
R
5a=(CH
2)
ncO
2r
4, n=0,1 or 2
R
5b=(CH
2)
ncO
2r
4, n=0,1 or 2
R
50=to the covalent linkage on a non-annularity connector, loop connecting thing or sequestrant
NON-ANNULATED LINKER(non-annularity connector)
R
6=H、OMe、F
R
7=H、OMe、F
R
8=H、OMe、F
R
9=H、OMe、F
R
51=at R
50place is to the covalent linkage on this fluorophore
R
52=to the covalent linkage on a non-annularity sequestrant
NON-ANNULATED CAP(non-annularity cap)
R
10=H、OMe、F
R
11=H、OMe、F
R
12=H, Me, F, Br, Cl, I, (CH
2)
ncO
2r
4[n=0,1 or 2], NO
2, CHO,
or
R
13=H、OMe、F
R
14=H、OMe、F
R
53=to the covalent linkage on a non-annularity sequestrant
ANNULATED LINKER(loop connecting thing)
N, O are the heteroatomss in this cyclic crown ether or cave ether
R
15=H、OMe、F
R
16=H、OMe、F
R
17=H、OMe、F
R
51=at R
50place is to the covalent linkage on this fluorophore
ANNULATED CAP(annular cap)
N, O are the heteroatomss in this cyclic crown ether or cave ether
R
18=H、OMe、F
R
19=H、OMe、F
R
20=H, Me, F, Br, Cl, I, (CH
2)
ncO
2r
4[n=0,1 or 2], NO
2, CHO,
R
21=H、OMe、F
The example that is used for the sequestrant of lithium comprises single azepine-12-crown ether-4, diaza-12-crown ether-4, dibenzo diaza-12-crown ether-4 and [1,1,1]-dibenzo cave ether.
R
22=(CH2)
ncH
3, n=0,1 or 2
The example that is used for the sequestrant of sodium comprises single azepine-15-crown ether-5, diaza-15-crown ether-5, dibenzo diaza-15-crown ether-5, [1,1,2]-dibenzo cave ether and [1,2,2]-dibenzo cave ether.
The example that is used for the sequestrant of potassium comprises single azepine-hexaoxacyclooctadecane-6-6, diaza-hexaoxacyclooctadecane-6-6, dibenzo diaza-hexaoxacyclooctadecane-6-6 and [2,2,2]-dibenzo cave ether.
The example that is used for the sequestrant of calcium comprises BAPTA and derivative thereof:
R
24=H、F、Br、Cl
R
25=H, Me, F, Br, Cl, I, NO
2, CHO, (CH
2)
ncO
2r
4[n=0,1 or 2],
R
26=H, F, Br, Cl, (CH
2)
ncO
2r
4[n=0,1 or 2],
R
51=at R
50place is to the covalent linkage on this fluorophore
The example that is used for the sequestrant of magnesium comprises APTRA and derivative thereof:
R
51=at R
50place is to the covalent linkage on this fluorophore
The example that is used for the sequestrant of thallium comprises partly-BAPTA and derivative thereof.
R
51=at R
50place is to the covalent linkage on this fluorophore
Synthetic
These synthetic schemess typically comprise synthetic, additional this fluorophore subsequently of this sequestrant/connector.This fluorophore causes the condensation of the phenyl aldehyde in compound 1 and this sequestrant/connector part
Z=OH or NR
5ar
5b
R
1=H, F, Cl, Br or (CH
2)
ncO
2r
4, n=0,1 or 2
R
2=H, F, Cl, Br or (CH
2)
ncO
2r
4, n=0,1 or 2
R
3=H、F、Cl、Br
R
4=H, salt (that is, TMA
+, K
+) or AM(CH
2oCOCH
3) or any other pharmacy acceptable salt and ester
R
5a=(CH
2)
ncO
2r
4, n=0,1 or 2
R
5b=(CH
2)
ncO
2r
4, n=0,1 or 2
a)NaOCl，KOH b)K
2CO
3，BnCl，NaI，DMF c)Br
-Ph
3P
+(CH
2CO
2Me)，K
2CO
3d)Pd/C/H
2AcOH，50psi
By commercially available 2,4-Dihydroxy benzaldehyde is used clorox chlorination in basic solution.These phenol protections, for benzyloxy ether, are then carried out to Wittig reaction (Wittig reaction) with the triphenylphosphine salt of monobromomethane acetic ester.Carry out subsequently catalytic reduction and debenzylation, provide desirable product.
a)NaOCl，KOH b)MnO
2，MeOH
By commercially available 2,4-Dihydroxy benzaldehyde is used clorox chlorination in basic solution.This aldehyde is oxidized to methyl esters with Manganse Dioxide in methyl alcohol.
a)NaOCl，KOH b)BnCl，NaI，K
2CO
3c)LAH d)SO
2Cl
2e)KCN f)1.KOH，2.HCl g)MeOH，H
2SO
4h)Pd/C/H
2AcOH，50psi
By commercially available 2,4-Dihydroxy benzaldehyde in basic solution with clorox chlorination and then carry out benzyl to protect these phenol.Carry out lithium aluminium hydride reduction reaction, provide this alcohol, this alcohol is converted into muriate with sulfuryl chloride.With potassium cyanide, replace this muriate, provide nitrile, this nitrile is hydrolyzed to carboxylic acid.Carry out esterification, then carry out catalysis debenzylation, provide desirable product.
Scheme 4 has been described the synthetic of compound 1 in following situation: Z=OH, R
1=(CH
2)
2cO
2me, R
2=Cl, and R
3=H.
a)Br
-Ph
3P
+(CH
2CO
2Me)，K
2CO
3b)Pd/C/H
2c)BBr
3d)MeOH，H
2SO
4e)NCS
By commercially available 2,6-dimethoxy benzaldehyde carries out condensation by the triphenylphosphine salt of monobromomethane acetic ester.This alkylidene group is carried out to catalytic reduction, then carry out the boron tribromide cutting of these methyl ethers.By this carboxylic acid resterification, and use N-chlorosuccinimide chlorination, provide desirable product.
Scheme 5 has been described the synthetic of compound 1 in following situation: Z=N (CH
2cH
2cO
2me)
2, R
1=R
2=R
3=H.
a)BrCH
2CO
2Me，DIPEA b)Pd/C/H
2AcOH
Commercially available 3-benzyloxy-aniline is carried out to alkylation with monobromomethane acetic ester, then carry out catalysis debenzylation to provide desirable product.
sodium
Because crown ether only or cave ether size (size) can change, for simplicity, for the sodium group of sequestrant, show the synthetic scheme for this sequestrant/connector, so that also represent lithium, potassium, rubidium or caesium.
Scheme 6 shows the synthetic of compound 2, R
6=R
7=R
8=R
9=H:
A) DMF backflow b) Pd/C/H
2c) NaNO
2/ HCl d) H
3pO
2e) DMF/POCl
3f) Z=OH, MeSO
3h or Z=NR
5r
6, propionic acid g) and to chloranil h) TMAOH i) AM-Br, DIPEA
Under DMF refluxes, by commercial azepine-15-crown ether-5 with fluorine nitre benzene is reacted to produce N-p-nitrophenyl-azepine-15-crown ether-5.By this nitroreduction duplicate removal nitrogenize to produce N-phenyl-azepine-15-crown ether-5.Carry out Wei Er David Smail reaction (Vilsmeier reaction) to produce phenyl aldehyde, this phenyl aldehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
Scheme 7a shows the synthetic of compound 2, R
6=R
7=R
8=R
9=H:
A) pyridine backflow b) Pd/C/H
2c) NaNO
2/ HCl d) H
3pO
2e) DMF/POCl
3f) Z=OH, MeSO
3h or Z=NR
5ar
5b, propionic acid g) and to chloranil h) TMAOH i) AM-Br, DIPEA
Under pyridine refluxes, by commercial diaza-15-crown ether-5 with fluorine nitre benzene is reacted to produce pair-N-p-nitrophenyl-diaza-15-crown ether-5.By these nitro catalytic reductions duplicate removal nitrogenize to produce two-N-phenyl-diaza-15-crown ether-5.Carry out Wei Er David Smail reaction (Vilsmeier reaction) to produce two phenyl aldehydes, this two phenyl aldehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
Scheme 7b shows the synthetic of compound 2, R
6=R
7=R
8=R
9=R
10=R
11=R
12=R
13=R
14=H.
E) DMF/POCl
3/ pyridine, room temperature f) Z=OH, MeSO
3h or NR
5ar
5b, propionic acid g) and to chloranil h) TMAOH i) AM-Br, DIPEA
From scheme 7a, two-N-phenyl-diaza-15-crown ether-5: carry out gentle Wei Er David Smail reaction, produce single phenyl aldehyde, this list phenyl aldehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
Scheme 8 shows the synthetic of compound 2, R
6=R
8=OMe, R
7=R
9=H.
A) chloroform/methanol backflow b) Pd/C/H
2c) Me
2sO
4, TMAOH d) and DMF/POCl
3e) Z=OH, MeSO
3h or Z=NR
5ar
5b, propionic acid f) and to chloranil g) TMAOH h) AM-Br, DIPEA
Quinone adducts is reacted to provide in commercial diaza-15-crown ether-5 with quinone, by its reduction and then carry out alkylation to provide this tetramethoxy-compound with methyl-sulfate.Carry out Wei Er David Smail formylation (Vilsmeier formylation), provide dialdehyde, this dialdehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
Be similar to scheme 6 contrast scheme 7a, with single azepine-15-crown ether-5, by scheme 8, provide list-nitric heterocyclic compound 2, R
6=R
8=Ome, R
7=R
9=H.Be similar to scheme 7b contrast scheme 7a, carry out gentle Wei Er David Smail formylation, provide single formylation compound rather than dialdehyde, this list formylation compound produces compound 2, R
6=R
8=R
10=R
13=OMe, R
7=R
9=R
11=R
12=R
14=H.
Scheme 9 shows the synthetic of compound 2, R
6=OMe, R
7=R
8=R
9=H.
A) 1, two (2-chloroethoxy) ethane of 2-, DIPEA b) chlorination dihydroxy acetyl, pyridine, high dilution benzene c) B
2h
6d) DMF/POCl
3e) Z=OH, MeSO
3h or Z=NR
5ar
5b, propionic acid f) and to chloranil g) TMAOH h) AM-Br, DIPEA
Two (2-chloroethoxy) ethane of commercially available o-anisidine and 1,2-is reacted to form " half crown ether ", and the addition by chlorination dihydroxy acetyl is " complete crown ether " by its closure, carries out subsequently the reduction reaction of diamide.Carry out Wei Er David Smail formylation, provide dialdehyde, this dialdehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
A variant; R
1=Cl; R
2=(CH
2)
ncOOR
4, n=2; R
3=R
7=R
8=R
9=H; R
6=OMe; Provide Asante NaTRIUM Green-3(ANG-3).
ANG-3TMA
+salt, R
4=(CH
3)
4n
+
ANG-3（AM），R
4=CH
2OCOCH
3
Enjoyably, with methyl alcohol, as unique solvent, replace 1:1 methyl alcohol/chloroform mixture to carry out the oxidation of chloranil to cause incomplete oxidation, final a kind of compound of output, wherein R
6=R
10=OMe, R
12=CHO, R
7=R
8=R
9=R
11=R
13=R
14=H.A variant, R
1=Cl; R
2=(CH
2)
ncOOR
4, n=2; R
3=H; Provide Asante NaTRIUM Green-1(ANG-1).
ANG-1TMA
+salt, R
4=(CH
3)
4n
+
ANG-1（AM），R
4=CH
2OCOCH
3
Be similar to scheme 7b contrast scheme 7a, carry out gentle Wei Er David Smail formylation, provide single formylation compound rather than dialdehyde, this list formylation compound produces a kind of compound, wherein R
6=R
10=OMe, R
7=R
8=R
9=R
11=R
12=R
13=R
14=H.
Scheme 10 shows the synthetic of compound 2, R
6=R
10=OMe, R
12=Me, R
7=R
8=R
9=R
11=R
13=R
14=H
A) Pd/C/H
2b) 1, two (2-chloroethoxy) ethane of 2-, DIPEA, NaI c) DIPEA, NaI d) chlorination dihydroxy acetyl, pyridine, high dilution benzene e) B
2h
6f) DMF/POCl
3g) Z=OH, MeSO
3h or Z=NR
5ar
5b, propionic acid h) and to chloranil i) TMAOH j) AM-Br, DIPEA
By commercially available 5-methyl-2-Nitroanisole catalytic reduction, be aniline, for reacting to form " 1/4th crown ethers " with two (2-chloroethoxy) ethane of 1,2-.It is reacted to be converted into o-anisidine " half crown ether ", the addition by chlorination dihydroxy acetyl is " complete crown ether " by its closure, carries out subsequently the reduction reaction of diamide.Carry out Wei Er David Smail formylation, provide dialdehyde, this dialdehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
A variant; R
1=Cl; R
2=(CH
2)
ncOOR
4, n=2; R
3=R
7=R
8=R
9=R
11=R
13=R
14=H; R
6=R
10=OMe; R
12=Me; Provide Asante NaTRIUM Green-2(ANG-2).
ANG-2TMA
+salt, R
4=(CH
3)
4n
+
ANG-2（AM），R
4=CH
2OCOCH
3
Scheme 11 shows the synthetic of compound 3, R
15=R
16=R
17=H, R
22=(CH
2)
ncH
3, n=1,2 or 3.
A) chlorination dihydroxy acetyl, pyridine, high dilution DCM b) B
2h
6c)R
23chloride of acid (R
23=(CH
2)
ncH
3, n=0,1 or 2) and d) B
2h
6(R
22=(CH
2)
ncH
3, n=1,2 or 3) and e) DMF/POCl
3f) Z=OH, MeSO
3h or Z=NR
5ar
5b, propionic acid g) and to chloranil h) TMAOH i) AM-Br, DIPEA
By 1, two (2-amino-benzene oxygen) ethane (biological chemistry (Biochemisty) 19:2396-2404 (1980)) of 2-reacts to produce diamide with chlorination dihydroxy acetyl (dyglycolyl) in high dilution, and diamide is reduced to obtain benzo-cyclic crown ether.In pyridine, react to produce the second diamide with alkyl acyl chlorine, and these acid amides are reduced to provide N-alkyl cyclic crown ether.Carry out Wei Er David Smail formylation, generate dialdehyde, this dialdehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
Be similar to scheme 7b contrast scheme 7a, carry out gentle Wei Er David Smail formylation, provide single formylation compound rather than dialdehyde, this list formylation compound produces compound 3, R
15=R
16=R
17=R
18=R
19=R
20=R
21=H; R
22=(CH
2)
ncH
3, n=1,2 or 3.
By using 1-(2-amino-5-methylphenoxy), 2-(2-amino-benzene oxygen) ethane (journal of biological chemistry (J.Biol.Chem.) 260:3440-3450; 1985) 1 in replacement scheme 11; two (2-amino-benzene oxygen) ethane of 2-; carry out Wei Er David Smail formylation; provide single formylation compound rather than dialdehyde; this list formylation compound produces compound 3, R
20=Me, R
15=R
16=R
17=R
18=R
19=R
21=H; R
22=(CH
2)
ncH
3, n=1,2 or 3.Similarly, any other precursor, to the variant (biological chemistry (Biochemisty) 19:2396-2404 (1980)) of BAPTA, comprises 5-halogen, 5,6-difluoro, 5-nitro, or any variant of the reduction of opposing diboron hexahydride, by producing the variant of compound 3, be mainly at R
20(that is, the R of place
20=F, Br, NO
2).
Scheme 12 shows the synthetic of compound 4, R
15=R
16=R
17=H.
A) chlorination dihydroxy acetyl, pyridine, high dilution DCM b) B
2h
6c) DMF/POCl
3d) Z=OH, MeSO
3h or Z=NR
5ar
5b, propionic acid e) and to chloranil f) TMAOH g) AM-Br, DIPEA
Benzo cyclic crown ether from scheme 11 is reacted to form diamide with chlorination dihydroxy acetyl, this diamide is reduced to produce [1,1,2] benzo ring-type cave ether with diboron hexahydride.Carry out Wei Er David Smail formylation to produce dialdehyde, this dialdehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
Be similar to scheme 7b contrast scheme 7a, carry out gentle Wei Er David Smail formylation, provide single formylation compound rather than dialdehyde, this list formylation compound produces compound 4, R
15=R
16=R
17=R
18=R
19=R
20=R
21=H.
Enter in scheme 12; with 1-(2-amino-5-methylphenoxy), 2-(2-amino-benzene oxygen) ethane (journal of biological chemistry (J.Biol.Chem.) 260:3440-3450; (1985)) replace 1; two (2-amino-benzene oxygen) ethane (scheme 11) of 2-; finally provide single formylation compound rather than dialdehyde; this list formylation compound produces compound 3, R
20=Me, R
15=R
16=R
17=R
18=R
19=R
21=H.Similarly, any other precursor, to the variant (biological chemistry (Biochemisty) 19:2396-2404 (1980)) of BAPTA, comprises 5-halogen, 5,6-difluoro, 5-nitro, or any variant of the reduction of opposing diboron hexahydride, by producing the variant of compound 3, be mainly at R
20(that is, the R of place
20=F, Br, NO
2).
With chlorination three hydroxyl acetyl, replace the chlorination dihydroxy acetyl in scheme 12, and subsequently with the chlorination dihydroxy acetyl in unsubstituted scheme 11 in chlorination three hydroxyl acetyl changes or replacement scheme 12, produce [1,2,2] benzo ring-type cave ether variant of this fluorescence ion indicator.
For the example synthetic schemes of sodium fluorescent indicator, can be modified to be provided for the scheme of following lithium, potassium, rubidium and caesium.
lithium
The example of lithium fluorescent indicator is based on 12-crown ether-4 and [1,1,1] cave ether sequestrant.
Scheme 13 shows synthesizing for the precursor of diaza-12-crown ether-4 fluorescent indicator.
A) chlorination dihydroxy acetyl, pyridine b) B
2h
6
Commercially available o-anisidine is reacted to provide diamide with chlorination dihydroxy acetyl, be reduced to " half-crown ether " that produce compound 5, R
6=OMe; R
7=R
8=R
9=H.
Can will should " complete-crown ether " leniently carry out formylation to provide the list-formyl radical precursor that produces compound 5, R
6=R
10=OMe, R
7=R
8=R
9=R
11=R
12=R
13=R
14=H.
In scheme 13, o-anisidine is reacted to provide the statistical distribution of dimethyl, monomethyl and non-methyl product with 4-methyl-o-methyl oxyaniline with chlorination dihydroxy acetyl.Due to the significant difference of the polarity among diformyl, single formyl radical/monomethyl and dimethyl product, this three can be easily separated after formylation.This list formyl radical/monomethylation compound finally produces compound 5, R
6=R
10=OMe, R
7=R
8=R
9=R
11=R
13=R
14=H, R
12=Me.
Scheme 14 shows synthesizing for the precursor of dibenzo ring-type 12-crown ether-4 or dibenzo-[1,1,1] and dibenzo-[1,1,2] cave ether.
A) DMI, K
2cO
3b) Pd/C/H
2
Commercially available o-NP and the chloro-2-oil of mirbane of 1-are at high temperature carried out to condensation to provide the compound of dinitrobenzene, are " half-crown ether " by its catalytic reduction.
potassium
The example of potassium fluorescent indicator is based on hexaoxacyclooctadecane-6-6 and [2,2,2] cave ether sequestrant.
Hexaoxacyclooctadecane-6-6 are replaced by 15-crown ether-5.
Chlorination three hydroxyl acetyl can be chlorinated dihydroxy acetyl and replace.Its synthetic reaction of the nitric acid oxidation to three oxyacetic acids and three oxyacetic acids and oxalyl chloride from triglycol.
These change application, in scheme 9, are produced to Asante Potassium Green(green)-3; R
1=Cl; R
2=(CH
2)
ncOOR
4, n=2; R
3=R
7=R
8=R
9=H; R
6=OMe:
APG-3TMA
+salt, R
4=(CH
3)
4n
+
APG-3（AM），R
4=CH
2OCOCH
3
By these change application in scheme 9, and with only in methyl alcohol rather than in 1:1 methyl alcohol/chloroform that chloranil oxidation is produced to Asante Potassium Green(is green)-1; R
1=Cl; R
2=(CH
2)
ncOOR
4, n=2; R
6=R
10=OMe; R
12=CHO; R
3=R
7=R
8=R
9=R
11=R
13=R
14=H
APG-1TMA
+salt, R
4=(CH
3)
4n
+
APG-1（AM），R
4=CH
2OCOCH
3
These change application, in scheme 10, are produced to Asante Potassium Green(green)-2; R
1=Cl; R
2=(CH
2)
ncOOR
4, n=2; R
3=R
7=R
8=R
9=R
11=R
13=R
14=H; R
6=R
10=OMe; R
12=Me
APG-2TMA
+salt, R
4=(CH
3)
4n
+
APG-2（AM），R
4=CH
2OCOCH
3
rubidium
The example of rubidium fluorescent indicator is based on heptaoxacycloheneicosane-7-7 and [2,2,3] and [2,3,3] cave ether sequestrant.
Heptaoxacycloheneicosane-7-7 are replaced by 15-crown ether-5.
Two [2-(2-chloroethoxy) ethyl] ether can be replaced by two (2-chloroethoxy) ethane of 1,2-.
3,6,9-trioxa undecane diacyl chloride can be chlorinated dihydroxy acetyl and replace.It is synthetic from 3,6,9-trioxa undecane diprotic acid and oxalyl chloride.
A) two [2-(2-chloroethoxy) ethyl] ether, NaI, K
2cO
3b) NaI, K
2cO
3
Commercially available o-NP is reacted to form with excessive two [2-(2-chloroethoxy) ethyl] ethers the ether that monoalkyl replaces.Do not need two excessive chlorine reagents, it directly forms symmetrical dinitro compound (ether of dialkyl group).The ether that this monoalkyl is replaced further reacts to produce this asymmetric dinitro compound with a variant (that is, 5-methyl-2-nitrophenols) of o-NP.
caesium
The example of caesium fluorescent indicator is based on octaoxacyclotetracosane-8-8 and [3,3,3] cave ether sequestrant.
Octaoxacyclotetracosane-8-8 are replaced by 15-crown ether-5.
Two [2-(2-chloroethoxy) ethyl] ether is replaced by two (2-chloroethoxy) ethane of 1,2-.
3,6,9-trioxa undecane diacyl chloride is chlorinated dihydroxy acetyl and replaces.It is synthetic from 3,6,9-trioxa undecane diprotic acid and oxalyl chloride.
calcium
The example of calcium fluorescent indicator based on as at journal of biological chemistry (J.Biol.Chem.) 260:3440-3450, synthetic BAPTA and derivative thereof in (1985), biological chemistry (Biochemisty) 19:2396-2404 (1980) and US5576433.
Scheme 16 shows the synthetic of compound 6.
A) Z=OH, MeSO
3h or Z=NR
5ar
5b, propionic acid b) and to chloranil c) 1.KOH, TMAOH, or Bu
4if NOH2.HCl(is used KOH) d) AM-Br, Na
2cO3
BAPTA aldehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
Variant a: R
1=Cl; R
2=(CH
2)
ncOOR
4, n=2; R
24=R
26=H; R
25=Me; Provide Asante Calcium Green-1(ACG-1).
ACG-1TMA
+salt, R
4=(CH
3)
4n
+
ACG-1（AM），R
4=CH
2OCOCH
3
magnesium
The example of magnesium fluorescent indicator is based on APTRA.
Scheme 16 shows the synthetic of compound 7.
A) monobromomethane acetic ester, NaI, DIPEA b) DMF/POCl
3c) Z=OH, MeSO
3h or Z=NR
5ar
5b, propionic acid d) and to chloranil e) 1.KOH2.HCl f) AM-Br, DIPEA
Ortho-Aminophenol is carried out to alkylation with monobromomethane acetic ester.Carry out Wei Er David Smail (Vilsmeir) reaction to produce aldehyde, this aldehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
Variant a: R
1=Cl; R
2=(CH
2)
ncOOR
4, n=2; Provide Asante Magnesium Green-1(AMG-1).
AMG-1TMA
+salt, R
4=(CH
3)
4n
+
AMG-1(AM)，R
4=CH
2OCOCH
3
thallium
The example of thallium fluorescent indicator is based on half-BAPTA.
Scheme 17 shows the synthetic of compound 8.
A) monobromomethane acetic ester, NaI, DIPEA b) DMF/POCl
3c) Z=OH, MeSO
3h or Z=NR
5ar
5b, propionic acid d) and to chloranil e) 1.KOH2.HCl f) AM-Br, DIPEA
O-anisidine is carried out to alkylation with monobromomethane acetic ester.Carry out Wei Er David Smail (Vilsmeir) reaction to produce aldehyde, this aldehyde is carried out to coupling with Resorcinol or amino-phenol variant respectively in methylsulfonic acid or propionic acid, and use subsequently chloranil is oxidized to provide fluorescence dye.Ester is hydrolyzed, provides salt form, and react with acetoxy-methyl, provide AM ester-formin.
Variant a: R
1=Cl; R
2=(CH
2)
ncOOR
4, n=2; Provide Asante Thallium Green-1(AMG-1).
ATG-1TMA
+salt, R
4=(CH
3)
4n
+
ATG-1(AM)，R
4=CH
2OCOCH
3
Example 1 to 5, scheme 1
Example arrives, scheme 9
Compound 103
O-anisidine (362mL) is added together together with diisopropylethylamine (233mL) and two (2-chloroethoxy) ethane (84mL) of 1,2-.This reaction is stirred and continued 3 days at 120 ° of C.This crude product is placed on the rotatory evaporator with oil bath and by high vacuum and is heated to >100 ° of C to remove most of excessive methyl oxyaniline.After this, by this resistates with ethyl acetate dilution and wash twice with water, then by dried over sodium sulfate and evaporate.By this crude product vaporising under vacuum, and use the ethyl acetate content of 6:1 hexane/ethyl acetate and increase gradually to carry out purifying by column chromatography.
Compound 104
Diglycollic acid (25g) is added in a dry flask.Add wherein thionyl chloride (85mL) and immediately this reaction is heated to and reflux and stir and continue 5 hours in this temperature.Meanwhile, this excessive thionyl chloride, under 85 ° of C and decompression, is at room temperature distilled until mixture precipitation goes out by high vacuum subsequently.Then by this thick slag at 40 ° of C, distill until collect whole liquid at high vacuum and the temperature that increases gradually.
Compound 107
By compound 103(17mmol) and 104(80mol%, with the 22mmol crossing) use dividually benzene (each 150mL) to be dissolved in flask.Through several hours, these are added in the flask that contains benzene (150mL) and pyridine (46mmol) with the speed equating simultaneously.After having added, flask is positioned in the flask in the oil bath in 75 ° of C and stirs and spend the night.Then benzene is fully evaporated and resistates is dissolved in methylene dichloride.With being mixed with the 1M HCl of some salt solution, then with sodium bicarbonate and finally use salt solution washed twice, then with sodium sulfate, be dried.After vaporising under vacuum, by column chromatography, be loaded with chloroform and use 5% methyl alcohol in chloroform and carry out purifying under the methanol content increasing gradually.
Compound 108
By compound 107(6.2g) be dissolved in 100mL THF, and add 6.0g sodium borohydride.Through 1 hour, add the solution of 35mL boron trifluoride-Anaesthetie Ether compound in 70mL THF.This reaction mixture is poured on ice, and the pH regulator of this slurry is arrived to neutral pH.This slurry is extracted fully with methylene dichloride, and by the salt water washing of these organism, with anhydrous magnesium sulfate drying, filter and vaporising under vacuum.
Compound 109
By compound 108(6.5g, from step above, use without further purification) be dissolved in 100mLDMF and in ice bath cooling 15 minutes.31mL phosphorus oxychloride is increased to the temperature of this reaction mixture speed to be no more than the several years is added in cooling solution.Once complete this interpolation, be about to this reaction mixture and stir and spend the night at 70 ° of C.Then this reaction mixture is poured on ice, and pH use KOH is adjusted to neutrality.This water paste is extracted fully with methylene dichloride, and by the salt water washing of these organism, with anhydrous magnesium sulfate drying, filtration vaporising under vacuum are to produce a kind of filemot solid.This solid is ground with methyl alcohol, to provide a kind of pale solid.
Compound 101
In the 4-methyl-o-methyl oxyaniline (6g) being dissolved in acetonitrile (30mL), add diisopropylethylamine (15mL), sodium iodide (1.8g) and l, two (2-chloroethoxy) ethane (14mL) of 2-.This reaction is stirred and spent the night at 110 ° of C.This mixture diluted is also used to salt water washing once three times in ethyl acetate and with the washing of pH2 damping fluid.This organic layer is evaporated by dried over sodium sulfate and under vacuum.By column chromatography, use 3:1 hexane/ethyl acetate to carry out purifying this crude product.
By compound 101(10.25g) be dissolved in 51mL acetonitrile.Add diisopropylethylamine (31mL), methyl oxyaniline (46mL) and sodium iodide (3.1g) and this mixture is stirred and spent the night at 120 ° of C.By this mixture diluted in ethyl acetate with in salt solution and separated these layer.By this organic layer salt solution washed twice, by dried over sodium sulfate, then by solvent vaporising under vacuum.The ethyl acetate content of this crude product being used to 6:1 hexane/ethyl acetate by column chromatography and increasing is gradually carried out purifying.
At 45 ° of C, in the mode of a plurality of small portions, add triglycol (18mL) to 70%HNO lentamente
3in (100g, d=l.4), before each interpolation, wait for the minimizing of being fuming.Then being heated to 80 ° of C stirs 1 hour.At 70 ° of C, by high vacuum, on rotatory evaporator, evaporate 2 hours.Add 120mL benzo and again by high vacuum, at 70 ° of C, be dried.In process of cooling, liquid does not solidify.
Compound 106
Be equipped with reflux exchanger and drying tube, by compound 105(32.7g) be dissolved in chloroform (500mL), and add in oil bath at 90 ° of C.Then add oxalyl chloride (58mL) and stir 5 hours in this temperature.Allow its more cooling and evaporation carefully on rotatory evaporator, at 40 ° of C, little by little increase vacuum, and final turn flask and shake complete drying in room temperature high vacuum.
example 1-sodium indicator ANG-1
Fig. 1 excites at 517nm place, green with the Asante Natrium Green(sodium of 1M sodium chloride solution titration)-1(ANG-1) salts solution fluorescence emission spectrum (1M TMACl, 10mM MOPS, pH=7.10), sodium titration.
Fig. 7 shows and is loaded with ANG-1(AM) HEK293 cell expressing TRPV1 passage.Capsaicine, a kind of agonist of TRPV1 passage, conduction Na
+and Ca
2+, be used to excite Na
+interior stream.
Fig. 8 shows and is loaded with ANG-1(AM) REF52 cell and application Na
+ionophore linear gramicidins promotes Na
+interior stream.The Na obtaining
+the rising of IC has caused the corresponding increase of ANG-1 fluorescence.
Fig. 9 shows and is loaded with ANG-1(AM) stellate cell, the crow bar glycosides showing due to this sodium pump suppresses the Na causing
+the increase of the fluorescence to this second frame certainly this first box that the rising of IC causes.
example 2-sodium indicator ANG-2
Fig. 2 excites, uses sodium chloride solution (1M NaCl at 517nm place, 10mM MOPS, pH=7.10) the Asante Natrium Green(sodium of titration is green)-2(ANG-2) salts solution fluorescence emission spectrum (140mM TMACl, 10mM MOPS, pH=7.11), sodium titration.
Figure 10 shows and is loaded with ANG-2(AM) REF52 inoblast and apply described Na
+ionophore SQI-Pr promotes Na
+interior stream.The Na obtaining
+the rising of IC has caused the corresponding increase of ANG-2 fluorescence.The further interpolation of 20 μ M amphotericin Bs has only provided faint fluorescence to be increased.
Figure 11 shows and will be loaded with ANG-2(AM) REF52 inoblast maintain in 145mM NMG-Gluconate.50 μ M amphotericin-Bs consume Na
+and K
+these cells.In cell and after the na concn balance of extracellular, extracellularly add the NaCl of increment, cause the corresponding increase of ANG-2 fluorescence.Then add 145mM K
+, cause the decline of ANG-2 fluorescence.
example 3-sodium indicator ANG-3
Fig. 3 excites, uses sodium chloride solution (1M NaCl at 517nm place, 10mM MOPS, pH=7.10) the Asante Natrium Green(sodium of titration is green)-3(ANG-3) salts solution fluorescence emission spectrum (1M TMACl, 10mM MOPS, pH=7.12), sodium titration.
example 4-potassium indicator APG-1
Fig. 4 excites at 517nm place, green with the Asante Potassium Green(potassium of 1M Klorvess Liquid titration)-1(APG-1) salts solution fluorescence emission spectrum (140mM TMACl, 10mM MOPS, pH=7.11), potassium titration.
Figure 12 shows and will be loaded with APG-1(AM) REF52 inoblast maintain in 145mM NMG-Gluconate.50 μ M amphotericin-Bs consume Na
+and K
+these cells.In cell and the K of extracellular
+after concentration balance, extracellularly add the KCl of increment, cause the corresponding increase of APG-1 fluorescence.Then add 10mM Na
+, only increase slightly APG-1 fluorescence.
example 5-potassium indicator APG-2
Fig. 5 excites at 517nm place, green with the Asante Potassium Green(potassium of 1M Klorvess Liquid titration)-2(APG-2) salts solution fluorescence emission spectrum (140mM TMACl, 10mM MOPS, pH=7.11), potassium titration.
example 6-calconcarboxylic acid ACG(K+ salt)
Fig. 6 excites, uses the ACG-1 salts solution fluorescence emission spectrum (10mM EGTA, 10mM MOPS, pH7.2), calcium titration of 10mM CaEGTA titration at 517nm place, once Ca
2+saturated, the fluorescence that >200X is shown increases.
It is green that Figure 13 shows Asante Calcium Green(calcium)-1 and fluorescent yellow etc. the fluorescence emission spectrum of absorbent solution, cause 0.495 Ca
2+the quantum yield of-border ACG-1.
Figure 14 shows rat vagus nerve (ganglion nodosum) neurone is loaded with to 50 μ M ACG-1K via full cell patch electrode
+salt.This neurone is received in from a series of depolarize steps in the lasting scope of 1-1000msec, from-70 to+10mV.By Ca2+ signal record in the cell bringing out by these depolarize steps, be the increase of ACG-1 fluorescence, be denoted as Δ F/F
0.
example 7-sodium indicator ANG-NM
Figure 15 shows in 130mM TMACl, 10mM MOPS, pH7.0, the Asante Natrium(sodium exciting with 492nm) titration of NM.Add the aliquot of 1M NaCl solution, to provide listed concentration.The ionic strength increasing is not proofreaied and correct.
example 8-sodium indicator ANG-TM
Figure 16 shows in 100mM TMACl, 5mM tris alkali, pH9.0, the Asante Natrium(sodium exciting with 500nm) titration of TM.Add the aliquot of 1M NaCl solution, to provide listed concentration.The ionic strength of dilution or increase is not proofreaied and correct.
Claims (52)
1. a fluorescence ion indicator, comprises a first hydroxyl xanthone fluorophore with following structure
For this structure, Z
1=OR
4and Z
2=O; R
1=H, F, Cl, Br or (CH
2)
ncO
2r
4, n=0,1 or 2; R
2=H, F, Cl, Br or (CH
2)
ncO
2r
4, n=0,1 or 2; R
3=H, F, Cl, Br; R
4=H, salt (that is, TMA
+, K
+) or AM (CH
2oCOCH
3) or any other pharmacy acceptable salt and ester; And R
50=H or to a covalent linkage on an organic molecule.
2. fluorescence ion indicator as claimed in claim 1, wherein R
50to a kind of sequestrant or to a covalent linkage on first connector being covalently bound on a kind of sequestrant.
3. fluorescence ion indicator as claimed in claim 2, wherein R
50by a first non-annularity connector, to a covalent linkage on a kind of non-annularity sequestrant.
4. fluorescence ion indicator as claimed in claim 3, wherein R
50be by a non-annularity connector, to a covalent linkage on a kind of sequestrant, this first non-annularity connector comprises
For it, R
6=H, OMe, F; R
7=H, OMe, F; R
8=H, OMe, F; R
9=H, OMe, F; R
51=to the covalent linkage R on this hydroxyl xanthone fluorophore
50; And R
52=to the covalent linkage on a kind of non-annularity sequestrant.
5. fluorescence ion indicator as claimed in claim 4, wherein this non-annularity sequestrant is a kind of crown ether, this crown ether is selected from lower group, and this group comprises: single azepine-12-crown ether-4, single azepine-15-crown ether-5, single azepine-hexaoxacyclooctadecane-6-6, single azepine-heptaoxacycloheneicosane-7-7 or single azepine-octaoxacyclotetracosane-8-8; And R
52the azepine official energy of=this crown ether.
6. fluorescence ion indicator as claimed in claim 5, wherein this non-annularity sequestrant is single azepine-15-crown ether-5, R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; R
3=R
6=R
7=R
8=R
9=H; R
50=from this hydroxyl xanthone fluorophore to the R on this non-annularity connector
51key; R
52the azepine on this list azepine-15-crown ether-5:
7. fluorescence ion indicator as claimed in claim 4, wherein this non-annularity sequestrant is a kind of crown ether, this crown ether is selected from lower group, this group comprises: diaza-12-crown ether-4, diaza-15-crown ether-5, diaza-hexaoxacyclooctadecane-6-6, diaza-heptaoxacycloheneicosane-7-7 or diaza-octaoxacyclotetracosane-8-8, make this sequestrant have a first azepine official energy and a second azepine official energy; This first non-annularity connector is at R
52the first azepine official that place is bonded to this sequestrant can go up and at R
51place is bonded on this first hydroxyl xanthone fluorophore; And have second connector and second fluorophore, this second connector comprises:
For it, R
6=H, OMe, F; R
7=H, OMe, F; R
8=H, OMe, F; R
9=H, OMe, F; R
51=to the covalent linkage R on second fluorophore
50; And R
52=to the organic covalent linkage of the second azepine of this non-annularity sequestrant.
8. fluorescence ion indicator as claimed in claim 7, wherein this non-annularity sequestrant is that diaza-15-crown ether-5 and this second fluorophore are identical with this first hydroxyl xanthone fluorophore; R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; R
3=R
7=R
8=R
9=H; R
6=OMe; R
50=from this fluorophore to the R on this non-annularity connector
51key; R
52each is these azepines on these diaza-15-crown ether-5 naturally:
9. fluorescence ion indicator as claimed in claim 4, wherein this non-annularity sequestrant is a kind of crown ether, this crown ether is selected from lower group, and this group comprises: diaza-12-crown ether-4, diaza-15-crown ether-5, diaza-hexaoxacyclooctadecane-6-6, diaza-heptaoxacycloheneicosane-7-7 or diaza-octaoxacyclotetracosane-8-8; R
52a first azepine official energy of=this crown ether, R
53a second azepine official energy of=this crown ether, as a substituting group on a cap, this cap comprises:
R
10=H, OMe, F; R
11=H, OMe, F; R
12=H, Me, F, Br, Cl, I, NO
2, CHO, (CH
2)
ncO
2r
4[
n=0,1 or 2],
r
13=H, OMe, F; R
14=H, OMe, F;
Make to exist one to pass through R
50be covalently bound to the R of this non-annularity connector
51on hydroxyl xanthone fluorophore, described non-annularity connector is at R
52place in conjunction with these azepines official of this diaza crown ether can one of; And there is one at R
53place is in conjunction with the non-annularity cap of other azepines official energy of this diaza crown ether.
10. fluorescence ion indicator as claimed in claim 9, wherein this non-annularity sequestrant is diaza-15-crown ether-5, R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; R
3=R
7=R
8=R
9=R
11=R
13=R
14=H; R
6=R
10=OMe; R
12=CHO; R
50=to the R on this non-annularity connector
51key; R
52it is first azepine on these diaza-15-crown ether-5; R
53second azepine on these diaza-15-crown ether-5:
11. fluorescence ion indicator as claimed in claim 9, wherein this non-annularity sequestrant is diaza-15-crown ether-5, R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; R
3=R
7=R
8=R
9=R
11=R
13=R
14=H; R
6=R
10=OMe; R
12=Me; R
50=to the R on this non-annularity connector
51key; R
52it is first azepine on these diaza-15-crown ether-5; R
53second azepine on these diaza-15-crown ether-5:
12. fluorescence ion indicator as claimed in claim 2, wherein R
50to a covalent linkage on a kind of ring-type sequestrant by a loop connecting thing.
13. fluorescence ion indicator as claimed in claim 12, wherein R
50be by a loop connecting thing to a covalent linkage on a kind of sequestrant, this loop connecting thing comprises
For it, N and O are the heteroatomss in this cyclic crown ether or cave ether; R
15=H, OMe, F; R
16=H, OMe, F; R
17=H, OMe, F; And R
51=to the covalent linkage R on this hydroxyl xanthone fluorophore
50.
14. fluorescence ion indicator as claimed in claim 13, wherein this ring-type sequestrant is a kind of dibenzo cyclic crown ether, this crown ether is selected from lower group, and this group comprises: dibenzo-diaza-12-crown ether-4, dibenzo-diaza-15-crown ether-5, dibenzo-diaza-hexaoxacyclooctadecane-6-6, dibenzo-diaza-heptaoxacycloheneicosane-7-7 or dibenzo-diaza-octaoxacyclotetracosane-8-8; Make to exist two identical hydroxyl xanthone fluorophores, each passes through R
50be covalently bound to the R of its corresponding loop connecting thing
51upper, each the loop connecting thing in this crown ether is in conjunction with its corresponding azepine/oxa-part, make these two azepine officials can each be covalently bound to its corresponding R
22go up, wherein R
22=H or (CH
2)
ncH
3(n=0,1,2).
15. fluorescence ion indicator as claimed in claim 14, wherein this ring-type sequestrant is dibenzo-diaza-15-crown ether-5, R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; R
3=R
15=R
16=R
17=H; R
22=(CH
2)
ncH
3, n=1, and R
51=to the covalent linkage R on this hydroxyl xanthone fluorophore
50:
16. fluorescence ion indicator as claimed in claim 13, wherein this ring-type sequestrant is a kind of dibenzo cyclic crown ether, this crown ether is selected from lower group, and this group comprises: dibenzo-diaza-12-crown ether-4, dibenzo-diaza-15-crown ether-5, dibenzo-diaza-hexaoxacyclooctadecane-6-6, dibenzo-diaza-heptaoxacycloheneicosane-7-7 or dibenzo-diaza-octaoxacyclotetracosane-8-8; Make this hydroxyl xanthone fluorophore pass through R
50be covalently bound to the R of its loop connecting thing
51upper, described loop connecting thing is in conjunction with the first azepine/oxa-part of this crown ether; And a second azepine/oxa-of this crown ether is partly bonded in an annular cap, and this cap comprises:
For it, N and O are two in the heteroatoms in this cyclic crown ether, R
18=H, OMe, F;
R
19=H, OMe, F; R
20=H, Me, F, Br, Cl, I, (CH
2)
ncO
2r
4[n=0,1 or 2], NO
2, CHO,
and R
21=H, OMe, F; Make this first and second azepines official can be covalently bound to R
22go up, wherein R
22=H or (CH
2)
ncH
3(n=0,1,2).
17. fluorescence ion indicator as claimed in claim 16, wherein this ring-type sequestrant is dibenzo-diaza-15-crown ether-5, R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; R
3=R
15=R
16=R
17=R
18=R
19=R
21=H; R
20=Me; R
22=(CH
2)
ncH
3, n=1, and R
51=to the covalent linkage R on this hydroxyl xanthone fluorophore
50:
18. fluorescence ion indicator as claimed in claim 13, wherein this ring-type sequestrant is a kind of dibenzo-cave ether, and this dibenzo-cave ether is selected from lower group, and this group comprises: dibenzo-[1,1,1], dibenzo-[1,1,2], dibenzo-[1,2,2], dibenzo-[2,2,2], dibenzo-[2,2,3], dibenzo-[2,3,3] and dibenzo-[3,3,3] cave ether, makes to exist two identical hydroxyl xanthone fluorophores, and each passes through R
50be covalently bound to the R of its corresponding loop connecting thing
51upper, each the loop connecting thing in this cave ether is in conjunction with its corresponding azepine/oxa-part.
19. fluorescence ion indicator as claimed in claim 18, wherein this ring-type sequestrant is dibenzo-[1,1,2]-cave ether, R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; R
3=R
15=R
16=R
17=H; And R
51=to the covalent linkage R on this hydroxyl xanthone fluorophore
50:
20. fluorescence ion indicator as claimed in claim 13, wherein this ring-type sequestrant is a kind of dibenzo ring-type cave ether, this dibenzo ring-type cave ether is selected from lower group, this group comprises: dibenzo-[1,1,1], dibenzo-[1,1,2], dibenzo-[1,2,2], dibenzo-[2,2,2], dibenzo-[2,2,3], dibenzo-[2,3,3] and dibenzo-[3,3,3] cave ether; Make this hydroxyl xanthone fluorophore pass through R
50be covalently bound to the R of its loop connecting thing
51upper, described loop connecting thing is in conjunction with the first azepine/oxa-part of this cave ether; And a second azepine/oxa-of this cave ether is partly bonded in this annular cap:
For it, N and O are two in the heteroatoms in this cyclic crown ether, R
18=H, OMe, F;
R
19=H, OMe, F; R
20=H, Me, F, Br, Cl, I, (CH
2)
ncO
2r
4[n=0,1 or 2], NO
2, CHO,
21. fluorescence ion indicator as claimed in claim 20, wherein R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; R
3=R
15=R
16=R
17=R
18=R
19=R
21=H; R
20=Me; And R
51=to the covalent linkage R on this hydroxyl xanthone fluorophore
50:
22. fluorescence ion indicator as claimed in claim 2, wherein this sequestrant is BAPTA and derivative thereof:
R
24＝H、F、Br、Cl
R
25=H, Me, F, Br, Cl, I, NO
2, CHO, (CH
2)
ncO
2r
4[n=0,1 or 2],
R
26=H, F, Br, Cl, (CH
2)
ncO
2r
4[n=0,1 or 2],
R
51=at R
50place is to the covalent linkage on this hydroxyl xanthone fluorophore.
23. fluorescence ion indicator as claimed in claim 22, wherein R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; R
3=R
24=R
26=H; R
25=Me; R
51=at R
50place is to the covalent linkage on this hydroxyl xanthone fluorophore:
24. fluorescence ion indicator as claimed in claim 2, wherein this sequestrant is APTRA and derivative thereof:
25. fluorescence ion indicator as claimed in claim 24, wherein R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; And R
51=at R
50place is to the covalent linkage on this hydroxyl xanthone fluorophore:
26. fluorescence ion indicator as claimed in claim 2, wherein this sequestrant is half-BAPTA and derivative thereof:
27. fluorescence ion indicator as claimed in claim 26, wherein R
1=Cl; R
2=(CH
2)
ncO
2r
4, n=2; And R
51=at R
50place is to the covalent linkage on this hydroxyl xanthone fluorophore:
28. 1 kinds of fluorescence ion indicator, comprise a rosamine fluorophore with following structure
For this structure Z
1=NR
5ar
5band Z
2=NR
5ar
5b; R
1=H, F, Cl, Br; R
2=H, F, Cl, Br; R
3=H, F, Cl, Br; R
4=H, salt (that is, TMA
+, K
+) or AM (CH
2oCOCH
3) or any other pharmacy acceptable salt and ester; R
5a=(CH
2)
ncO
2r
4, n=0,1 or 2; R
5b=(CH
2)
ncO
2r
4, n=0,1 or 2 and R
50=H or to a covalent linkage on an organic molecule.
29. fluorescence ion indicator as claimed in claim 28, wherein R
50to a kind of sequestrant or to a covalent linkage on first connector being covalently bound on a kind of sequestrant.
30. fluorescence ion indicator as claimed in claim 29, wherein R
50to a covalent linkage on a kind of non-annularity sequestrant by a first non-annularity connector.
31. fluorescence ion indicator as claimed in claim 30, wherein R
50be by a non-annularity connector to a covalent linkage on a kind of sequestrant, this first non-annularity connector comprises
For it, R
6=H, OMe, F; R
7=H, OMe, F; R
8=H, OMe, F; R
9=H, OMe, F; R
51=be bonded to the covalent linkage R on this rosamine fluorophore
50; And R
52=to the covalent linkage on a kind of non-annularity sequestrant.
32. fluorescence ion indicator as claimed in claim 31, wherein this non-annularity sequestrant is a kind of crown ether, this crown ether is selected from lower group, and this group comprises: single azepine-12-crown ether-4, single azepine-15-crown ether-5, single azepine-hexaoxacyclooctadecane-6-6, single azepine-heptaoxacycloheneicosane-7-7 or single azepine-octaoxacyclotetracosane-8-8; And R
52the azepine official energy of=this crown ether.
33. fluorescence ion indicator as claimed in claim 32, wherein this non-annularity sequestrant is single azepine-15-crown ether-5, R
1=R
2=R
3=R
6=R
7=R
8=R
9=H; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; R
50=from this rosamine fluorophore to the R on this non-annularity connector
51key; R
52the azepine on this list azepine-15-crown ether-5:
34. fluorescence ion indicator as claimed in claim 31, wherein this non-annularity sequestrant is a kind of crown ether, this crown ether is selected from lower group, this group comprises: diaza-12-crown ether-4, diaza-15-crown ether-5, diaza-hexaoxacyclooctadecane-6-6, diaza-heptaoxacycloheneicosane-7-7 or diaza-octaoxacyclotetracosane-8-8, make this sequestrant have a first azepine official energy and a second azepine official energy; This first non-annularity connector is at R
52the first azepine official that place is bonded to this sequestrant can go up and at R
51place is bonded on a rosamine fluorophore; And have second connector and second fluorophore, this second connector comprises:
For its R
6=H, OMe, F; R
7=H, OMe, F; R
8=H, OMe, F; R
9=H, OMe, F; R
51=to the covalent linkage R on second fluorophore
50; And R
52=to the organic covalent linkage of the second azepine of this non-annularity sequestrant.
35. fluorescence ion indicator as claimed in claim 34, wherein this non-annularity sequestrant is that diaza-15-crown ether-5 and this second fluorophore are identical with a rosamine fluorophore; R
1=R
2=R
3=R
6=R
7=R
8=R
9=H; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; R
50=from this fluorophore to the R on this non-annularity connector
51key; R
52each is these azepines on these diaza-15-crown ether-5 naturally:
36. fluorescence ion indicator as claimed in claim 31, wherein this non-annularity sequestrant is a kind of crown ether, this crown ether is selected from lower group, and this group comprises: diaza-12-crown ether-4, diaza-15-crown ether-5, diaza-hexaoxacyclooctadecane-6-6, diaza-heptaoxacycloheneicosane-7-7 or diaza-octaoxacyclotetracosane-8-8; R
52a first azepine official energy of=this crown ether, R
53a second azepine official energy of=this crown ether, as a substituting group on a cap, this cap comprises:
R
10=H, OMe, F; R
11=H, OMe, F; R
12=H, Me, F, Br, Cl, I, NO
2, CHO, (CH
2)
ncO
2r
4[n=0,1 or 2],
r
13=H, OMe, F; R
14=H, OMe, F;
Make to exist one to pass through R
50be covalently bound to the R of this non-annularity connector
51on rosamine fluorophore, described non-annularity connector is at R
52place in conjunction with these azepines official of this diaza crown ether can one of; And there is one at R
53place is in conjunction with the non-annularity cap of other azepines official energy of this diaza crown ether.
37. fluorescence ion indicator as claimed in claim 36, wherein this non-annularity sequestrant is diaza-15-crown ether-5, R
1=R
2=R
3=R
6=R
7=R
8=R
9=R
11=R
13=R
14=H; R
6=R
10=OMe; R
12=CHO; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; R
50=to the R on this non-annularity connector
51key; R
52it is first azepine on these diaza-15-crown ether-5; R
53second azepine on these diaza-15-crown ether-5:
38. fluorescence ion indicator as claimed in claim 36, wherein this non-annularity sequestrant is diaza-15-crown ether-5, R
1=R
2=R
3=R
6=R
7=R
8=R
9=R
11=R
13=R
14=H; R
6=R
10=OMe; R
12=Me; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; R
50=to the R on this non-annularity connector
51key; R
52it is first azepine on these diaza-15-crown ether-5; R
53second azepine on these diaza-15-crown ether-5:
39. fluorescence ion indicator as claimed in claim 29, wherein R
50to a covalent linkage on a kind of ring-type sequestrant by a loop connecting thing.
40. fluorescence ion indicator as claimed in claim 39, wherein R
50be by a loop connecting thing to a covalent linkage on a kind of sequestrant, this loop connecting thing comprises
For its N and O, it is the heteroatoms in this cyclic crown ether or cave ether; R
15=H, OMe, F; R
16=H, OMe, F; R
17=H, OMe, F; And R
51=to the covalent linkage R on this rosamine fluorophore
50.
41. fluorescence ion indicator as claimed in claim 40, wherein this ring-type sequestrant is a kind of dibenzo cyclic crown ether, this crown ether is selected from lower group, and this group comprises: dibenzo-diaza-12-crown ether-4, dibenzo-diaza-15-crown ether-5, dibenzo-diaza-hexaoxacyclooctadecane-6-6, dibenzo-diaza-heptaoxacycloheneicosane-7-7 or dibenzo-diaza-octaoxacyclotetracosane-8-8; Make to exist two identical rosamine fluorophores, each passes through R
50be covalently bound to the R of its corresponding loop connecting thing
51upper, each the loop connecting thing in this crown ether is in conjunction with its corresponding azepine/oxa-part, make these two azepine officials can each be covalently bound to its corresponding R
22go up, wherein R
22=H or (CH
2)
ncH
3(n=0,1,2).
42. fluorescence ion indicator as claimed in claim 41, wherein this ring-type sequestrant is dibenzo-diaza-15-crown ether-5, R
1=R
2=R
3=R
15=R
16=R
17=H; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; R
22=(CH
2)
ncH
3, n=1; And R
51=to the covalent linkage R on this rosamine fluorophore
50:
43. fluorescence ion indicator as claimed in claim 40, wherein this ring-type sequestrant is a kind of dibenzo cyclic crown ether, this crown ether is selected from lower group, and this group comprises: dibenzo-diaza-12-crown ether-4, dibenzo-diaza-15-crown ether-5, dibenzo-diaza-hexaoxacyclooctadecane-6-6, dibenzo-diaza-heptaoxacycloheneicosane-7-7 or dibenzo-diaza-octaoxacyclotetracosane-8-8; Make this rosamine fluorophore pass through R
50be covalently bound to the R of its loop connecting thing
51upper, described loop connecting thing engages the first azepine/oxa-part of this crown ether; And a second azepine/oxa-of this crown ether is partly bonded in an annular cap, and this cap comprises:
For it, N and O are two in the heteroatoms in this cyclic crown ether, R
18=H, OMe, F;
R
19=H, OMe, F; R
20=H, Me, F, Br, Cl, I, (CH
2)
ncO
2r
4[n=0,1 or 2], NO
2, CHO,
and R
21=H, OMe, F; Make this first and second azepines official can be covalently bound to R
22go up, wherein R
22=H or (CH
2)
ncH
3(n=0,1,2).
17. fluorescence ion indicator as claimed in claim 16, wherein this ring-type sequestrant is dibenzo-diaza-15-crown ether-5, R
1=R
2=R
3=R
15=R
16=R
17=R
18=R
19=R
21=H; R
20=Me; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; R
22=(CH
2)
ncH
3, n=1; And R
51=to the covalent linkage R on this rosamine fluorophore
50:
18. fluorescence ion indicator as claimed in claim 13, wherein this ring-type sequestrant is a kind of dibenzo-cave ether, and this dibenzo-cave ether is selected from lower group, and this group comprises: dibenzo-[1,1,1], dibenzo-[1,1,2], dibenzo-[1,2,2], dibenzo-[2,2,2], dibenzo-[2,2,3], dibenzo-[2,3,3] and dibenzo-[3,3,3] cave ether, makes to exist two identical rosamine fluorophores, and each passes through R
50be covalently bound to the R of its corresponding loop connecting thing
51upper, each the loop connecting thing in this cave ether is in conjunction with its corresponding azepine/oxa-part.
46. fluorescence ion indicator as claimed in claim 45, wherein this ring-type sequestrant is dibenzo-[1,1,2]-cave ether, R
1=R
2=R
3=R
15=R
16=R
17=H; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; And R
51=to the covalent linkage R on this rosamine fluorophore
50:
47. fluorescence ion indicator as claimed in claim 40, wherein this ring-type sequestrant is a kind of dibenzo ring-type cave ether, this dibenzo ring-type cave ether is selected from lower group, this group comprises: dibenzo-[1,1,1], dibenzo-[1,1,2], dibenzo-[1,2,2], dibenzo-[2,2,2], dibenzo-[2,2,3], dibenzo-[2,3,3] and dibenzo-[3,3,3] cave ether; Make this rosamine fluorophore pass through R
50be covalently bound to the R of its loop connecting thing
51upper, described loop connecting thing is in conjunction with the first azepine/oxa-part of this cave ether; And a second azepine/oxa-of this cave ether is partly bonded in this annular cap:
For its N and O, be two in the heteroatoms in this cyclic crown ether, R
18=H, OMe, F;
R
19=H, Ome, F; R
20=H, Me, F, Br, Cl, I, (CH
2)
ncO
2r
4[n=0,1 or 2], NO
2, CHO,
and R
21=H, Ome, F.
48. fluorescence ion indicator as claimed in claim 47, wherein R
1=R
2=R
3=R
15=R
16=R
17=R
18=R
19=R
21=H; R
20=Me; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; R
22=(CH
2)
ncH
3, n=1; And R
51=to the covalent linkage R on this rosamine fluorophore
50:
49. fluorescence ion indicator as claimed in claim 29, wherein this sequestrant is BAPTA and derivative thereof:
R
24＝H、F、Br、Cl
R
25=H, Me, F, Br, Cl, I, NO
2, CHO, (CH
2)
ncO
2r
4[n=0,1 or 2],
R
26=H, F, Br, Cl, (CH
2)
ncO
2r
4[n=0,1 or 2],
R
51=at R
50place is to the covalent linkage on this rosamine fluorophore.
50. fluorescence ion indicator as claimed in claim 49, wherein R
1=R
2=R
3=H; R
25=Me; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; R
51=at R
50place is to the covalent linkage on this rosamine fluorophore:
51. fluorescence ion indicator as claimed in claim 29, wherein this sequestrant is APTRA and derivative thereof:
r
51=at R
50place is to the covalent linkage on this rosamine fluorophore.
52. fluorescence ion indicator as claimed in claim 51, wherein R
1=R
2=R
3=H; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; And R
51=at R
50place is to the covalent linkage on this rosamine fluorophore:
53. fluorescence ion indicator as claimed in claim 29, wherein this sequestrant is half-BAPTA and derivative thereof:
54. fluorescence ion indicator as claimed in claim 53, wherein R
1=R
2=R
3=H; R
5a=(CH
2)
ncO
2r
4, n=1; R
5b=(CH
2)
ncO
2r
4, n=1; And R
51=at R
50place is to the covalent linkage on this rosamine fluorophore.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| US61/384,695 | 2010-09-20 | ||
| PCT/US2011/052353 WO2012040204A2 (en) | 2010-09-20 | 2011-09-20 | Cytosolic fluorescent ion indicators |
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| CN103687857A true CN103687857A (en) | 2014-03-26 |
| CN103687857B CN103687857B (en) | 2016-11-09 |
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| CN (1) | CN103687857B (en) |
| WO (1) | WO2012040204A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112939997A (en) * | 2021-02-03 | 2021-06-11 | 山西大学 | Spiropyran-crown ether derivative SP-CE and synthetic method and application thereof |
| CN115215878A (en) * | 2022-07-08 | 2022-10-21 | 中国科学院理化技术研究所 | Fluorescent probe for detecting millimole free calcium ions and synthetic method thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014085568A2 (en) * | 2012-11-27 | 2014-06-05 | Saint Louis University | Hbv rnase h purification and enzyme inhbitors |
| US10590155B2 (en) | 2016-07-15 | 2020-03-17 | Arizona Board Of Regents On Behalf Of Arizona State University | Mitochondria-targeting fluorescent potassium+ sensor and method of making the same |
| US20210072222A1 (en) * | 2019-09-05 | 2021-03-11 | University Of Massachusetts | Compositions and methods for visualization of extracellular activity |
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| US5049673A (en) * | 1987-10-30 | 1991-09-17 | The Regents Of The University Of California | Fluorescent indicator dyes for calcium working at long wavelengths |
| US20060030054A1 (en) * | 2002-07-08 | 2006-02-09 | Tetsuo Nagano | Fluorescent probe |
| CN101056961A (en) * | 2004-09-22 | 2007-10-17 | 独立行政法人科学技术振兴机构 | Water-soluble fluorescent material and method for producing same |
| WO2008151303A1 (en) * | 2007-06-05 | 2008-12-11 | Invitrogen Corporation | Long wavelength fluorogenic intercellular ion indicators |
| US20100047821A1 (en) * | 2000-12-20 | 2010-02-25 | Life Technologies Corporation | Crown ether derivatives |
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| US7842823B2 (en) * | 2005-10-27 | 2010-11-30 | The Regents Of The University Of California | Fluorogenic probes for reactive oxygen species |
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2011
- 2011-09-20 WO PCT/US2011/052353 patent/WO2012040204A2/en active Application Filing
- 2011-09-20 EP EP11827351.5A patent/EP2619199A4/en not_active Withdrawn
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| US5049673A (en) * | 1987-10-30 | 1991-09-17 | The Regents Of The University Of California | Fluorescent indicator dyes for calcium working at long wavelengths |
| US20100047821A1 (en) * | 2000-12-20 | 2010-02-25 | Life Technologies Corporation | Crown ether derivatives |
| US20060030054A1 (en) * | 2002-07-08 | 2006-02-09 | Tetsuo Nagano | Fluorescent probe |
| CN101056961A (en) * | 2004-09-22 | 2007-10-17 | 独立行政法人科学技术振兴机构 | Water-soluble fluorescent material and method for producing same |
| WO2008151303A1 (en) * | 2007-06-05 | 2008-12-11 | Invitrogen Corporation | Long wavelength fluorogenic intercellular ion indicators |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112939997A (en) * | 2021-02-03 | 2021-06-11 | 山西大学 | Spiropyran-crown ether derivative SP-CE and synthetic method and application thereof |
| CN115215878A (en) * | 2022-07-08 | 2022-10-21 | 中国科学院理化技术研究所 | Fluorescent probe for detecting millimole free calcium ions and synthetic method thereof |
| CN115215878B (en) * | 2022-07-08 | 2024-03-01 | 中国科学院理化技术研究所 | Fluorescent probe for detecting millimole free calcium ions and synthesis method thereof |
Also Published As
| Publication number | Publication date |
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| WO2012040204A2 (en) | 2012-03-29 |
| WO2012040204A3 (en) | 2014-04-17 |
| EP2619199A4 (en) | 2015-08-12 |
| CN103687857B (en) | 2016-11-09 |
| EP2619199A2 (en) | 2013-07-31 |
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