EP2619199A2 - Cytosolic fluorescent ion indicators - Google Patents

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

TITLE: CYTOSOLIC FLUORESCENT ION INDICATORS

AUTHORS: AK.WASI MINTA and P. ROGELIO ESCAMILLA

ASSIGNEE: ASANTE RESEARCH, LLC FILING DATE: September 20, 201 1 RELATED APPLICATIONS

This application is related to US Provisional Patent Application No. 61/384,695 filed September 20, 2010, and claims priority from that application. This application is related to US Provisional Patent Application No. 61/453,664 filed March 17, 201 1 , which is incorporated by reference herein.

FIELD OF INVENTION

The current invention relates to a fluorophore and the use of the fluorophore in combination with a chelating portion to provide new fluorescent ion indicators.

BACKGROUND

Fluorescent ion indicators typically comprise a chelating portion linked to a fluorophore such that a change in excitation and/or emission wavelengths, or a change in excitation and/or emission intensity, or both, occurs upon binding of the ion. To sense ion concentrations in aqueous media, the fluorescent ion indicator should be water soluble. For intracellular measurements, the indicator should not leak from the cell at a rate that will interfere with the analysis. For convenience, the indicator would be loaded non-invasively, for example as an acetoxymethyl ester.

Many chelators, such as crowns and cryptands, suffer from poor solubility in water, thereby limiting ion measurement to organic solvents. If these chelators are linked to a water-soluble fluorophore, however, they can produce a fluorescent ion indicator for aqueous media.

For chelators that are readily soluble in water, such as BAPTA, the water-soluble fluorophore can enhance water-solubility and, for intracellular measurements, cellular retention of the fluorescent ion indicator.

Two such water-soluble fluorophores, a hydroxyxanthenone variant and a rosamine variant, are Asante Green™ (I) and Asante Orange™ (II).

SUMMARY OF INVENTION The current invention discloses new fluorescent ion indicators for measuring concentrations of metal ions.

The fluorescent ion indicators comprise a visible-wavelength fluorophore that incorporates water- solubilizing functional groups, so that the chelating portion of the indicator need not incorporate the same groups. Such a fluorophore therefore enables visible-wavelength analysis of ion concentrations with chelators that are insoluble or poorly soluble in water.

Since one application of these indicators comprises measurement of cytosolic concentrations of ions, the water-solubilizing groups should be maskable as hydrophobic moieties that can permeate the lipophilic cell membrane. Such a hydrophobic moiety is typically susceptible to cleavage by a cellular component in order to return the indicator to its water-soluble form. The most popular hydrophobic mask is the acetoxymethyl ester, which is cleaved to the carboxylate by non-specific esterases in the cytosol.

For the same cellular application, the fluorescent ion indicator should remain in the cell long enough so that its leakage does not adversely affect experimental results. Water-solubilizing carboxylates help to retain the indicator in the cell.

For the calcium ion, a BAPTA chelating portion of the indicator contains four water-solubilizing and cell-retentive carboxylates. For the alkali earth metals, the chelating portion typically comprises crowns or cryptands without carboxylate functional groups. In this case, the fluorophore provides the necessary carboxylate functional groups.

In the case of chelators such as BAPTA that do contain water-solubilizing and cell-retentive moieties, the fluorophore merely adds to the hydrophilicity and enhances the retention of the indicator.

Some prior-art UV excitation fluorophores have acetoxymethyl ester-maskable, water-solubilizing functional groups; such as those in Fura-2, Indo-1 , SBF1, PBFI, or SBFO. Few visible excitation fluorophores exist with these properties, and even fewer have been covalently linked to a chelator.

A green version of the fluorophore comprises a putative fluorescein that is halogenated to ensure pH insensitivity at physiological pH and that exhibits two carboxylate functions at physiological pH.

An orange version of the fluorophore comprises a putative rhodamine with the amino/immino functions alkylated with carboxylate salts.

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 or its analogs produces a new calcium indicator family that strongly resists leakage from the cell due to the extra carboxylates on the fluorophore. The calcium indicators exhibit greater brightness and dynamic range than prior art indicators such as Fluo-4.

Attaching the fluorophore to APTRA or its analogs produces a new magnesium indicator family that strongly resists leakage from the cell due to the extra carboxylates on the fluorophore.

Attaching the fluorophore to half-BAPTA or its analogs produces a new indicator family that strongly resists leakage from the cell due to the extra carboxylates on the fluorophore.

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows the response of fluorescent sodium indicator ANG-1 to increasing sodium concentration in an acellular aqueous titration.

Figure 2 shows the response of fluorescent sodium indicator ANG-2 to increasing sodium concentration in an acellular aqueous titration.

Figure 3 shows the response of fluorescent sodium indicator ANG-3 to increasing sodium concentration in an acellular aqueous titration.

Figure 4 shows the response of fluorescent potassium indicator APG- 1 to increasing potassium concentration in an acellular aqueous titration.

Figure 5 shows the response of fluorescent potassium indicator APG-2 to increasing potassium concentration in an acellular aqueous titration.

Figure 6 shows the response of fluorescent calcium indicator ACG- 1 to increasing calcium concentration in an acellular aqueous titration.

Figure 7 shows the response of fluorescent sodium indicator ANG-1 in HEK.293 cells to increasing intracellular Na⁺ concentration due to capsaicin agonization of TRPVl channels.

Figure 8 shows the response of fluorescent sodium indicator ANG-1 in REF52 cells to increasing intracellular Na⁺ concentration due to Na⁺ ionophore gramicidin-promoted influx.

Figure 9 shows the response of fluorescent sodium indicator ANG- 1 in astrocytes to increasing intracellular Na⁺ concentration due to ouabain inhibition of the sodium pump. Figure 10 shows the response of fluorescent sodium indicator ANG-2 in REF52 fibroblasts to increasing intracellular Na⁺ concentration due to Na⁺ ionophore SQI-Pr-promoted influx.

Figure 1 1 shows the response of fluorescent sodium indicator ANG-2 in REF52 fibroblasts to amphotericin-B depletion of intracellular Na⁺ and then to increasing increments of intracellular Na⁺ concentration.

Figure 12 shows the response of fluorescent sodium indicator APG-1 in REF52 fibroblasts to amphotericin-B depletion of intracellular K⁺ and then to increasing increments of intracellular ⁺ concentration.

Figure 13 compares the quantum efficiency of Ca²⁺-saturated ACG- 1 to fluorescein.

Figure 14 shows the response of fluorescent calcium indicator ACG- 1 in rat vagal sensory neurons to step depolarizations ranging from 1 -1000 msec.

Figure 15 shows the response of a fluorescent sodium indicator (diaza- 15-crown-5 chelator; Z¹ = O^{" +}TMA ; Z² = O; R¹ = CI; R² = (CH₂)_nCOO ⁺TMA, n = 2; R³ = R⁶ = R⁷ = R⁸ = R⁹ = H) to increasing sodium concentration in an acellular aqueous titration.

Figure 16 shows the response of a fluorescent sodium indicator (diaza- 15-crown-5 chelator; Z' = O^{" +}TMA ; Z² = O; R¹ = H; R² = (CH₂)nCOO ⁺TMA, n = 2; R³ = R⁷ = R⁸ = R⁹ = H, R⁶ = OMe) to increasing sodium concentration in an acellular aqueous titration.

DESCRIPTION OF EMBODIMENT

Asante Green™ and Asante Orange™ are covalently linked to certain chelators to produce water- soluble and cell-retentive fluorescent ion indicators, including those for lithium, sodium, potassium, rubidium, cesium, magnesium, calcium, and thallium.

I. Asante Green II. Asante Orange

X = F, CI, Br R¹ = (CH₂)_nCOO-, n = 0, 1 , 2, 3

R¹ = n = 0, 1 , 2, 3 R² = H or (CH₂)nCOO-, n = 0, 1 , 2, 3

In this embodiment, the ion indicators comprise the Asante Green or Asante Orange fluorophore (FLUOROPHORE) connected via a linker (LINKER) to a chelator. When the chelator comprises a diaza crown, the fluorophore may be connected to one aza or amine function of the crown via a non- annulated linker (NON-ANNULATED LINKER), whereas the other aza or amine function of the crown is capped with an aromatic function (NON-ANNULATED CAP) that may contain moieties to increase or decrease chelator affinity, to enhance cellular retention, to localize the indicator at the cell membrane or other cellular compartments, or to enable a reactive site for attachment to synthetic or natural polymers. When the chelator comprises an annulated diaza crown or cryptand, the fluorophore may be connected to one aza function of the crown via an annulated linker (ANNULATED LINKER), whereas the other aza function of the crown is capped with an annulated aromatic function (ANNULATED CAP) that may contain moieties to increase or decrease chelator affinity, to enhance cellular retention, to localize the indicator at the cell membrane or other cellular compartment, or to enable a reactive site for attachment to synthetic or natural polymers.

FLUOROPHORE

Z¹ = OR⁴ or NR^SaR^5b

Z² = O or NR^5aR^5b

R¹ = H, F, CI, Br, or (CH₂)„C0₂R⁴, n = 0, 1 , or 2

R² = H, F, CI, Br, or (CH₂)_nC0₂R⁴, n = 0, 1 , or 2

R³ = H, F, CI, Br R⁴ = H, salt (i.e., TMA⁺, ⁺) or AM (CH2OCOCH3), or any other pharmaceutically acceptable salts and esters

R^5a = (CH2)nC0₂R⁴, n = 0, I , or 2

R^5b = (CH2)„C0₂R⁴, n = 0, 1 , or 2

R⁵⁰ = covalent bond to a Non-Annulated Linker, Annulated Linker, or Chelator

NON-ANNULATED LINKER

R⁶ = H, OMe, F

R⁷ = H, OMe, F

R⁸ = H, OMe, F

R⁹ = H, OMe, F

R⁵¹ = covalent bond to the Fluorophore at R⁵⁰

R⁵² = covalent bond to a non-annulated chelator

NON-ANNULATED CAP

R'° = H, OMe, F

R" = H, OMe, F

R'² = H, (CH₂)_nC0₂R⁴ [n = 0, 1 , or 2], N0₂, CHO,

R¹³ = H, OMe, F

R¹⁴ = H. OMe. F

R⁵³ = covalent bond to a non-annulated chelator

ANNULATED LINKER

N, O are heteroatoms of the annulated crown or cryptand

R^I5 = H, OMe, F

R^I6=H, OMe, F

R¹⁷ = H, OMe, F

R⁵¹ = covalent bond to the Fluorophore at R⁵⁰

ANNULATED CAP

N, O are heteroatoms of the annulated crown or cryptand

R^I8 = H. OMe. F

R¹⁹ = H, OMe, F

R²⁰ = H, ⁴ [n = 0, 1, or 2], N0₂, CHO,

₂₅

R²' = H, OMe, F

Example chelators for lithium include monoaza-l2-crown-4, diaza-12-crown-4, dibenzodiaza-12- crown-4, and [l,l,l]-dibenzocryptand.

Non-Annulated- O Non-Annulated-N N- -Non -Annulated

Linker 1 \ Linker I \ Cap

Non-Annulaled-N N · Non-Annulated

Linker I \ Linker

1

Example chelators for sodium include monoaza-15-cro\vn-5, diaza-15-crown-5, dibenzodiaza-15- crown-5, [l,l,2]-dibenzocryptand, and [l,2,2]-dibenzocr ptand.

ap

Example chelators for potassium include monoaza-18-crown-6, diaza-18-crown-6, dibenzodiaza- l i crovvn-6, and [2,2,2]-dibenzocryptand.

Non-Annulated Cap

r

Example chelators for calcium include BAPTA and its derivatives:

R²⁴ = H, F, Br, CI

R²⁵ = H, Me, F, Br, CI, I, NO:, CHO, (CH₂)nC0₂R⁴ [n = 0, 1. or 2], R²⁶ = H,

covalent bond to the Fluorophore at R^so

Example chelators for magnesium include APTRA and its derivatives:

R⁵' = covalent bond to the Fluorophore at R⁵⁰

Example chelators for thallium include Half-BAPTA and its derivatives.

covalent bond to the Fluorophore at R⁵⁰

SYNTHESIS

The synthetic schemes typically comprise the synthesis of the chelator/linker, followed by appending the fluorophore. The fluorophore results from condensation of Compound 1 with the benzaldehyde on the chelator/linker portion. Z = OH or NR^5oR^5b

R¹ = H, F, CI, Br, or (CH₂)„C0₂R⁴. n = 0, 1 , or 2

R² = H, F, CI, Br, or (CH₂)_nC0₂R\ n = 0, I , or 2

R³ = H, F, CI, Br

R" = H, salt (i.e., TMA⁺, ⁺) or AM (CH₂OCOCH₃), or any other pharmaceutically acceptable salts and esters

R^5a = (CH₂)„C0₂R⁴, n = 0, I , or 2

R^5b = (CH₂)_nC0₂R⁴, n = 0, 1 , or 2

Scheme 1 delineates the synthesis of Compound 1 for the case of Z = OH, R¹ = CI, R² = (CH^CC Me, and R³ = H.

a) NaOCI, KOH b) K₂C0₃, BnCl, Nal, DMF c) BrPh₃P⁺(CH₂C0₂Me), K₂C0₃ d) Pd/C/H₂ AcOH, 50 psi

Commercially available 2,4-dihydroxybenzaldehyde is chlorinated with sodium hypochlorite in basic solution. The phenols are protected as benzyloxy ethers, followed by Witlig reaction with the triphenylphosphonium salt of methylbromoacetate. Subsequent catalytic reduction and debenzylation gives the desired product.

Scheme 2 delineates the synthesis of Compound 1 for the case of Z = OH, R¹ = CI, R² = C0₂Me, and R³ = H. a) NaOCI, KOH b) MnO¾ MeOH

Commercially available 2,4-dihydroxybenzaldehyde is chlorinated with sodium hypochlorite in basic solution. The aldehyde is oxidized to the methyl ester with manganese dioxide in methanol.

Scheme 3 delineates the synthesis of Compound I for the case of Z = OH, R¹ = CI, R² = CH₂C0₂Me, and R³ = H.

a) NaOCI, KOH b) BnCI, Nal, K2CO3 c) LAH d) SO2CI2 e) CN f) 1. OH, 2. HC1 g) MeOH, H2SO4 h) Pd/C/H₂ AcOH, 50 psi

Commercially available 2,4-dihydroxybenzaldehyde is chlorinated with sodium hypochlorite in basic solution and then benzylated to protect the phenols. Lithium aluminum hydride reduction gives the alcohol, which is converted to chloride with sulfuryl chloride. Displacement of the chloride with potassium cyanide gives the nitrile, which is hydrolyzed to the carboxylic acid. Esterification followed by catalytic debenzylation gives the desired product.

Scheme 4 delineates the synthesis of Compound 1 for the case of Z = OH, R¹ = (CH₂)₂C02Me, R² = CI, and R³ = H.

a) BrPh₃P⁺(CH₂C02 e), K2CO3 b) Pd/C/H₂ c) BBn d) MeOH, H2SO4 e) NCS

Commercially available 2,6-dimethoxybenzaldehyde is condensed with the triphenylphosphonium salt of methylbromoacetate. The alkene is catalytically reduced, followed by boron tribromide cleavage of the methyl ethers. The carboxylic acid is re-esterified, and chlorination with N-chlorosuccinimide gives the desired product. Scheme 5 delineates the synthesis of Compound 1 for the case of Z = NiChhCr- CC Meh, Ri = R2 ^: R₃ = H. a) BrCI- CC Me, DIPEA b) Pd/C H₂ AcOH

Commercially available 3-benzyloxyaniline is alkylated with methyibromoacetate, followed by catalytic debenzylation to give the desired product.

Sodium

For simplicity, schemes for the synthesis of the chelator/1 inker are shown for the sodium group of chelators so as to also represent lithium, potassium, rubidium, or cesium, since only the crown or cryptand size will change.

Scheme 6 shows the synthesis of compound 2, R⁶ = R⁷ = R⁸ = R⁹ = H:

a) D F reflux b)Pd/C/H₂ cJNalWHCl d)H₃P0₂ e)DMF/POCl₃ 0 Z = OH, eS0₃H or Z = NR⁵R⁶, propionic acid g) p-chloranil h) TMAOH i) AM-Br, DIPEA Commercial aza- 1 5-crown-5 reacts with p-fluoronitrobenzene at DMF reflux to produce N-p- nitrophenyl-aza- 15-crown-5. The nitro group is reduced and dediazotized to produce N-phenyl-aza- 15- crown-5. Vilsmeier reaction produces the benzaldehyde, which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

Scheme 7a shows the synthesis of compound 2, R⁶ = R⁷ = R⁸ = R⁹ = H:

a) Pyridine reflux b) Pd/C/H₂ c) NaNOz/HCl d) H3PO2 e) DMF POCI3 f) Z = OH, MeS0₃H or Z = NR^5aR^5b, propionic acid g) p-chloranil h) TMAOH i) AM-Br, DIPEA

Commercial diaza- 15-crown-5 reacts with p-fluoronitrobenzene at pyridine reflux to produce bis-N-p- nitrophenyl-diaza- 15-crown-5. The nitro groups are catalytically reduced and dediazotized to produce bis-N-phenyl-diaza-15-crown-5. Vilsmeier reaction produces the dibenzaldehyde, which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

Scheme 7b shows the synthesis for Compound 2, R⁶ = R⁷ = R⁸ = R⁹ = R¹⁰ = R" = R¹² = Rⁿ = R¹⁴ = H.

e) DMF/POCb/Pyridine, room temperature f) Z = OH, MeSCbH or NR^5aR^sb, propionic acid g) p- chloranil h) TMAOH i) AM-Br, DIPEA

From Scheme 7a, bis-N-phenyl-diaza-15-crown-5: Mild Vilsmeier reaction produces the monobenzaldehyde, which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

Scheme 8 shows the synthesis for Compound 2, R⁶ = R⁸ = OMe, R⁷ = R⁹ = H.

a) chloroform/methanol reflux b) Pd/C/H₂ c) Me₂S04, TMAOH d) D F/POCb e) Z = OH, MeS0₃H or Z = NR^5aR^5b, propionic acid 0 p-chloranil g) TMAOH h) AM-Br, DIPEA

Commercial diaza- 15-crown-5 reacts with quinone to give the quinone adduct, which is reduced and then alkylated with dimethyl sulfate to give the tetramethoxy compound. Vilsmeier formylation gives the dialdehyde, which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

Similar to Scheme 6 versus Scheme 7a, taking monoaza-15-crovvn-5 through Scheme 8 gives mono-aza Compound 2, R⁶ = R^s = OMe, R⁷ = R⁹ = H. Similar to Scheme 7b versus Scheme 7a, mild Vilsmeier formylation gives the monoformyl compound instead of the dialdeyde, which leads to Compound 2, R⁶ = R⁸ = R¹⁰ = R¹³ = OMe, R⁷ = R⁹ = R" = R¹² = R¹⁴ = H.

Scheme 9 shows the synthesis for Compound 2, R⁶ = OMe, R⁷ = R⁸ = R⁹ = H.

a) l ,2-Bis(2-chloroethoxy)ethane, DIPEA b) diglycolyl chloride, pyridine, high dilution benzene c) B₂H₆ d) DMF/POCb e) Z = OH, MeS0₃H or Z = NR^5aR^5b, propionic acid p-chloranil g) TMAOH h) AM-Br, DIPEA

Commercially available o-anisidine reacts with l ,2-Bis(2-chloroethoxy)ethane to form the "half crown", which is closed to the "full crown" by addition of diglycolyl chloride, with subsequent reduction of the diamide. Vilsmeier formylation gives the dialdehyde, which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

One variant; R¹ = CI; R² = (CH²)„COOR⁴, n = 2; R³ = R⁷ = R⁸ = R⁹ = H; R⁶ = OMe; gives Asante NaTRIUM Green

ANG-3 TMA⁺ Salt, R⁴ = (CH₃)4N⁺

ANG-3 (AM), R⁴ = CH2OCOCH3

Interestingly, p-chloranil oxidation with methanol as the only solvent instead of a 1 : 1 methanol/ chloroform mixture results in incomplete oxidation, eventually yielding a compound wherein R⁶ = R¹⁰ = OMe, R¹² = CHO, R⁷ = R⁸ =R⁹ = R" = R¹³ = R¹⁴ = H. One variant, R¹ = CI; R² = (CH²)_nCOOR⁴, n = 2; R³ = H; gives Asante NaTRIUM Green -1 (ANG-1).

ANG-1 TMA⁺ Salt, R⁴ = (CH₃)4N⁺

ANG- 1 (AM), R⁴ = CH2OCOCH3

Similar to Scheme 7b versus Scheme 7a, mild Vilsmeier formylation gives the monoformyl compound instead of the dialdehyde, which leads to a compound wherein R⁶ = R¹⁰ = OMe, R⁷ = R⁸ =R⁹ = R" = R¹² = R^{I 3} = R^I = H.

Scheme 10 shows the synthesis for Compound 2, R⁶ = R¹⁰ = OMe, R¹² = Me, R⁷ = R⁸ =R⁹ = R" = R¹³ = R^l4 = H

a) Pd/C/H₂ b) l ,2-Bis(2-chloroethoxy)ethane, DIPEA, Nal c) DIPEA, Nal d) diglycolyl chloride, pyridine, high dilution benzene e) B₂H₆ DMF/POCb g) Z = OH, MeS0₃H or Z = NR^5aR^5b, propionic acid h) p-chloranil i) TMAOH j) AM-Br, DIPEA

Commercially available 5-methyl-2-nitroanisole is catalytically reduced to the aniline for reaction with l ,2-Bis(2-chloroethoxy)ethane to form the "quarter crown". Conversion to the "half crown" entails reaction with o-anisidine, which is closed to the "full crown" by addition of diglycolyl chloride, with subsequent reduction of the diamide. Vilsmeier formylation gives the dialdehyde. which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

One variant; R¹ = CI; R² = (CH²)„ COOR⁴, n = 2; R³ = R⁷ = R⁸ = R⁹ = R" = R¹³ = R¹⁴ = H; R⁶ = R¹⁰ = O e; R¹² = Me; gives

ANG-2 TMA⁺ Salt, R⁴ = (CH₃),N⁺

ANG-2 (AM), R⁴ = CH2OCOCH3 R²² = (CH₂)„CH₃, n

a) diglycolyl chloride, pyridine, high dilution DCM b) B₂H₆ c) R²³ acid chloride (R²³ = (CH₂)_nCH₃, n = 0, 1 , or 2) d) B₂H₆ (R²² = (CH₂)„CH₃, n = 1 , 2, or 3) e) DMF/POCI₃ f) Z = OH, MeS0₃H or Z = NR^SaR^5b, propionic acid g) p-chloranil h) TMAOH i) AM-Br, DIPEA l ,2-bis(2-aminophenoxy)ethane (Biochemisty 19: 2396-2404 (1980)) reacts with dyglycolyl chloride in high dilution to produce the diamide, which is reduced to the benzo-annulated crown. Reaction with alkyl acid chloride in pyridine produces the second diamide, and reduction of the amides gives the N- alkyl annulated crown. Vilsmeier formylation results in the dialdehyde, which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

Similar to Scheme 7b versus Scheme 7a, mild Vilsmeier formylation gives the monoformyl compound instead of the dialdehyde, which leads to Compound 3, R^{l s} = R¹⁶ = R¹⁷ = R¹⁸ = R¹⁹ = R²⁰ = R²¹ = H; R²² = (CH₂)„CH₃, n = 1 , 2, or 3.

By substituting l -(2-amino-5-methylphenoxy),2-(2-aminophenoxy)ethane (J.Biol.Chem. 260: 3440-3450, 1985)) for l ,2-bis(2-aminophenoxy)ethane in Scheme I I , Vilsmeier formylation gives the monoformyl compound instead of the dialdehyde, which leads to Compound 3, R²⁰ = Me, R¹⁵ = R¹⁶ = R¹⁷ = R¹⁸ = R¹⁹ = R²¹ = H, R²² = (CH₂)nCH₃, n = 1 , 2, or 3. Similarly, any other precursors to variants of BAPTA (Biochemisty 19: 2396-2404 ( 1980)), including 5-halogen, 5,6-difluoro, 5-nitro, or any variants, that are resistant to reduction by diborane will lead to variants of compound 3, primarily at R²⁰ (i.e., R²⁰ = F, Br, N0₂).

Scheme 12 shows the synthesis for Compound 4, R¹⁵ = R¹⁶ = R'⁷ = H.

a) diglycolyl chloride, pyridine, high dilution DCM b) B₂H₆ c) DMF POCb d) Z = OH, MeSCbH or Z = NR^5aR^5b, propionic acid e) p-chloranil TMAOH g) AM-Br, D1PEA

Benzoannulated crown from Scheme 1 1 reacts with diglycolyl chloride to form the diamide, which is reduced with diborane to yield the [1 , 1 ,2] benzoannulated cryptand. Vilsmeier formylation results in the dialdehyde, which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

Similar to Scheme 7b versus Scheme 7a, mild Vilsmeier formylation gives the monoformyl compound instead of the dialdehyde, which leads to Compound 4, R¹⁵ = R¹⁶ = R¹⁷ = R¹⁸ = R¹⁹ = R²⁰ = R²¹ = H.

Substitution of 1 -(2-amino-5-methylphenoxy).2-(2-aminophenoxy)ethane (J.Biol .Chem. 260: 3440-3450. ( 1985)) for l ,2-bis(2-aminophenoxy)ethane (Scheme 1 1 ) into Scheme 12. eventually gives the monoformyl compound instead of the dialdehyde, which leads to Compound 3, R²⁰ = Me, R¹⁵ = R¹⁶ = R¹⁷ = R¹⁸ = R¹⁹ = R²' = H. Similarly, any other precursors to variants of BAPTA (Biochemisty 19: 2396-2404 ( 1980)), including 5-halogen, 5,6-difluoro, 5-nitro, or any variants that are resistant to reduction by diborane will lead to variants of compound 3, primarily at R²⁰ (i.e., R²⁰ = F, Br, NC ).

Substitution of triglycolyl chloride for diglycolyl chloride in Scheme 12 and subsequent variations or substitution of triglycolyl chloride for diglycolyl chloride in Scheme 1 1 with no substitution in Scheme 12, results in the [1 ,2,2] benzoannulated cryptand variant of the fluorescent ion indicator. The example synthetic schemes for the fluorescent sodium indicators may be modified to provide schemes for lithium, potassium, rubidium, and cesium follow.

Example fluorescent lithium indicators are based on 12-crown-4 and [1 , 1 ,1 ] cryptand chelators. fluorescent indicator.

a) diglycolyl chloride, pyridine b) E He

Commercially available o-anisidine reacts with diglycolyl chloride to give the diamide, which is reduced to the "half-crown" that yields Compound 5, R⁶ = OMe; R⁷ = R⁸ = R⁹ = H .

The "full-crown" may be mildly formylated to give the mono-formyl precursor that yields Compound 5, R⁶ = R'° = OMe, R⁷ = R⁸ = R⁹ = R" = R¹² = R¹³ = R¹⁴ = H.

Reaction of o-anisidine and 4-methyl-o-anisidine with diglycolyl chloride in Scheme 13 gives a statistical distribution of dimethyl, monomethyl, and nonmethyl products. These three may be easily separated after formylation, due to the significant differences in polarity among diformyl, monoformyl/ monomethyl, and dimethyl products. The monoformyl/monomethyl compound eventually yields Compound 5, R⁶ = R'° = OMe, R⁷ = R⁸ = R⁹ = R" = R¹³ = R¹⁴ = H, R¹² = Me.

Scheme 14 shows the synthesis of the precursor for dibenzoannulated 12-crown-4 or dibenzo- [ 1 , 1 , 1] and dibenzo-[l , l ,2] cryptands.

a) l ,3-dimethyl-2-imidazolidinone, K.2CO3 b) Pd/C/f

Commercially available o-nitrophenol and l -chloro-2-nitrobenzene is condensed at high temperature to give the dinitro compound, which is catalytically reduced to the "half-crown".

Potassium

Example fluorescent potassium indicators are based on 18-crown-6 and [2,2,2] cryptand chelators. 18-crown-6 is substituted for 15-crown-5. Triglycolyl chloride may be substituted for diglycolyl chloride. It is synthesized from the nitric acid oxidation of triethyleneglycol to triglycolic acid and reaction of triglycolic acid and oxalyl chloride.

Applying these changes to Scheme 9 produces Asante Potassium Green - 3; R¹ = CI; R² = (CH²)„COOR⁴, n = 2; R³ = R⁷ = R⁸ = R⁹ = H; R⁶ = OMe:

APG-3 TMA⁺ Salt, R₄ = (CH₃)4N⁺

APG-3 (AM), R₄ = CH2OCOCH3

Applying these changes to Scheme 9 and oxidizing with p-chloranil in methanol only, not 1 : 1 methanol/chloroform, produces Asante Potassium Green - 1 ; R¹ = CI; R² = (CH )„COOR⁴, n = 2; R⁶ = R¹⁰ = OMe; R¹² = CHO; R³ = R⁷ = R⁸ =R⁹ = R" = R¹³ = R¹⁴ = H

APG-1 TMA⁺ Salt, R4 = (ΟΗ₃)₄Ν⁺

APG- 1 (AM), R₄ = CH2OCOCH3

Applying these changes to Scheme 10 produces Asante Potassium Green - 2, R¹ = CI; R² = (CH²)„ COOR⁴, n = 2; R³ = R⁷ = R⁸ = R⁹ = R" = R¹³ = R¹⁴ = H; R⁶ = R¹⁰ = OMe; R'² = Me

APG-2 TMA⁺ Salt, = (CH

APG-2 (AM), R₄ = CH2OCOCH3

Rubidium

Example fluorescent rubidium indicators are based on 21-crown-7 and [2,2,3] and [2,3,3] cryptand chelators.

21-cro\vn-7 is substituted for 15-crown-5.

Bis[2-(2-chloroethoxy)ethyl]ether may be substituted for l ,2-Bis(2-chloroethoxy)ethane.

3,6,9-trioxaundecanedioyl chloride may be substituted for diglycolyl chloride. It is synthesized form 3,6,9-trioxaundecanedioic acid and oxalyl chloride.

Scheme 15 shows the synthesis of a precursor for 21-crovvn-7 and [2,2,3] and [2,3,3] cryptands. a) Bis[2-(2-chloroethoxy)ethyl]ether, Nal, 2CO3 b) Nal, 2CO3

Commercially available o-nitrophenol reacts with an excess of bis[2-(2-chloroethoxy)ethyl]ether to form monoalkylated ether. Without an excess of the dichloro reagent, it forms the symmetric dinitro compound (dialkylated ether) directly. The monoalkylated ether further reacts with a variant of o- nitrophenol (i.e., 5-methy-2-nitrophenol) to produce the asymmetric dinitro compound.

Cesium

Example fluorescent cesium indicators are based on 24-crown-8 and [3,3,3] cryptand chelators. 24-cro\vn-8 is substituted for 15-crown-5.

Bis[2-(2-chloroethoxy)ethyl]ether is substituted for l ,2-Bis(2-chloroethoxy)ethane. 3,6,9-trioxaundecanedioyl chloride is substituted for diglycolyl chloride. It is synthesized form 3,6,9- trioxaundecanedioic acid and oxalyl chloride.

Scheme 15 gives the precursor for dibenzoannulated 24-crown-8 and [3,3,3] cryptand. Calcium

Example fluorescent calcium indicators are based on BAPTA and its derivatives, as synthesized in J.Bioi.Chem. 260: 3440-3450, ( 1985), Biochemisty 19: 2396-2404 ( 1980), and US 5576433.

Scheme 16 shows the synthesis of Compound 6.

a) Z = OH, MeS0₃H or Z = NR⁵R⁶, proprionic acid b) p-chloranil c) 1 . OH, TMAOH, or Βα,ΝΟΗ 2. HC1 if KOH is used d) AM-Br, Na₂C03

BAPTA aldehyde is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

One variant: R¹ = CI; R² = (CH²)„C00R⁴, n = 2; R²⁴ = R²⁶ = H; R²⁵ = Me; gives Asante Calcium Green - 1 (ACG- 1 ).

ACG- 1 TMA⁺ Salt, R₄ = (CH₃)4N⁺

ACG-1 (AM), R, = CH2OCOCH3 Magnesium

Example fluorescent magnesium indicators are based on APTRA.

Scheme 16 shows the synthesis of Compound 7.

a) methylbromoacetate, Nal, D1PEA b) DMF/POCb c) Z = OH, MeS0₃H or Z = NR⁵R⁶, proprionic acid d) p-chloranil e) l . OH 2. HC1 f) AM-Br, DIPEA o-aminophenol is alkylated with methylbromoacetate. Vilsmeir reaction produces the aldehyde, which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

One variant: R' = CI; R² = (CH²)_nCOOR«, n = 2; gives Asante Magnesium Green - 1 (AMG- 1 ).

AMG- 1 TMA⁺ Salt, R. = (CH₃)₄N⁺

AMG- 1 (AM), = CH2OCOCH3

Thallium

Example fluorescent thallium indicators are based on Half-BAPTA. Scheme 17 shows the synthesis of Compoi

a) methylbromoacetate, Nal, D1PEA b) DMF/POC c) Z = OH, MeSCbH or Z = NR⁵R⁶, proprionic acid d) p-chloranil e) I . OH 2. HC1 f) AM-Br, DIPEA o-anisidine is alkylated with methylbromoacetate. Vilsmeir reaction produces the aldehyde, which is coupled with a resorcinol or aminophenol variant in methanesulfonic acid or propionic acid, respectively, and subsequently oxidized with p-chloranil to give the fluorescent dye. Hydrolysis of esters gives the salt form and reaction with acetoxymethyl bromide gives the AM ester form.

One variant: R¹ = CI; R² = (CH²)„COOR⁴. n = 2; gives Asante Thallium Green - 1 (AMG- 1 ).

ATG-1 TMA⁺ Salt, R4 = (CH₃)4N⁺

ATG-1 (AM), R4 = CH2OCOCH3

Examples 1 to 5, Scheme 1

Examples to , Scheme 9

Compound 103 o-Anisidine (362 mL) was added along with diisopropylethylamine (233 mL) and l ,2-bis(2- chloroethoxy)ethane (84 mL). The reaction was stirred at I 20°C for 3 days. The crude was placed on rotatory evaporator with an oil bath and heated to >100°C with high vacuum in order to remove most of the excess anisidine. After this, the residue was diluted in ethyl acetate and washed with water twice, then dried over sodium sulfate and evaporated. The crude was evaporated under vacuum and purified by column chromatography using 6: 1 hexane/ethyl acetate and increasing ethyl acetate content.

Compound 104

Diglycolic acid (25 g) was added to a dry flask. To this was added thionyl chloride (85 mL) and the reaction was immediately heated to reflux and stirred at this temperature for 5 hours. At this time, the excess thionyl chloride was distilled at 85°C and reduced pressure, followed by high vacuum at room temperature until mixture precipitated. Then the crude sludge was distilled at 40°C under high vacuum and increasing temperature until all liquid was collected.

Compound 107

Compounds 103 (17 mmol) and 104 (80 mol%, used 22 mmol) were dissolved separately in flasks with benzene ( 150 mL in each). These were concurrently added at equal rate, over several hours, to a flask containing benzene ( 150 mL) and pyridine (46 mmol). After addition was complete, placed flask in oil bath at 75°C to stir overnight. Then evaporated the benzene completely and dissolved residue in dichloromethane. Washed twice with 1 HCI mixed with some brine, then with sodium bicarbonate, and finally with brine, then dried over sodium sulfate. After evaporation under vacuum, purified by column chromatography, loading with chloroform and using 5% methanol in chloroform and increasing methanol content.

Compound 108

Compound 107 (6.2 g) was dissolved in 100 mL THF, and 6.0 g sodium borohydride were added. A solution of 35 mL boron trifluoride-diethyletherate in 70 mL THF was added over 1 hr. The reaction mixture was poured over ice, and the pH of the slurry was adjusted to neutral pH. The slurry was extracted thoroughly with dichloromethane, and the organics were washed with brine, dried over anhydrous magnesium sulfate, filtered, and evaporated under vacuum.

Compound 109

Compound 108 (6.5 g, used without purification from the previous step) was dissolved in 100 mL DMF, and cooled in an ice bath for 15 minutes. 3 1 mL phosphorous oxychloride were added to the cooled solution at a rate to not increase the temperature of the reaction mixture by more than a few degrees. Upon completion of the addition, the reaction mixture was stirred at 70 °C overnight. The reaction mixture was then poured over ice, and the pH was adjusted to neutral with aqueous OH. The aqueous slurry was extracted thoroughly with dichloromethane, and the organics were washed with brine, dried over anhydrous magnesium sulfate, filtered, and evaporated under vacuum to yield a tan solid. The solid was triturated with methanol to give an off-white solid.

Compound 101

To 4-methyl-o-anisidine (6 g) dissolved in acetonitrile (30 mL) was added diisopropylethylamine ( 15 mL), sodium iodide (1.8 g), and l ,2-bis(2-chloroethoxy)ethane ( 14 mL). The reaction was stirred at 1 10°C overnight. The mixture was diluted in ethyl acetate and washed three times with pH2 buffer and once with brine. The organic layer was dried over sodium sulfate and evaporated under vacuum. The crude was purified by column chromatography using 3: 1 hexane/ethyl acetate.

Compound I 0 I ( 10.25 g) was dissolved in 5 1 mL acetonitrile. Diisopropylethylamine (3 1 mL), anisidine (46 mL), and sodium iodide (3.1 g) were added and the mixture stirred at 120°C overnight. The mixture was diluted into ethyl acetate and brine and the layers separated. The organic layer was washed twice with brine and dried over sodium sulfate, followed by evaporation of the solvent under vacuum. The crude was purified by column chromatography using 6: 1 hexane/ethyl acetate and increasing ethyl acetate content.

Triethylene glycol (18 mL) was added slowly, in small portions, to 70% HNO3 ( 100 g, d=l .4) at 45°C, waiting for fuming to decrease before each successive addition. Then heated to 80°C to stir for 1 hour. Evaporated on rotatory evaporator for 2 hours at 70°C with high vacuum. Added 120 mL benzene and dried again by high vacuum at 70°C. Liquid did not solidify upon cooling.

Compound 106

Dissolved compound 105 (32.7 g) in chloroform (500 mL) and added to an oil bath at 90°C with reflux condenser and drying tube equipped. Then added oxalyl chloride (58 mL) and stirred at this temperature for 5 hours. Let cool some and evaporated carefully on a rotatory evaporator, gradually increasing vacuum at 40°C, and finally swirling flask and pulling a high vacuum at room temperature to dry completely.

Example 1- sodium indicator ANG-1

FIG. 1 is a fluorescence emission spectra of a sodium titration of Asante Natrium Green- 1 (ANG- I ) salt solution ( 1 M T ACI, l Om MOPS, pH = 7.10) titrated with 1 sodium chloride solution, excitation at 517 nm.

FIG. 7 shows HE 293 cells expressing TRPV 1 channels were loaded with ANG-1 (AM). Capsaicin, an agonist for TRPV 1 channels, which conduct Na⁺ and Ca²⁺, was used to stimulate Na+ influx.

FIG. 8 shows REF52 cells were loaded with ANG- 1 (AM) and the Na⁺ ionophore gramicidin was applied to promote Na⁺ influx. The resulting rise in Na⁺ intracellular concentration caused a corresponding increase in ANG-1 fluorescence.

FIG. 9 shows astrocytes loaded with ANG- 1 (AM) showed an increase in fluorescence from the first frame to the second frame as a result of a rise in Na⁺ intracellular concentration due to ouabain inhibition of the sodium pump.

Example 2 - sodium indicator ANG-2

FIG. 2 is a fluorescence emission spectra of a sodium titration of Asante Natrium Green-2 (ANG-2) salt solution ( 140 111M TMACI, lOmM MOPS, pH = 7.1 1 ), excitation at 517 nm, titrated with sodium chloride solution ( I NaCI, l OmM MOPS, pH = 7.10). FIG. 10 shows REF52 fibroblasts loaded with ANG-2 (AM) and the Na⁺ ionophore SQI-Pr was applied to promote Na⁺ influx. The resulting rise in Na⁺ intracellular concentration caused a corresponding increase in ANG-2 fluorescence. Further adition of 20 μΜ amphotericin B gave only a small increase of fluorescence.

FIG. 1 1 shows REF52 fibroblasts loaded with ANG-2 (AM) were maintained in 145 mM NMG- gluconate. 50 μΜ amphotericin-B depleted the cells of Na⁺ and ⁺. Increments of NaCl were added extracellularly, resulting in a corresponding increase in ANG-2 fluorescence after equilibration of intracellular and extracellular sodium concentrations. 145 mM ⁺ was then added, resulting in a decrease of ANG-2 fluorescence.

Example 3- sodium indicator ANG-3

FIG. 3 is a fluorescence emission spectra of a sodium titration of Asante Natrium Green-3 (ANG-3) salt solution (1 M TMAC1, l OmM MOPS, pH = 7.12) titrated with sodium chloride solution (1 M NaCl, 10 mM MOPS, pH = 7.10), excitation at 517 nm.

Example 4- potassium indicator APG- 1

FIG. 4 is a fluorescence emission spectra of a potassium titration of Asante Potassium Green- 1 (APG- 1 ) salt solution ( 140mM TMACI, lOmM MOPS, pH = 7.1 1 ), excitation at 517 nm, titrated with 1 M potassium chloride solution.

FIG. 12 shows REF52 fibroblasts loaded with APG- 1 (AM) were maintained in 145 mM NMG- gluconate. 50 μΜ amphotericin-B depleted the cells of Na⁺ and K⁺. Increments of KC1 were added extracellularly, resulting in a corresponding increase in APG-1 fluorescence after equilibration of intracellular and extracellular K⁺ concentrations. 10 mM Na⁺ was then added, only slightly increasing APG-1 fluorescence.

Example 5- potassium indicator APG-2

FIG. 5 is a fluorescence emission spectra of a potassium titration of Asante Potassium Green-2 (APG-2) salt solution (140mM TMACI, l OmM MOPS, pH = 7.1 1 ), excitation at 517 nm, titrated with 1 M potassium chloride solution. Example 6- calcium indicator ACG (K.+ salt)

FIG. 6 is a fluorescence emission spectra of calcium titration of ACG- 1 salt solution (10 mM EGTA, 10 mM MOPS, pH 7.2) titrated with 10 mM CaEGTA, excitation at 517 nm, showing a >200X increase in fluorescence upon saturating with Ca²⁺.

FIG. 13 shows fluorescence emission spectra for equi-absorbing solutions of Asante Calcium Green- 1 and fluorescein, resulting in a quantum efficiency of 0.495 for Ca²⁺-bound ACG- 1 .

FIG. 14 shows rat vagal sensory (nodose ganglion) neurons were loaded with 50 μΜ ACG- 1 K⁺ salt via whole-cell patch electrode. The neuron received a series of step depolarizations from -70 to + 10 mV that ranged in duration from 1 -1000 msec. Intracellular Ca2+ signals evoked by the depolarizing steps were recorded as increases in ACG- 1 fluorescence, reported as AF/Fo.

Example 7-sodium indicator ANG-NM

FIG. 15 shows titration of Asante Natrium NM in 130 mM TMACl, 10 mM MOPS, pH 7.0 with an excitation of 492 nm. Aliquots of 1 M NaCl solution were added to give the listed concentrations. No corrections were made for increasing ionic strength.

Example 8-sodium indicator ANG-TM

FIG. 16 shows titration of Asante Natrium TM in 100 mM TMACl, 5 mM tris base, pH 9.0 with an excitation of 500 nm. Aliquots of 1 M NaCl solution were added to give the listed concentrations. No corrections were made for dilution or increasing ionic strength.

Claims

What is claimed is:

1. A fluorescent ion indicator comprising a first hydroxyxanthenone fluorophore of the structure

for which Z¹ = OR⁴ and Z² = O; R¹ = H, F, CI, Br, or (CH₂)„C0₂R⁴, n = 0, 1 , or 2; R² = H, F, CI, Br, or (CH₂)nC0₂R⁴, n = 0, 1 , or 2; R³ = H, F, CI, Br; R⁴ = H, salt (i.e., TMA⁺, ⁺) or AM (CH₂OCOCH₃), or any other pharmaceutically acceptable salts and esters; and R⁵⁰ = H or a covalent bond to an organic molecule.

2. The fluorescent ion indicator of Claim 1 , wherein R^so is a covalent bond to a chelator or to a first linker covalently bonded to a chelator.

3. The fluorescent ion indicator of Claim 2. wherein R⁵⁰ is a covalent bond to a non-annulated chelator through a first non-annulated linker.

4. The fluorescent ion indicator of Claim 3, wherein R⁵⁰ is a covalent bond to a chelator through a non- annulated linker, the first non-annulated linker comprising

for which R⁶ = H, OMe, F; R⁷ = H, OMe, F; R⁸ = H, OMe, F; R⁹ = H, OMe, F; R^{S I} = the covalent bond R⁵⁰ to the hydroxyxanthenone fluorophore; and R⁵² = covalent bond to a non-annulated chelator.

5. The fluorescent ion indicator of Claim 4, wherein the non-annulated chelator is a crown selected from the group comprising monoaza- 12-crown-4, monoaza- 15-crown-5, monoaza- 18-crown-6, monoaza-21-crown-7, or monoaza-24-crown-8; and R⁵² = the aza function of the crown.

6. The fluorescent ion indicator of Claim 5. wherein the non-annulated chelator is a monoaza- 15-crown-5, R¹ = CI; R² = (CH₂)„C02R⁴, n = 2; R³ = R⁶ = R⁷ = R⁸ = R⁹=H; R⁵⁰ = the bond from the hydroxyxanthenone fluorophore to R⁵¹ on the non-annulated linker; R^S2 is the aza on the monoaza- 15-crown-5:

7. The fluorescent ion indicator of Claim 4, wherein the non-annulated chelator is a crown selected from the group comprising diaza- l 2-crown-4, diaza-l 5-crown-5, diaza- l 8-crown-6, diaza-21-crown-7, or diaza-24- crown-8, such that the chelator has a first aza function and a second aza function; the first non-annulated linker bonds to the first aza function of the chelator at R⁵² and to the first hydroxyxanthenone fluorophore at R⁵¹; and there is a second linker and a second fluorophore, the second linker comprising:

for which R⁶ = H, OMe, F; R⁷ = H, OMe, F; R⁸ = H, OMe, F; R⁹ = H, OMe, F; R⁵¹ = the covalent bond R⁵⁰ to a second fluorophore; and R⁵² = covalent bond to the second aza function of the non-annulated chelator.

8. The fluorescent ion indicator of Claim 7, wherein the non-annulated chelator is diaza-l 5-crown-5 and the second fluorophore is identical to the first hydroxyxanthenone fluorophore; R¹ = CI; R² = (CFhJnCOzR , n = 2; R³ = R⁷ = R⁸ = R⁹= H; R⁶ = OMe; R⁵⁰ = the bond from the fluorophore to R⁵¹ on the non-annulated linker; R⁵² are each of the azas on the diaza-l 5-crown-5:

Non-annulated

9. The fluorescent ion indicator of Claim 4, wherein the non-annulated chelator is a crown selected from the group comprising diaza-12-crown-4, diaza-15-crown-5, diaza- 18-crown-6, diaza-21-crown-7, or diaza-24- crovvn-8 ; R⁵² = a first aza function of the crown, R⁵³ = a second aza function of the crown as a substituent on a cap, the cap comprising:

R'° = H, OMe, F; R" = H, OMe, = H, Me, F, Br, CI, I, N0₂, CHO, (CH₂)„C0₂R⁴ [n = 0, 1 , or 2],

25;

R¹³ = H, OMe, F; R¹⁴ = H, OMe, F;

such that there is one hydroxyxanthenone fluorophore covalently bonded through R⁵⁰ to R⁵¹ of the non- annulated linker, said non-annulated linker incorporating one of the aza functions of the diaza crown at R⁵²; and there is one non-annulated cap incorporating the other aza function of the diaza crown at R⁵³.

10. The fluorescent ion indicator of Claim 9, wherein the non-annulated chelator is diaza- 15-crown-5, R¹ = CI; R² = (CH₂)_nC0₂R⁴, n = 2; R³ = R⁷ = R⁸ = R⁹= R" = R¹³ = R¹⁴ = H; R⁶ = R¹⁰ = OMe; R¹² = CHO ; R⁵⁰ = the bond to R⁵¹ on the non-annulated linker; R⁵² is a first aza on the diaza- 15-crown-5; R⁵³ is a second aza on the diaza- 15-crown-5 :

1 1. The fluorescent ion indicator of Claim 9, wherein the non-annulated chelator is diaza- 15-crown-5, R¹ = CI; R² = (CH₂)nC0₂R⁴, n = 2; R³ = R⁷ = R⁸ = R⁹= R" = R¹³ = R¹⁴ = H; R⁶ = R¹⁰ = OMe; R¹² = Me ; R⁵⁰ = the bond to R⁵¹ on the non-annulated linker; R⁵² is a first aza on the diaza- 15-crown-5; R⁵³ is a second aza on the diaza-15-crown-5:

12. The fluorescent ion indicator of Claim 2, wherein R⁵⁰ is a covalent bond to an annulated chelator through an annulated linker.

13. The fluorescent ion indicator of Claim 12, wherein R⁵⁰ is a covalent bond to a chelator through an annulated linker, the annulated linker comprising

for which N and O are heteroatoms in the annulated crown or cryptand; R¹⁵ = H, O e, F; R'⁶ = H, OMe, F; R¹⁷ = H, OMe, F; and R⁵¹ = the covalent bond R⁵⁰ to the hydroxyxanthenone fluorophore.

14. The fluorescent ion indicator of Claim 13, wherein the annulated chelator is a dibenzo annulated crown selected from the group comprising dibenzo-diaza- 12-crown-4, dibenzo-diaza- l 5-crown-5, dibenzo-diaza-18- crown-6, or dibenzo-diaza-24-crown-8 ; such that there are two identical hydroxyxanthenone fluorophores, each covalently bonded through R^so to R⁵¹ of its respective annulated linker, each annulated linker incorporating its respective aza oxa moiety in the crown, such that each of the two aza functions is covalently bonded to its respective R²², wherein R²² = H or (CFhinCFb (n = 0, 1 , 2).

15. The fluorescent ion indicator of Claim 14, wherein the annulated chelator is dibenzo-diaza-15-crown-5, R¹ = CI; R² = (CH2)nC0₂R⁴, n = 2; R³ = R¹⁵ = R¹⁶ = R¹⁷ = H; R²² = (CH₂)nCH₃, n = 1 , and R⁵¹ = the covalent bond R^so to the hyd

16. The fluorescent ion indicator of Claim 13, wherein the annulated chelator is a dibenzo annulated crown selected from the group comprising dibenzo-diaza- 12-crovvn-4, dibenzo-diaza-15-crown-5, dibenzo-diaza- 18- crown-6, dibenzo-diaza-2 l -crown-7, or dibenzo-diaza-24-crown-8 ; such that the hydroxyxanthenone fluorophore is covalently bonded through R⁵⁰ to R⁵¹ of its annulated linker, said annulated linker incorporating a first aza oxa moiety of the crown; and a second aza/oxa moiety of the crown is incorporated in an annulated cap comprising:

for which N and O are two of the heteroatoms in the annulated crown, R'⁸ = H, OMe, F;

R = H, Me, F, Br, CI, I, (CH₂)_nC0₂R⁴ [n = 0, l , or 2], N0₂, CHO,

; and R²¹ = H, OMe, F; such that the first and second aza functions are covalently bonded to R²², wherein R²² = H or (CH2)nCH3 (n = 0, 1 , 2).

17. The fluorescent ion indicator of Claim 16, wherein the annulated chelator is dibenzo-diaza- 15-crown-5, R¹ = CI; R² = (CH₂)„C0₂R⁴, n = 2; R³ = R¹⁵ = R¹⁶ = R¹⁷ = R¹⁸ = R¹⁹ = R²¹ = H; R²⁰ = Me; R²² = (CH₂)_nCH₃, n = 1 , and R⁵¹ = the covalent bond R⁵⁰ to the hydroxyxanthenone fluorophore:

18. The fluorescent ion indicator of Claim 13, wherein the annulated chelator is a dibenzo-cryptand selected from the group comprising dibenzo-[ 1 , 1 , 1 ], dibenzo-[l , l ,2], dibenzo-[ 1 ,2,2], dibenzo-[2,2,2], dibenzo-[2,2,3], dibenzo-[2,3,3], and dibenzo-[3,3.3] cryptands such that there are two identical hydroxyxanthenone fluorophores, each covalently bonded through R^so to R⁵¹ of its respective annulated linker, each annulated linker incorporating its respective aza/oxa moiety in the cryptand.

19. The fluorescent ion indicator of Claim 18, wherein the annulated chelator is a dibenzo-[l , l ,2]-cryptand, R¹ = CI; R² = (CH₂)nC0₂R⁴, n = 2; R³ = R¹⁵ = R¹⁶ = R¹⁷ = H; and R⁵' = the covalent bond R⁵⁰ to the hydroxyxanthenone fluorophore:

20. The fluorescent ion indicator of Claim 13, wherein the annulated chelator is a dibenzo annulated cryptand selected from the group comprising dibenzo-[ 1 , 1 , 1], dibenzo-[ l , l ,2], dibenzo-[ l ,2,2], dibenzo-[2,2,2], dibenzo- [2,2,3], dibenzo-[2,3,3], and dibenzo-[3,3,3] cryptands; such that the hydroxyxanthenone fluorophore is covalently bonded through R⁵⁰ to R⁵¹ of its annulated linker, said annulated linker incorporating a first aza/oxa moiety of the cryptand; and a second aza/oxa moiety of the cryptand is incorporated in the annulated cap:

for which N and O are two of the heteroatoms in the annulated crown, R¹⁸ = H, OMe, F; R¹⁹ = CI, I, (CH₂)„C0₂R⁴ [n = 0, 1 , or 2], N0₂, CHO,

5 ; and R²' = H, OMe, F.

21. The fluorescent ion indicator of Claim 20, wherein R¹ = CI; R² = (CFhJnCO.R⁴, n = 2; R³ = R^{I S} = R'⁶ = R¹⁷ = R¹⁸ = R¹⁹ = R²¹ = H; R²⁰ = Me; and R^{S I} = the covalent bond R⁵⁰ to the hydroxyxanthenone fiuorophore:

22. The fluorescent ion indicator of Claim 2, wherein the chelator is BAPTA and its derivatives:

R²⁴ = H, F, Br, CI

R²⁵ = H, Me, F, Br, CI, I, N0₂, CHO, (CH₂)„C0₂R⁴ [n = 0, 1 , or 2], R²⁵ = H,

R⁵¹ = covalent bond to the hydroxyxanthenone fluorophore at R⁵⁰

23. The fluorescent ion indicator of Claim 22, wherein R' = CI; R² = (CH₂)_nC02R⁴, n = 2; R³ = R²⁴ = R²⁶ = H; R^2S = Me; R⁵¹ = covalent bond to the hydroxyxanthenone fluorophore at R⁵⁰:

M0

24. The fluorescent ion indicator of Claim 2, wherein the chelator is APTRA and its derivatives: R⁵¹ = covalent bond to the hydroxyxanthenone fluorophore at R^so.

25. The fluorescent ion indicator of Claim 24, wherein R¹ = CI; R² = (CH₂)„C0₂R⁴, n = 2; and R⁵¹ = covalent bond to the hydroxyxanthenone fluorophore at R^so:

26. The fluorescent ion indicator of Claim 2, wherein the chelator is Half-BAPTA and its derivatives:

R⁵' = covalent bond to the hydroxyxanthenone fluorophore at R⁵⁰

27. The fluorescent ion indicator of Claim 26, wherein R¹ = CI; R² = (CfbinCCh ⁴, n = 2; and R⁵¹ = covalent bond to the hydroxyxanthenone fluorophore at R⁵⁰:

R^5bR^5aN _{N R}5a_R5b

R R

for which Z¹ = NR^5aR^5b and Z² = NR^SaR^5b; R¹ = H, F, CI, Br; R² = H, F, Ci, Br; R³ = H, F, CI, Br; R⁴ = H, salt (i.e., TMA⁺, ⁺) or AM (CH2OCOCH3), or any other pharmaceutically acceptable salts and esters; R^5a = (CH₂)nC0₂R⁴, n = 0, 1 , or 2; R^5b = (CH2)nCC>2R⁴, n = 0, 1 , or 2 and R⁵⁰ = H or a covalent bond to an organic molecule.

29. The fluorescent ion indicator of Claim 28, wherein R⁵⁰ is a covalent bond to a chelator or to a first linker covalently bonded to a chelator.

30. The fluorescent ion indicator of Claim 29, wherein R⁵⁰ is a covalent bond to a non-annulated chelator through a first non-annulated linker.

31. The fluorescent ion indicator of Claim 30, wherein R⁵⁰ is a covalent bond to a chelator through a non- annulated linker, the first non-annulated linker comprising

for which R⁶ = H, OMe, F; R⁷ = H, OMe, F; R⁸ = H, OMe, F; R⁹ = H, OMe, F; R⁵¹ = the covalent bond R⁵⁰ to the rosamine fluorophore; and R⁵² = covalent bond to a non-annulated chelator.

32. The fluorescent ion indicator of Claim 31 , wherein the non-annulated chelator is a crown selected from the group comprising monoaza- 12-crown-4, monoaza-15-crown-5, monoaza-1 8-crown-6, monoaza-21-crown-7, or monoaza-24-crown-8; and R⁵² = the aza function of the crown.

33. The fluorescent ion indicator of Claim 32, wherein the non-annulated chelator is a monoaza- 15-crown-5, R¹ = R² = R³ = R⁶ = R⁷ = R⁸ = R⁹= H; R^5a = (CH2)nC0₂R⁴, n = 1 ; R^5b = (CH₂)nC0₂R⁴, n = 1 ; R^so = the bond from the rosamine fluorophore to R⁵¹ on the non-annulated linker; R⁵² is the aza on the monoaza-15-crown-5:

34. The fluorescent ion indicator of Claim 31 , wherein the non-annulated chelator is a crown selected from the group comprising diaza-12-crown-4, diaza- 15-crown-5, diaza- 18-crown-6, diaza-21 -crown-7, or diaza-24- crown-8, such that the chelator has a first aza function and a second aza function; the first non-annulated linker bonds to the first aza function of the chelator at R⁵² and to the first rosamine fluorophore at R⁵¹; and there is a second linker and a second fluorophore, the second linker comprising:

for which R⁶ = H, OMe, F; R⁷ = H, OMe, F; R⁸ = H, OMe, F; R⁹ = H, OMe, F; R⁵¹ = the covalent bond R⁵⁰ to a second fluorophore; and R⁵² = covalent bond to the second aza function of the non-annulated chelator.

35. The fluorescent ion indicator of Claim 34, wherein the non-annulated chelator is diaza-15-crown-5 and the second fluorophore is identical to the first rosamine fluorophore; R¹ = R² = R³ = R⁶ = R⁷ = R⁸ = R⁹= H; R^Sa = (CH₂)nC0₂R⁴, n = 1 ; R^5b = (CH₂)nC0₂R⁴, n = 1 ; R⁵⁰ = the bond from the fluorophore to R⁵¹ on the non- annulated linker; R⁵² are each of the azas on the diaza- 15-crown-5:

36. The fluorescent ion indicator of Claim 3 1, wherein the non-annulated chelator is a crown selected from the group comprising diaza-12-crown-4, diaza- 15-crown-5, diaza- 18-crown-6, diaza-21-crown-7, or diaza-24- crown-8 ; R⁵² = a first aza function of the crown, R⁵³ = a second aza function of the crown as a substituent on a cap, the cap comprising:

¹⁰ = H, OMe, F; R" = H, OMe, F; R¹² = H, Me, F, Br, CI, I, N0₂, CHO, (CH2)nC0₂R⁴ [n = 0, 1 , or 2],

25;

Rⁿ = H, OMe, F; R¹ = H, OMe, F;

such that there is one rosamine fluorophore covalently bonded through R⁵⁰ to R⁵¹ of the non-annulated linker, said non-annulated linker incorporating one of the aza functions of the diaza crown at R⁵²; and there is one non- annulated cap incorporating the other aza function of the diaza crown at R⁵³.

37. The fluorescent ion indicator of Claim 36, wherein the non-annulated chelator is diaza- 15-crown-5, R¹ = R² = R³ = R⁶ = R⁷ = R⁸ = R⁹ = R" = R¹³ = R¹ = H; R⁶ = R'° = OMe; R¹² = CHO; R^5a = (CH₂)„C0₂R⁴, n = 1 ; R^5b = (CH2)nC02R⁴, n = 1 ; R^so = the bond to R^{S I} on the non-annulated linker; R⁵² is a first aza on the diaza-15- crown-5; R⁵³ is a second aza on the diaza- 15-crown-5:

38. The fluorescent ion indicator of Claim 36, wherein the non-annulated chelator is diaza- 15-crown-5, R¹ = R² = R³ = R⁶ = R⁷ = R⁸ = Rⁱ> = R^{l l} = R^{l 3} = R^l = H; R⁶ = R¹⁰ = OMe; R¹² = Me; R^5a = (CH2)nC0₂R⁴, n = 1 ; R^5b = (CH2)_nC02R⁴, n = I ; R⁵⁰ = the bond to R^{S I} on the non-annulated linker; R⁵² is a first aza on the diaza- 15- crown-5; R⁵³ is a second aza on the diaza- 15-crown-5:

39. The fluorescent ion indicator of Claim 29, wherein R^so is a covalent bond to an annulated chelator through an annulated linker.

40. The fluorescent ion indicator of Claim 39, wherein R^so is a covalent bond to a chelator through an annulated linker, the annulated linker comprising

for which N and O are heteroatoms in the annulated crown or cryptand; R¹⁵ = H, OMe, F; R¹⁶ = H, O e, F; R¹⁷ = H, OMe. F; and R⁵¹ = the covalent bond R⁵⁰ to the rosamine fluorophore.

41. The fluorescent ion indicator of Claim 40, wherein the annulated chelator is a dibenzo annulated crown selected from the group comprising dibenzo-diaza- 12-crown-4, dibenzo-diaza- 15-crown-5, dibenzo-diaza- 18- crown-6, dibenzo-diaza-21-crown-7, or dibenzo-diaza-24-crown-8 ; such that there are two identical rosamine fluorophores, each covaiently bonded through R⁵⁰ to R⁵¹ of its respective annulated linker, each annulated linker incorporating its respective aza/oxa moiety in the crown, such that each of the two aza functions is covaiently bonded to its respective R²², wherein R²² = H or (CH₂)„CH3 (n = 0, 1 , 2).

42. The fluorescent ion indicator of Claim 41 , wherein the annulated chelator is dibenzo-diaza- 15-crown-5, = R² = R³ = R¹⁵ = R¹⁶ = R¹⁷ = H ; R^5a = (CH₂)„C0₂R⁴, n = 1 ; R^5b = (CH₂)„C0₂R<, n = 1 ; R²² = (CH₂)„CH3, 1 ; and R⁵¹ = the covalent bond R⁵⁰ to the rosamine fluorophore:

43. The fluorescent ion indicator of Claim 40, wherein the annulated chelator is a dibenzo annulated crown selected from the group comprising dibenzo-diaza- l 2-crown-4, dibenzo-diaza-15-crown-5, dibenzo-diaza-18- crown-6, dibenzo-diaza-21-crown-7, or dibenzo-diaza-24-crown-8 ; such that the rosamine fluorophore is covaiently bonded through R⁵⁰ to R⁵¹ of its annulated linker, said annulated linker incorporating a first aza oxa moiety of the crown; and a second aza/oxa moiety of the crown is incorporated in an annulated cap comprising:

for which N and O are two of the heteroatoms in the annulated crown, R¹⁸ = H, OMe, F; R¹⁹ Br, CI, I, (CH₂)nC02R⁴ [n = 0, 1 , or 2], N0₂, CHO,

; _and R²¹ = H, OMe, F; such that the first and second aza functions are covalently bonded to R²², wherein R²² = H or (CH₂)_nCH3 (n = 0, 1 , 2).

17. The fluorescent ion indicator of Claim 16, wherein the annulated chelator is dibenzo-diaza-15-crown-5, R' = R² = R³ = R¹⁵ = R¹⁶ = R¹⁷ = R¹⁸ = R¹⁹ = R²¹ = H; R²⁰ = Me; R^5a = (CH₂)nC0₂R⁴, n = 1 ; R^5b = (CH₂)_nC0₂R⁴, n = 1 ; R²² = (CH2)nCH3, n = 1 ; and R⁵¹ = the covalent bond R⁵⁰ to the rosamine fluorophore:

18. The fluorescent ion indicator of Claim 13, wherein the annulated chelator is a dibenzo-cryptand selected from the group comprising dibenzo-[ 1 ,1 , 1 ], dibenzo-[l , l ,2], dibenzo-[l ,2,2], dibenzo-[2,2,2], dibenzo-[2,2,3], dibenzo-[2,3,3], and dibenzo-[3,3,3] cryptands such that there are two identical rosamine fluorophores, each covalently bonded through R⁵⁰ to R⁵¹ of its respective annulated linker, each annulated linker incorporating its respective aza/oxa moiety in the cryptand.

46. The fluorescent ion indicator of Claim 45, wherein the annulated chelator is a dibenzo-[l , l ,2]-cryptand, R¹ = R² = R³ = R¹⁵ = R¹⁶ = R¹⁷ = H; R^5a = (CH₂)nC0₂R⁴, n = 1 ; R^5b = (CH₂)„C0₂R⁴, n = 1 ; and R⁵¹ = the covalent bond R⁵⁰ to the rosamine fluorophore:

47. The fluorescent ion indicator of Claim 40, wherein the annulated chelator is a dibenzo annulated cryptand selected from the group comprising dibenzo-[ 1 , 1 , 1], dibenzo-[l , l ,2], dibenzo-[l ,2,2], dibenzo-[2,2,2], dibenzo- [2,2,3], dibenzo-[2,3,3], and dibenzo-[3,3,3] cryptands; such that the rosamine fiuorophore is covalentiy bonded through R⁵⁰ to R^{S I} of its annulated linker, said annulated linker incorporating a first aza oxa moiety of the cryptand; and a second aza/oxa moiety of the cryptand is incorporated in the annulated cap:

for which N and O are two of the heteroatoms in the annulated crown, R¹⁸ = H, OMe, F;

R'⁹ = ²⁰ = H, Me, F, Br, CI, I, (CH₂)nC0₂R⁴ [n = 0, 1 , or 2], NC , CHO.

5 ; and R²¹ = H, OMe, F.

48. The fluorescent ion indicator of Claim 47, wherein R¹ = R² = R³ = R¹⁵ = R¹⁶ = R'⁷ = R¹⁸ = R¹⁹ = R²¹ = H; R²⁰ = Me; R^5a = (CH₂)„C0₂R⁴, n = 1 ; R^5b = (CH₂)„C0₂R⁴, n = 1 ; R²² = (CH₂)_nCH₃, n = 1 ; and R⁵' = the covalent bond R^so to the rosamine fiuorophore:

49. The fluorescent ion indicator of Claim 29, wherein the chelator is BAPTA and its derivatives:

R²⁴ = H. F, Br, CI

R²⁵ = H, Me, F, Br, CI, I, NO2, CHO, (CH₂)„C0₂R⁴ [n = 0, 1 , or 2], H,

R^SI = covalent bond to the rosamine fluorophore at R⁵⁰

50. The fluorescent ion indicator of Claim 49, wherein R¹ = R² = R³ = H; R^2S = e; R^5a = (CH₂)_nC0₂R⁴, n = I ; R^5b = (CH₂)_nC0₂R⁴. fluorophore at R⁵⁰:

51. The fluorescent ion indicator of Claim 29, wherein the chelator is APTRA and its derivatives:

R⁵¹ = covalent bond to the rosamine fluorophore at R^so.

52. The fluorescent ion indicator of Claim 51 , wherein R¹ = R² = R³ = H; R^5a = (CH₂)„C0₂R⁴, n = 1 ; R^5b = (CH₂)_nC0₂R⁴, n = 1 ; and R⁵¹ = covalent bond to the rosamine fluorophore at R⁵⁰:

53. The fluorescent ion indicator of Claim 29, wherein the chelator is Half-BAPTA and its derivatives:

R⁵¹ = covalent bond to the rosamine fluorophore at R⁵⁰

54. The fluorescent ion indicator of Claim 53, wherein R¹ = R² = R³ = H; R^5a

(CH2)nC02R⁴, n = 1 ; and R^{s l} = covalent bond to the rosamine fluorophore at R⁵⁰.