Fluorescent indicators for metal ion detection as substrates for a self-labeling enzyme

Abstract

The present disclosure describes fluorescent probes for cations. Such probes remain responsive to their corresponding analyte after binding to a self-labeling enzyme, thus allowing the detection of changes of metal ion concentration at particular intracellular locations to which the self-labeling enzyme has been expressed or in specific cells expressing the enzyme in a co-culture.

Description

FLUORESCENT INDICATORS FOR METAL ION DETECTION AS SUBSTRATES FOR A SELF-LABELING ENZYME TECHNICAL FIELD

[0001] The present invention is related to fluorescent indicators for ion detection, which are substrates for a self-labeling enzyme. BACKGROUND

[0002] Metal ions are essential to all forms of life since these ions are involved in critical processes such as metabolism and respiration. Sodium, potassium, calcium and magnesium play important roles in dynamic events including osmotic regulation and signaling. Since the early 80s, fluorescent indicators have been designed to sense dynamic pools of accessible metal ions in live cells (See Tsien, R. Y. A nondisruptive technique for loading calcium buffers and indicators into cells. Nature 290, 527 (1981)).

[0003] Small molecule probes are important tools for sensing ions in cells; however, the poor control over specific localization, both to cell type and to intracellular compartments, limits their applicability. Specific cell localization of fluorophores and fluorescent indicators can be achieved by derivatizing the small molecules into substrates for self-labeling enzymes such as HaloTag (See Los et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol.3, 373 (2008)). SUMMARY OF THE INVENTION

[0004] The present disclosure describes fluorescent indicators for cations, including thallium, calcium, potassium, and sodium. Such indicators remain responsive to their corresponding analyte after binding to a self-labeling enzyme, thus allowing the detection of changes of metal ion concentration at particular intracellular locations to which the self-labeling enzyme has been expressed or in specific cells expressing the enzyme in a co-culture. In the case of thallium, the fluorescent probes of the present invention are believed to be the first of their kind to be reported.

[0005] In one embodiment, a HaloTag ligand can be appended to xanthene-based fluorescent indicators to generate targetable probes for various metal ions. Chemically reactive handles introduced at various positions enable functionalization to proteins (e.g. the HALOTAG® ligand) without disabling the fluorescence-based detection of the analyte.

[0006] Fluorescent indicators selective for thallium ions include a compound having the chemical structure (I) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (I) can exist in the following tautomeric forms:.

[0007] Fluorescent indicators selective for thallium ions include a compound having the chemical structure (II) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; where R11is H, an alkyl group, or Ac; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;Linker is a linking group; and L represents a coupling agent; wherein, when R1is CO2R’, the compound (II) can exist in the following tautomeric forms:.

[0008] Fluorescent indicators selective for thallium ions include a compound having the chemical structure (III) or a salt thereof:where:each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; R11 is H, an alkyl group, or Ac; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (III) can exist in the following tautomeric forms:.

[0009] Fluorescent indicators selective for calcium ions include a compound having the chemical structure (IVA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation;R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; each R12independently represents H, Me, halogen, or NO2; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (IVA) can exist in the following tautomeric forms:.

[0010] The terms “heterocycle” and “heterocyclic group” as used herein generally refers to a closed-ring structure, in which one or more of the atoms in the ring is an element other than carbon. Heterocycles and heterocyclic groups may include aromatic compounds or non-aromatic compounds.

[0011] Fluorescent indicators selective for calcium ions include a compound having the chemical structure (IVB) or a salt thereof:(IVB) where:each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (IVB) can exist in the following tautomeric forms:.

[0012] Fluorescent indicators selective for calcium ions include a compound having the chemical structure (VA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl;R11is H, an alkyl group, or Ac; R12represents H, Me, halogen, or NO2; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (VA) can exist in the following tautomeric forms:.

[0013] Fluorescent indicators selective for calcium ions include a compound having the chemical structure (VB) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cationeach R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (VB) can exist in the following tautomeric forms:.

[0014] Fluorescent indicators selective for calcium ions include a compound having the chemical structure (VIA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R12represents H, Me, halogen, or NO2;each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (VIA) can exist in the following tautomeric forms:L.

[0015] Fluorescent indicators selective for calcium ions include a compound having the chemical structure (VIB) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (VIB) can exist in the following tautomeric forms: L.

[0016] Fluorescent indicators selective for sodium or potassium ions include a compound having the chemical structure (VII) or a salt thereof:Lwhere: R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R3independently represent H, F, or Cl; R4represents H, a counter cation, an alkyl, or an acyloxyalkyl group R6represents H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4 each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; each R independently represent H, Me, Et,–CH2OAc, or a counter cation; Linker1and Linker2are linking groups; “Na / K Chelator” is a chelator selective for sodium or potassium ions; and L represents a coupling agent.

[0017] In some embodiments the Linker of any of fluorescent indicators (I), (II), (III), (IV), (V), (VI) and (VII) include, but are not limited to: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group.

[0018] In some embodiments the Linker the of any of fluorescent indicators (I), (II), (III), (IV), (V), (VI) and (VII) include, but are not limited to: –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s–,where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; and where Y1is Het and Y2is –C(O)–, –NH–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, or –C(O)–NR11–.

[0019] In some embodiments the Het group the of any of fluorescent indicators (I), (II), (III), (IV), (V), (VI) and (VII) include, but are not limited to a 1,2,3-triazole heterocycle including, but not limited to:

[0020] In some embodiments the Het group the of any of fluorescent indicators (I), (II), (III), (IV), (V), (VI) and (VII) include, but are not limited to a pyridazine or dihydropyridazine heterocycle including, but not limited to:where R10is H or Me.

[0021] In some embodiments the coupling agent “L” of any of fluorescent indicators (I), (II), (III), (IV), (V), (VI) and (VII) is a ligand for a self-labeling enzyme. Exemplary ligands for a self-labeling enzyme include HALOTAG® ligands having the structure:, where t is 2-8 and u is 2 to 10. Specific examples of HALOTAG® ligands include, but are not limited to:.Other ligands for a self-labeling enzyme include benzylguanine or chloropyrimidine. Exemplary benzylguanine or chloropyrimidine ligands include, but are not limited to:. Other ligands for a self-labeling enzyme include benzylcytosine. An exemplary benzylcytosine ligand is:.

[0022] In some embodiments the coupling agent “L” of any of fluorescent indicators (I), (II), (III), (IV), (V), (VI) and (VII) is a group capable of reacting with a side chain of an amino acid. Exemplary ligands that can react with a side chain of an amino acid include, but are not limited to a maleimide or a N-hydroxysuccinimide.

[0023] In some embodiments the coupling agent “L” of any of fluorescent indicators (I), (II), (III), (IV), (V), (VI) and (VII) comprises a biotin a group capable of reacting with streptavidin to couple the fluorescent indicator to a protein or a solid substrate.

[0024] In some embodiments the coupling agent “L” of any of fluorescent indicators (I), (II), (III), (IV), (V), (VI) and (VII) comprises a chemical handle that is capable of reacting with a complementary chemical handle to form a heterocyclic group. Examples of chemical handles include, but are not limited to:.

[0025] In some embodiments the coupling agent “L” of any of fluorescent indicators (I), (II), (III), (IV), (V), (VI) and (VII) comprises a functional group capable of forming a bond with a complementary functional group on a biomolecule or a solid substrate. Examples of functional groups include, but are not limited to: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)– OR9, –C(O)–NHR11, –NR11–C(O)–OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

[0026] In some embodiments the sodium or potassium chelator of fluorescent indicators (VII) include a cryptand selective for sodium or potassium ions. Examples of cryptands selective for sodium or potassium ions include, but are not limited to:

[0027] In some embodiments the sodium or potassium chelator of fluorescent indicators (VII) is a crown ether or aza-crown ether selective for sodium or potassium ions. Examples of crown ethers selective for sodium or potassium ions include, but are not limited to, monoaza-15-crown- 5, monoaza-18-crown-6, diaza-15-crown-5, diaza-18-crown-6. Specific examples of crown ethers include, but are not limited to: ;R6is H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4; and R7is H or Me.

[0028] In another embodiment, a fluorescent probe includes a fluorescent indicator having the structure of any of general formulas (I), (II), (III), (IV), (V), (VI) and (VII) coupled to a biomolecule or a solid support. BRIEF DESCRIPTION OF THE FIGURES

[0029] Advantages of the present invention will become apparent to those skilled in the art with the benefit of the following detailed description of embodiments and upon reference to the accompanying drawings in which: FIG.1 shows the fluorescence polarization of compound I-2 and compound II-2 vs. control when exposed to HALOTAG® protein; FIG.2 shows the fluorescence polarization of compound IVA-7 and compound VIA-1 vs. control when exposed to HALOTAG® protein;FIG.3 shows sensitivity of compound I-2 and compound II-2 to thallium ions. FIG.4A and 4B shows the calcium sensitivity of compound IVA-7 and compound VIA-1. FIG.5 shows sensitivity of compound VII-2 to sodium ions. FIG.6 shows a comparison of relative fluorescence (F / F0) over time for HT and WT cells in the presence of compound IVA-7 ; and FIG.7 shows the potassium sensitivity of the biotinylated compound VII-11 vs a non- biotinylated control when coated on a slide using streptavidin.

[0030] While the invention may be susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. The drawings may not be to scale. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.DETAILED DESCRIPTION OF THE INVENTION

[0031] Described here is a fluorescent indicator for cations that can be coupled to a biomolecule and / or a solid substrate. The fluorescent indicator includes a fluorescence component, a metal ion chelating component, and a coupling agent that can interact with a biomolecule and / or a solid support to form an attachment between the fluorescent indicator and the biomolecule and / or the solid support.

[0032] The florescent indicators described herein are composed of a metal ion chelating component, a fluorescence component, and a coupling agent. The metal ion chelating component can chelate cations including, but not limited to, thallium, calcium, potassium, and sodium. The fluorescent indicators can be coupled to a biomolecule or a solid substrate and remain responsive to their corresponding metal ion while coupled to the biomolecule or the solid substrate. The metal ion chelating component in combination with the florescence component allows the detection of changes of metal ion concentration in the vicinity of where the indicator has been attached. For example, when the florescent indicator is coupled to a biomolecule, the fluorescent indicator can be used to monitor metal ion concentration at a particular intracellular location in which the biomolecule is present or in specific cells expressing the biomolecule in a co-culture. In the case of thallium, it is believed that the fluorescent probes described herein are the first of their kind to be reported.

[0033] In some embodiments, the fluorescent indicators are coupled to a self-labeling enzyme. Such enzyme can be fused to a protein of interest and thus the fusion can be localized at discrete locations inside cells. The fusion locates to the biological site where the fluorescent indicator can chelate metal ions and provide a fluorescent signal corresponding to the concentration of metal ions.

[0034] In an embodiment, a florescent indicator is provided with a coupling agent for a self- labeling enzyme. The fluorescent indicator can be coupled to the self-labeling enzyme through the coupling agent, according to the specific ligand that is chosen. In one embodiment, the ligand is an alkylhalide ligand which allows self-labeling of a haloalkane dehalogenase enzyme (e.g., using the HALOTAG® linking strategy). In some embodiments, the enzyme can be expressed as a fusion to a protein of interest, allowing this labeling technique to monitor a variety of biological processes at precise locations.

[0035] A self-labeling enzyme is an enzyme that can catalyze the covalent attachment of a compound to the enzyme through a ligand recognized and processed by the enzyme. Thecovalent attachment of the compound to the enzyme is highly specific, happens rapidly under physiological conditions in living cells, or in chemically fixed cells, and is irreversible. The three main self-labeling enzymes that are in current use include HALOTAG® (Promega), SNAP-tag and CLIP-tag (New England Biolabs). HaloTag is a haloalkane dehalogenase that reacts irreversibly with primary alkylhalide ligands. SNAP-tag is an O6-alkylguanine-DNA- alkyltransferase that reacts with O6-benzylguanine derivatives. CLIP-tag is an O2-alkylcytosine- DNA-alkyltransferase, reacts with O2-benzylcytosine derivatives. Compounds are attached to the self-labeling enzyme by reaction of a specific synthetic ligand that is covalently coupled to the compound. The self-labeling reactions are highly specific for the ligand and enable a variety of compounds to be coupled to the self-labeling enzyme. Further details about coupling compounds to self-labeling enzymes are described in U.S. Patent No.7,429,472 to Darzins et al., which is incorporated herein by reference.

[0036] In an embodiment, a ligand for a self-labeling enzyme can be introduced at discrete locations into xanthene-based ion indicators. The placement of the ligand does not prohibit the irreversible coupling catalyzed by the self-labeling enzyme, nor the fluorescent response to binding the metal ion (the analyte). Introduction of the ligand has little to no effect on the mechanism (photoinduced electron transfer, internal charge transfer, etc.) that governs the sensing ability of the sensors.

[0037] In some embodiments, the fluorescent indicators are tethered to a chemical handle. The chemical handle allows for the coupling of the fluorescent indicators to biomolecule (proteins, nucleic acids, sugars, etc.) or a solid substrate (e.g. formed from glass or a polymer). In one embodiment, the chemical handle is selected for coupling using click chemistry.

[0038] Fluorescent indicators, as described in some embodiments herein, have the general structures (FI-1) and (FI-2). Metal Ion Chelating Com onentAgent(FI-1) (FI-2) The fluorescent indicators are composed of three general components. The fluorescence component, the metal ion chelating component and the coupling agent. The metal ion couplingcomponent and the coupling agent are coupled to the fluorescence component either directly or through a linker. Fluorescence Component

[0039] The fluorescence component can be any compound that exhibits or changes fluorescence when a metal ion is bound to the metal ion chelating component. In a preferred embodiment, the fluorescence component is a xanthene fluorescence compound.

[0040] Xanthene fluorescence compounds useful in the present fluorescent indicators include fluorescence compounds having the general structure (FC-1):where: R1is H, Me, CO2R’, SO3R’; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl. In the structures set forth in this application the use of a dashed line [ ] in the structures denotes that the bond can be either a single bond or a double bond. Specific examples of xanthene fluorescence compounds include the compounds set forth in Table 1.TABLE 1

[0041] Many xanthene fluorescent compounds can exist in tautomeric forms. The specific tautomeric form adopted by the xanthene dye can depend on the solution conditions that the compound is exposed to (e.g., pH). For example, for fluorescein and similar xanthene fluorescent compound having a carboxylate group in the R1position (e.g., Oregon Green), the fluorescent compound can exist in either an open form (FC-2) or a spirolactone form (FC-3)..

[0042] Xanthene fluorescent compounds having an amine group in the W1and / or the W2position, such as rhodamine B, and a carboxylate group in the R1position, can exist in either an open form (FC-4), (FC-6) or a spirolactone form (FC-5), (FC-7).Throughout the specification, for readability and efficiency of describing the fluorescent indicators, only one of the various tautomeric chemical structures will be displayed. However, it should be understood that the non-displayed tautomeric chemical structure is also encompassed by the displayed chemical structure. Metal Ion Chelating Component

[0043] Fluorescence, from the fluorescent compound, involves the emission of photons that occurs after an absorption event. The fluorescent indicators described herein include a metal ion chelating component. The fluorescent indicator can function as a metal ion sensor when metal binding to the metal ion chelating compound, for example, alters the electronic structure or the molecular structure of the sensor. Changes in the electronic structure can lead to a change in the intensity or wavelength of light absorption or emission. In the compounds disclosed herein, the metal ion chelating compound is attached to the fluorescent compound such that the fluorescence of the fluorescent compound is changed in the presence of the appropriate metal ion. In preferred embodiments, the metal ion chelating component can bind to thallium, calcium, sodium or potassium. Thallium Ion Chelating Component

[0044] Examples of groups capable of chelating thallium cations include derivatives of 2,2'-((2- alkoxyphenyl)azanediyl)diacetic acid and their cell-permeable, esterase-hydrolyzable ester forms, preferably methyl, ethyl, and acyloxyalkyl ester derivatives (e.g., acetoxymethyl). An example of a thallium ion chelating compound is depicted as (T-1).Each R of (T-1) can independently represent H, Me, Et, CH2OAc, or a counter cation. R’ is typically methyl. However, it has been surprisingly discovered that the thallium ion chelating component retains the ability to bind to thallium ions when R’ is a methylene (CH2) group which is used to connect the coupling agent to the chelating component (as shown schematically in structure (FI-2)). Calcium Ion Chelating Component

[0045] Calcium ion chelating groups include derivatives of 1,2-bis(o-aminophenoxy)ethane- N,N,N’-N’-tetraacetic acid and 5-methyl 1,2-bis(o-aminophenoxy)ethane-N,N,N’-N’-tetraacetic acid and their cell-permeable, esterase-hydrolyzable ester forms, with methyl, ethyl, and acyloxyalkyl ester (e.g., acetoxymethyl) derivatives preferred. Examples of calcium ion chelating components that are suitable for use in the described fluorescent indicators are described in U.S. Patent Application Publication No.2012 / 0183986 and U.S. Patent No. 9,810,700, both of which are incorporated herein by reference.

[0046] Embodiments of a calcium ion chelating compound have the structures (CA-1) or (CA- 2).(CA-1) (CA-2) where each R independently represent H, Me, Et,–CH2OAc, or a counter cation; and R12represents H, halogen, or Me. Sodium and Potassium Ion Chelating Components

[0047] Sodium and potassium ion chelating groups include cryptands and crown ethers that are specific for sodium or potassium ions. Additional sodium and potassium ion chelating groups include ethylene glycol tetracetic acid and 2,2'-((2-(2-(2-(bis(carboxymethyl)amino)ethoxy) ethoxy)phenyl)-azanediyl)diacetic acid, and ester derivates thereof.

[0048] Cryptand sodium and potassium ion chelating groups include cryptands having the structures (C-1), (C-2), (C-3), or (C-4).

[0049] Additional sodium and potassium ion chelating groups include crown ethers and aza- crown ethers. Exemplary crown ethers and aza-crown ethers that can be coupled to the fluorescent indicator include, but are not limited to, monoaza-15-crown-5, monoaza-18-crown-6, diaza-15-crown-5, diaza-18-crown-6. Crown ethers and aza-crown ethers can be tailored to a specific metal cation (e.g., sodium or potassium) by altering the size of the crown ether. Exemplary crown ether sodium or potassium ion chelating groups include crown ethers having the structures (C-5), (C-6), or (C-7) ;R6is H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4; and R7is H or Me. Coupling Agent

[0050] The coupling agent represents a means for connecting the fluorescent compound / metal ion chelating component to a biomolecule or a solid substrate. The term “biomolecule” encompasses polypeptides, polynucleotides, and polysaccharides. Exemplary biomolecules include proteins, enzymes, antibodies, DNA and RNA. Solid substrates can include glass or polymeric substrates.

[0051] A “coupling agent” is a functional group that can be used to couple the indicator / chelating agent to a biomolecule or a solid substrate. For example, a coupling agent can include a functional group capable of reacting with a protein or polypeptide, or a complementary functional group attached to the protein or polypeptide, to couple the protein or polypeptide to the indicator / chelating agent. Alternatively, the coupling agent can include a functional group capable of reacting with a complementary functional group on a solid substrate (e.g., a polymeric substrate) to couple the indicator / chelating agent to the solid substrate.

[0052] In one embodiment, the coupling agent (L) represents a ligand for a self-labeling enzyme. The introduction of a ligand for a self-labeling enzyme enables the precise localization of the fluorescent indicators in cells. Examples of self-labeling enzymes include HALOTAG® ligands, SNAP-tag ligands, and CLIP-tag ligands.

[0053] HALOTAG® ligands have the general structure:, where t is 2-8 and u is 2 to 10. Exemplary HALOTAG® ligands include [2-(2-((6- chlorohexyl)oxy)ethoxy)ethanamino group (Amine O2chloroalkane) and 21-chloro-3,6,9,12,15- pentaoxahenicosan-1-amino group (Amine O4chloroalkane)].e O4chloroalkane).

[0054] SNAP-tag ligands include benzylguanine and chloropyrimidine derivatives. Exemplary SNAP-tag ligands include:(benzylguanine) (chloropyrimidine).

[0055] CLIP-tag ligands include benzylcitosine derivatives. The benzylcitosine ligand is typically attached to the coupling group, –C(O)–, as an amide.(benzylcytosine).

[0056] An alternate method of binding the fluorescent compound / metal ion chelating component onto a biomolecule is through the use of a covalent bond. A number of covalent bond forming ligands can be used to couple the fluorescent compound / metal ion chelating component to a biomolecule. In an embodiment, the coupling agent (L) is a maleimide group. Maleimide ligands have the general structure:Maleimide groups react with thiol groups of a polypeptide(e.g. a protein) to form a covalent bond linking the polypeptide to the fluorescent compound / metal ion chelating component, as shown in the reaction scheme below:

[0057] In an embodiment, the coupling agent (L) is an N-hydroxysuccinimide group (NHS). NHS ligands have the general structure:NHS groups activate the adjacent carbonyl coupling group to allow amine groups of a polypeptide(e.g. a protein) to form a covalent bond (an amide) linking the polypeptide to the fluorescent compound / metal ion chelating component, as shown in the reaction scheme below:

[0058] In an embodiment, coupling agent (L) is an alkyne group or an azido group. The copper(I) catalyzed azide alkyne cycloaddition reaction (CuAAC) between an alkyne group and an azide forms a 1,4-disubstituted 1,2,3-triazole, which can be used to couple the fluorescent compound / metal ion chelating component to the biomolecule or other compounds. The biomolecule (e.g., a protein) and the fluorescent compound / metal ion chelating component have complementary groups, azide and alkyne, attached which react to form a disubstituted 1,2,3- triazole. Exemplary coupling schemes based on azide-alkyne cycloaddition reaction are shown below. In one embodiment, the alkyne group is coupled to the fluorescent compound / metal ion chelating component and an azido group is coupled to the biomolecule (protein). In an alternate embodiment, the azido group is coupled to the fluorescent compound / metal ion chelating component and the alkyne group is coupled to the biomolecule (protein). Ruthenium catalyzed azide-alkyne cycloaddition can also be used as a coupling scheme with the only difference being the generation of the isomeric 1,5-disubstituted 1,2,3-triazole.(1) The 1,4-disubstituted 1,2,3-triazole forms a stable covalent attachment between the fluorescent compound / metal ion chelating component and the biological macromolecule, as shown in reaction scheme (1).

[0059] In an embodiment, coupling agent (L) comprises a biotin group:Biotin is a small hapten molecule with high binding affinity for streptavidin. The streptavidin- biotin complex is one of the strongest non-covalent interactions between a protein and its ligand. This strong binding can be used to couple the fluorescent compound / metal ion chelating component to a biomolecule through streptavidin which is coupled to the biomolecule.

[0060] Click chemistry can also be used to couple the fluorescent compound / metal ion chelating component to a biomolecule or a solid substrate. Click chemistry uses the formation of a heterocycle from highly reactive precursors coupled to the fluorescent compound / metal ion chelating component and the biomolecule or solid substrate.

[0061] Heterocycle groups can be synthesized by reaction of complementary chemical handle groups (T, T’) coupled to the fluorescent compound / metal ion chelating component and the biomolecule. Alternatively, as depicted in the reaction scheme below, a chemical handle (T’) can be coupled to a linker (X) of a coupling agent (L) (e.g., a ligand for a self-labeling enzyme)where T and T’ combine to form the Het group. Examples of chemical handles (T, T’) include, but are not limited to:

[0062] The click chemistry reaction forms a heterocycle ring that couples the fluorescent compound / metal ion chelating component to the biomolecule. In one embodiment, a 1,2,3- triazole heterocycle is formed by the reaction of an azido group with an ethynyl group, a cyclooctyne, a cyclopropylcyclooctyne, or a dibenzoazacyclooctyne. Exemplary 1,2,3-triazole heterocyles formed using click chemistry include, but are not limited to:. In other embodiments, a pyridazine or dihydropyridazine heterocycle is formed by the reaction of a tetrazine (e.g., 1,2,4,5-tetrazine; and 6-methyl-1,2,4,5-tetrazine) with a strained alkene or alkyne (e.g., norbornene, (methyl)cyclopropene, cyclopropyloctyne, and trans-cyclooctene). Exemplary pyridazine or dihydropyridazine heterocycles formed using click chemistry include, but are not limited to:where R10is H or Me. In some embodiments, the coupling agent is a functional group capable of reacting with a complementary functional group on the biomolecule or solid substrate to form a covalent bond linking the biomolecule or solid substrate to the fluorescent compound / metal ion chelating component. Exemplary reactive functional groups include, but are not limited to: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)–OR9, or –NR11– C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac. Linking Groups

[0063] Linking groups, designated as “Linker” in the structures, can, optionally, be used to connect the various components of the fluorescent indicator together. A “linking group” describes one or more atoms that directly links components of the fluorescent indicator. Linkinggroups can be used to: connect the metal ion chelating component to the fluorescent compound; connect the coupling agent to the fluorescent compound; or connect the coupling agent to the metal ion chelating compounds. Linking groups can be used to create space between the components and / or provide chemical handles to facilitate the formation of chemical bonds linking the components of the fluorescent indicator.

[0064] A “linking group” describes one or more atoms that directly links components of the fluorescent indicator. A linking group is interposed between the indicator / chelating agent and the coupling agent to create a link that allows the indicator / chelating agent to be spaced art from a biomolecule or a polymeric support.

[0065] Exemplary linking groups include, but are not limited to: –(CH2)m–, –(CH2)m–Y1– (CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group. Methods of Detecting Ions

[0066] Generally, the fluorescent indicator compounds described herein can be used to determine the presence and / or concentration of metal ions in a sample. In one embodiment, the fluorescent indicator compounds described herein can be used to monitor metal ions in a biological system. In this method, the fluorescent indicator compound is coupled to a biomolecule through the coupling agent. The tagged biomolecule is then used to monitor the presence and / or concentration of metal ions in a sample.

[0067] In another embodiment, the fluorescent indicator compounds described herein can be used to monitor metal ions in a sample using a solid substrate. In this method, the fluorescent indicator compound is coupled to a solid substrate through the coupling agent. The tagged solid substrate is then used to monitor the presence and / or concentration of metal ions in a sample. Thallium Specific Fluorescent Indicators

[0068] Thallium specific fluorescent indicators, as described in some embodiments herein, have the general structures (FI-3) and (FI-4). Thallium Ion Chelating ComponentCoupling AgentThe thallium specific fluorescent indicators are composed of three general components. The fluorescence component, the thallium ion chelating component and the coupling agent. The thallium ion chelating component and the coupling agent are coupled to the fluorescence component either directly or through a linker.

[0069] In one embodiment, a thallium specific fluorescent indicator has the structure (I) [where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;O U represents or ; Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:; where R10is H or Me; L represents a ligand for coupling to a protein selected from the group consisting of:,where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting ofL represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (I) can exist in the following tautomeric forms:

[0071] Structure (I) can exist in two isomeric forms (Ia) and (Ib).

[0072] A specific example of the compound of Structure (I) includes compounds having the structure (IA):wherein each X1and X2independently represent: a bond, –(CH2)m–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–O–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–NH–, –NH–(CH2)m–(CH2–CH2–O)p–(CH2)n–, –O–(CH2)m–(CH2–CH2–O)p–(CH2)n–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–O–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–NH–, –NH–(CH2)m–(O–CH2–CH2)p–(CH2)n–, or –O–(CH2)m–(O–CH2–CH2)p–(CH2)n–; and where m is 0-4; n is 0-4; and p is 0-4 further, when R1is CO2R’, the compound (I) can exist in the following tautomeric forms:.

[0073] Specific examples of compound (I) include, but are not limited to:;;;W;

[0074] The compounds encompassed by chemical structure (I) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate.

[0075] In one embodiment, a thallium specific fluorescent indicator has the structure (II)where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; where R11is H, an alkyl group, or Ac; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; O U represents or ; Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; and Het represents a heterocyclic group selected from the group consisting of:where R10is H or Me; L represents a ligand for coupling to a protein selected from the group consisting of: l ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (II) can exist in the following tautomeric forms:.

[0076] A specific example of the compound of Structure (II) includes compounds having the structure (IIA):wherein each X1and X2independently represent: a bond, –(CH2)m–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–O–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–NH–, –NH–(CH2)m–(CH2–CH2–O)p–(CH2)n–, –O–(CH2)m–(CH2–CH2–O)p–(CH2)n–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–O–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–NH–, –NH–(CH2)m–(O–CH2–CH2)p–(CH2)n–, or –O–(CH2)m–(O–CH2–CH2)p–(CH2)n–; and where m is 0-4; n is 0-4; and p is 0-4 wherein, when R1is CO2R’, the compound (IIA) can exist in the following tautomeric forms:.

[0077] Specific examples of compound (II) include, but are not limited to:

[0078] The compounds encompassed by chemical structure (II) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate.

[0079] In one embodiment, a thallium specific fluorescent indicator has the structure (III)where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;where Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; R11 is H, an alkyl group, or Ac; Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: ,where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (III) can exist in the following tautomeric forms:.

[0080] A specific example of the compound of Structure (III) includes a compound having the structure (III-1):

[0081] The compounds encompassed by chemical structure (III) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate. Calcium Specific Fluorescent Indicators

[0082] Calcium specific fluorescent indicators, as described in some embodiments herein, have the general structures (FI-5).The calcium specific fluorescent indicators are composed of three general components. The fluorescence component, the calcium ion chelating component and the coupling agent. The calcium ion chelating component and the coupling agent are coupled to the fluorescence component either directly or through a linker.

[0083] In one embodiment, a calcium specific fluorescent indicator has the structure (IVA)where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; R12represents H, Me, halogen, or NO2; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: l ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (IVA) can exist in the following tautomeric forms:.

[0084] Specific examples of compound (IVA) include, but are not limited to:AA

[0085] The compounds encompassed by chemical structure (IVA) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate.

[0086] In one embodiment, a calcium specific fluorescent indicator has the structure (IVB)where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents: ,where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (IVB) can exist in the following tautomeric forms:.

[0087] The compounds encompassed by chemical structure (IVB) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate.

[0088] In one embodiment, a calcium specific fluorescent indicator has the structure (VA):where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; R12represents H, Me, halogen, or NO2; Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: l ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (VA) can exist in the following tautomeric forms:.

[0089] Specific examples of compound (VA) include, but are not limited to:A

[0090] The compounds encompassed by chemical structure (VA) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate.

[0091] In one embodiment, a calcium specific fluorescent indicator has the structure (VB)where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–,where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (VB) can exist in the following tautomeric forms:.

[0092] The compounds encompassed by chemical structure (VB) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate.

[0093] In one embodiment, a calcium specific fluorescent indicator has the structure (VIA)where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R12represents H, Me, halogen, or NO2; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: l ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (VIA) can exist in the following tautomeric forms:.

[0094] Specific examples of compound (VIA) include, but are not limited to:.

[0095] The compounds encompassed by chemical structure (VIA) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate.

[0096] In one embodiment, a calcium specific fluorescent indicator has the structure (VIB) Lwhere: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4;each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (VIB) can exist in the following tautomeric forms:.

[0097] The compounds encompassed by chemical structure (VIB) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate. Sodium / Potassium Fluorescent Indicators

[0098] Sodium and potassium specific fluorescent indicators, as described in some embodiments herein, have the general structures (FI-6).The sodium and potassium specific fluorescent indicators are composed of three general components. The fluorescence component, the sodium and potassium ion chelating component and the coupling agent. The sodium and potassium ion chelating component and the coupling agent are coupled to the fluorescence component either directly or through a linker.

[0099] In one embodiment, a sodium and potassium specific fluorescent indicator has the structure (VII) Lwhere: R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R3independently represent H, F, or Cl; R4represents H, a counter cation, an alkyl, or an acyloxyalkyl group R6represents H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4 each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; each R independently represent H, Me, Et,–CH2OAc, or a counter cation;where “Na / K Chelator” is a chelator selective for sodium or potassium ions, wherein the chelator is selected from the group consisting of ;R6is H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4; and R7is H or Me; Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac;Het represents a heterocyclic group selected from the group consisting of:; where R10 is H or Me; L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

[0100] Specific examples of compound (VII) include, but are not limited to:(VII-1);

[0101] The compounds encompassed by chemical structure (VII) can be used as a fluorescent probe when coupled to a biological macromolecule or solid substrate.

[0102] The compounds represented by the formulas (I), (II), (III), (IV), (V), (VI), and (VII) can also exist as salts. Examples of salts include hydrochloride, sulfate, nitrate, methanesulfonate, etc. Examples of metal salts include sodium, potassium, calcium, magnesium. Examples of organic salts include ammonium and triethylamine. The compounds presented here may exist as a hydrate or a solvate.

[0103] Methods of preparing representative compounds are specifically illustrated in Examples herein. Therefore, those skilled in the art can adapt the methods here described to prepare any compound represented by the formulas (I), (II), (III), (IV), (V), (VI), and (VII).

[0104] The compounds represented by the formulas (I), (II), (III), (IV), (V), (VI), and (VII) of the present invention are useful as fluorescent indicators to detect ion analytes.

[0105] Another aspect of the present invention is a fluorescent indicator including a compound represented by the formulas (I), (II), (III), (IV), (V), (VI), and (VII) or a salt thereof.

[0106] The compounds represented by the formulas (I), (II), (III), (IV), (V), (VI), and (VII) or their salts thereof according to the present invention undergo a change in fluorescence intensity and / or fluorescence lifetime once they are bound to the corresponding metal ion. Accordingly, the compounds of the present invention represented by the formulas (I), (II), (III), (IV), (V), (VI), and (VII) or salts thereof are useful for the detection of metal ions (e.g., thallium, calcium, sodium, and potassium ions) in cells.EXAMPLES

[0107] Hereinafter, the present invention will be described with examples, however it is not limited thereto. Example 1. Synthesis of bis(acetoxymethyl) 2,2'-((2-((1-(2-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-((3',6'- diacetoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5- yl)carbamoyl)phenyl)azanediyl)diacetate (compound I-2).

[0108] Compound I-2, one of the compounds of the present invention, was synthesized according to the procedures depicted in the following reaction scheme.Example 2. Synthesis of bis(acetoxymethyl) 2,2'-((2-((1-(21-chloro-8-oxo-3,6,12,15-tetraoxa- 9-azahenicosyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-((3',6'-diacetoxy-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthen]-5-yl)carbamoyl)phenyl)azanediyl)diacetate (compound 14).

[0109] Compound 14, one of the compounds of the present invention, was synthesized according to the procedures depicted in the following reaction scheme.Example 3. Synthesis of bis(acetoxymethyl) 2,2'-((4-((3',6'-diacetoxy-4'-((((1-(2-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4- yl)methyl)(methyl)amino)methyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5- yl)carbamoyl)-2-methoxyphenyl)azanediyl)diacetate (compound 19)

[0110] Compound 19, one of the compounds of the present invention, was synthesized according to the procedures depicted in the following reaction scheme.Example 4. Synthesis of bis(acetoxymethyl) 2,2'-((4-((3',6'-diacetoxy-4'-((((1-(21-chloro-8- oxo-3,6,12,15-tetraoxa-9-azahenicosyl)-1H-1,2,3-triazol-4- yl)methyl)(methyl)amino)methyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5- yl)carbamoyl)-2-methoxyphenyl)azanediyl)diacetate (compound 21)

[0111] Compound 21, one of the compounds of the present invention, was synthesized according to the procedures depicted in the following reaction scheme.Example 5. Synthesis of bis(acetoxymethyl) 2,2'-((4-((3',6'-diacetoxy-4'-(18-chloro-2- methyl-5-oxo-9,12-dioxa-2,6-diazaoctadecyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]- 5-yl)carbamoyl)-2-methoxyphenyl)azanediyl)diacetate (compound 27)

[0112] Compound 27, one of the compounds of the present invention, was synthesized according to the procedures depicted in the following reaction scheme.Example 6. Synthesis of bis(acetoxymethyl) 2,2'-((4-(3-(((3',6'-diacetoxy-5-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-4'- yl)methyl)(methyl)amino)propanamido)-2-methoxyphenyl)azanediyl)diacetate (compound 32)

[0113] Compound 32, one of the compounds of the present invention, was synthesized according to the procedures depicted in the following reaction scheme.Example 7. Synthesis of bis(acetoxymethyl) 2,2'-((4-(6-(acetoxymethoxy)-5-(2-acetyl-18- chloro-5-oxo-9,12-dioxa-2,6-diazaoctadecyl)-2,7-difluoro-3-oxo-3H-xanthen-9-yl)-2-(2-(2- (bis(2-(acetoxymethoxy)-2-oxoethyl)amino)-5- methylphenoxy)ethoxy)phenyl)azanediyl)diacetate and bis(acetoxymethyl) 2,2'-((4-(6- (acetoxymethoxy)-4-(2-acetyl-18-chloro-5-oxo-9,12-dioxa-2,6-diazaoctadecyl)-2,7-difluoro- 3-oxo-3H-xanthen-9-yl)-2-(2-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)-5- methylphenoxy)ethoxy)phenyl)azanediyl)diacetate (compounds 36-1 and 36-2).

[0114] Compounds 36-1 and 36-2 were synthesized according to the procedures depicted in the following reaction scheme.Example 8: One-pot synthesis of according to the following reaction scheme: a) 1 eq. Ac2O, 8 eq DIPEA, DMF, RT, 30 min; b) Amino (2O) chloroalkane halo ligand, RT, 30 min; and c) AMBr, RT, 30 min.Synthesis of 1-nitro-2-(prop-2-yn-1-yloxy)benzene (compound 1)

[0115] To a round bottom flask were added 2-nitrophenol (0.500 g, 3.594 mmol, 1.0 eq.), dry DMF (5 mL) and potassium carbonate (0.993 g, 7.189 mmol, 2.0 eq.). Then, the resulting mixture was stirred at rt for 15 min. Next, propargyl bromide (0.51 mL, 4.673 mmol, 1.3 eq.) was added and the mixture was stirred at 65 °C for 45 min. upon cooling to rt, the crude mixture was diluted with EtOAc and the heterogenous mixture was decanted. The solution was put into a sep funnel and washed with water. The aqueous phase was back extracted with EtOAc and the combined organic layers were washed with brine. The organic phase was then dried over Na2SO4, filtered and concentrated in vacuo to give a yellow solid, 0.600 g (94% crude yield). Synthesis of 2-(prop-2-yn-1-yloxy)aniline (compound 2)

[0116] To a solution of compound 1 (0.600 g, 3.387 mmol, 1.0 eq.) in EtOH (18 mL) were added 6 M HCl (5.6 mL, 0.034 mol, 10.0 eq.) and tin (0.905 g, 7.620 mmol, 2.3 eq.). The resulting heterogenous mixture was then stirred at reflux for 2 h. After cooling to rt, the crude mixture was poured into icy water and the pH was adjusted to around 9 by adding aqueous KOH. The mixture was extracted three times with CHCl3. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. Analysis by MS of the crude mixture showed the desired compound constituted 60% of the mass. The crude mixture was used in the next step without any purification. Synthesis of dimethyl 2,2'-((2-(prop-2-yn-1-yloxy)phenyl)azanediyl)diacetate (compound 3)

[0117] Compound 2 (0.300 g, 2.038 mmol, 1.0 eq.), proton sponge (1.136 g, 5.300 mmol, 2.6 eq.) and NaI (0.229 g, 1.523 mmol, 0.8 eq.) were added to a heavy wall pressure vessel. Then, dry CH3CN (1.5 mL) and methyl 2-bromoacetate (0.58 mL, 6.115 mmol, 3.0 eq.) were addedand the reaction mixture was stirred overnight at 95 °C. After cooling to rt, the crude mixture was diluted with CH3CN and filtered off. The filtrate was concentrated in vacuo. Then, the residue was dissolved with a mixture of CHCl3and MeOH, adsorbed on silica and chromatographed (SiO2, 2:1 Hex:EtOAc). Compound 3 was recovered as yellow solid, 0.535 g (90% isolated yield). LRMS (ESI) calcd from C15H18NO5[M+H]+292.12, found 292.30. Synthesis of dimethyl 2,2'-((4-nitro-2-(prop-2-yn-1-yloxy)phenyl)azanediyl)diacetate (compound 4)

[0118] To a vial containing compound 3 (0.535 g, 1.837 mmol, 1.0 eq.) was added glacial acetic acid (80 uL, 1,377 mmol, 0.8 eq.). Then, the mixture was cooled down in an ice bath and nitric acid (90 uL, 1.377 mmol, 0.8 eq.) was added dropwise. The reaction mixture was stirred at 0-5 °C for 30 min. Next, the crude mixture was diluted with EtOAc and washed with saturated NaHCO3 aqueous solution. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved with CHCl3, adsorbed on silica and chromatographed (SiO2, 2:1 Hex:EtOAc) to give a yellow solid (0.288 g, 51% isolated yield). LRMS (ESI) calcd from C15H17N2O7[M+H]+337.10, found 337.20. Synthesis of 2,2'-((4-nitro-2-(prop-2-yn-1-yloxy)phenyl)azanediyl)diacetic acid (compound 5)

[0119] To a solution of compound 4 (0.055 g, 0.164 mmol, 1.0 eq.) in a mixture of MeOH (1.5 mL) and 1,4-dioxane (1.5 mL) was added an aqueous solution (1.5 mL) of KOH (0.092 g, 1.635 mmol, 10.0 eq.). The reaction mixture was stirred at rt overnight. To the crude mixture were added 1 M HCl (5 mL) and brine, then the mixture was extracted twice with CHCl3. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give a yellow solid (full conversion by MS). LRMS (ESI) calcd from C13H13N2O7[M+H]+309.07, found 309.10. Synthesis of bis(acetoxymethyl) 2,2'-((4-nitro-2-(prop-2-yn-1- yloxy)phenyl)azanediyl)diacetate (compound 6)

[0120] A solution of compound 5 (0.045 g, 0.146 mmol, 1.0 eq.) and DIPEA (0.25 mL, 1.022 mmol, 7.0 eq.) in dry DCM (1 mL) was cooled down in an ice bath. Then, bromomethyl acetate (60 uL, 0.584 mmol, 4.0 eq) was added dropwise and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved with CHCl3and applied to a preparatory TLC plate, using 1:1 Hex:EtOAc as mobile phase.Compound 6 was recovered as a yellow solid, 0.035 g (53% isolated yield). LRMS (ESI) calcd from C19H21N2O11 [M+H]+453.11, found 453.40. Synthesis of 2-(4-((2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)-5-nitrophenoxy)methyl)- 1H-1,2,3-triazol-1-yl)acetic acid (compound 7)

[0121] Compound 6 (0.044 g, 0.097 mmol, 1.0 eq.), azidoacetic acid (0.020 g, 0.195 mmol, 2.0 eq.) and CuI (0.002 g, 0.010 mmol, 0.1 eq.) were added to a Schlenk flask, then the flask was placed under vacuum. Next, the flask was back-filled with Ar using a balloon. THF (1 mL) and DIPEA (0.03 mL, 0.165 mmol, 1.7 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was then diluted with CHCl3and washed with brine two times. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved with CHCl3and filtered through a PTFE syringe filter. The filtrate was concentrated in vacuo to give a yellow solid, 0.040 g (74% crude yield). The crude mixture was used in the next step without any purification. LRMS (ESI) calcd from C21H24N5O13 [M+H]+554.14, found 554.40. Synthesis of bis(acetoxymethyl) 2,2'-((2-((1-(2-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-4- nitrophenyl)azanediyl)diacetate (compound 8)

[0122] Compound 7 (0.040 g, 0.072 mmol, 1.0 eq.), 2-(2-((6- chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (0.019 g, 0.072 mmol, 1.0 eq.) and HATU (0.027 g, 0.072 mmol, 1.0 eq.) were added to a Schlenk flask and the flask was connected to a vacuum line. Then, the flask was back-filled with inert atmosphere using an Ar-filled balloon. Dry DMF (2 mL) and DIPEA (0.06 mL, 0.325 mmol, 4.5 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed twice with brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved with CHCl3and the crude mixture was subjected to purification via preparatory TLC using EtOAc as eluent. Compound 8 was obtained as a yellow solid, 0.038 g (69% isolated yield). LRMS (ESI) calcd from C31H44ClN6O14[M+H]+759.26, found 759.50. Synthesis of bis(acetoxymethyl) 2,2'-((4-amino-2-((1-(2-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4- yl)methoxy)phenyl)azanediyl)diacetate (compound 9)

[0123] To a solution of compound 8 (0.030 g, 0.040 mmol) in DCM (3 mL) was added Pd / C (0.030 g). Then, the mixture was bubbled with H2using a balloon. The reaction mixture was stirred at rt for 2 h. Next, the crude mixture was diluted with CHCl3and filtered through a PTFEsyringe filter. The filtrate was concentrated in vacuo to give a pale yellow solid (full conversion by MS). LRMS (ESI) calcd from C31H46ClN6O12 [M+H]+729.29, found 729.60. Synthesis of bis(acetoxymethyl) 2,2'-((2-((1-(2-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-((3',6'- diacetoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5- yl)carbamoyl)phenyl)azanediyl)diacetate (compound 10)

[0124] Compound 9 (0.026 g, 0.036 mmol, 1.0 eq.), 5-FAM diacetate (0.016 g, 0.036 mmol, 1.0 eq.) and HATU (0.014 g, 0.036 mmol, 1.0 eq.) were placed in a Schlenk flask. Then the flask was connected to a vacuum line and back-filled with Ar using a balloon. Dry DMF (2 mL) and DIPEA (0.02 mL, 0.125 mmol, 3.5 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed two times with brine. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The desired product was purified by reverse phase preparatory HPLC (10-95% CH3CN / H2O, linear gradient). Compound 10 was recovered as a white-off solid (0.018 g, 43% isolated yield). LRMS (ESI) calcd from C56H60ClN6O20[M+H]+1171.75, found 1171.70. Synthesis of 2-(2-(2-azidoethoxy)ethoxy)-N-(2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)acetamide (compound 11)

[0125] Compound 11. HATU (0.201 g, 0.529 mmol, 1.0 eq.), 2-[2-(2-azidoethoxy)ethoxy]acetic acid (0.100 g, 0.529 mmol, 1.0 eq.) and .), 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (0.138 g, 0.529 mmol, 1.0 eq.) were added to a Schlenk flask and the flask was connected to a vacuum line. Then, the flask was back-filled with Ar using a balloon. Dry DMF (4.5 mL) and DIPEA were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed two times with brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved with CHCl3, adsorbed on silica and chromatographed (SiO2, 6% MeOH in CHCl3) to give a yellow oil, 0.162 g (78% isolated yield). LRMS (ESI) calcd from C16H32ClN4O5 [M+H]+395.21, found 395.30. Synthesis of bis(acetoxymethyl) 2,2'-((2-((1-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9- azahenicosyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-nitrophenyl)azanediyl)diacetate (compound 12)

[0126] Compound 6 (0.035 g, 0.077 mmol, 1.0 eq.), compound 11 (0.061 g, 0.155 mmol, 2.0 eq.) and CuI (0.001 g, 0.008 mmol, 0.1 eq.) were put into a Schlenk flask. Then, the flask was placed under vacuum and back-filled with Ar using a balloon. THF (1 mL) and DIPEA were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted withCHCl3and washed with brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved with CHCl3and applied to a preparatory TLC plate, using 6% MeOH in CHCl3as mobile phase. Compound 12 was obtained as a yellow solid, 0.052 g (79% isolated yield). LRMS (ESI) calcd from C35H52ClN6O16 [M+H]+847.31, found 847.60. Synthesis of bis(acetoxymethyl) 2,2'-((4-amino-2-((1-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9- azahenicosyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)azanediyl)diacetate (compound 13)

[0127] To a solution of compound 12 (0.044 g, 0.051 mmol) in DCM (4 mL) was added Pd / C (0.044 g). Then, the mixture was bubbled with H2 using a balloon. The reaction mixture was stirred at rt for 2 h. Next, the crude mixture was filtered through a PTFE syringe filter. The filtrate was concentrated in vacuo to give a pale yellow oil (quantitative by MS). LRMS (ESI) calcd from C35H54ClN6O14 [M+H]+817.34, found 817.60. Synthesis of bis(acetoxymethyl) 2,2'-((2-((1-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9- azahenicosyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-((3',6'-diacetoxy-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthen]-5-yl)carbamoyl)phenyl)azanediyl)diacetate (compound 14)

[0128] Compound 13 (0.040 g, 0.049 mmol, 1.0 eq.), 5-FAM diacetate (0.023 g, 0.049 mmol, 1.0 eq.) and HATU (0.019 g, 0.049 mmol, 1.0 eq.) were added to a Schlenk flask. Then, the flask was placed under vacuum followed by back-filling with Ar using a balloon. Dry DMF (2.5 mL) and DIPEA (0.03 mL, 0.171 mmol, 3.5 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3, dried over Na2SO4, filtered and concentrated in vacuo. The desired product was purified by reverse phase preparatory HPLC (10-95% CH3CN / H2O, linear gradient). Compound 14 was obtained as a an off-white solid (0.028 g, 45% isolated yield. LRMS (ESI) calcd from C60H68ClN6O22 [M+H]+1259.41, found 1259.80. Synthesis of 2-azido-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide (compound 15)

[0129] Azidoacetic acid (0.019 g, 0.192 mmol, 1.0 eq.), 2-(2-((6- chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (0.050 g, 0.192 mmol, 1.0 eq.) and HATU (0.073 g, 0.192 mmol, 1.0 eq.) were added to a Schlenk flask. After placing the flask under high vacuum, the flask was back-filled with Ar using a balloon. Then, dry DMF (2 mL) and DIPEA (0.15 mL, 0.865 mmol, 4.5 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed two times with brine. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved withCHCl3, adsorbed on silica and chromatographed (SiO2, 6% MeOH in CHCl3) to give a colorless oil (0.033 g, 56% isolated yield). LRMS (ESI) calcd from C12H24ClN4O3 [M+H]+307.15, found 307.30. Synthesis of bis(acetoxymethyl) 2,2'-((4-amino-2-methoxyphenyl)azanediyl)diacetate (compound 16)

[0130] To a solution of nitro derivative (0.025 g, 0.058 mmol) in DCM (2 mL) was added Pd / C (0.025 g) and the mixture was bubbled with H2 using a balloon. The reaction mixture was stirred at rt for 2 h. Then, the crude mixture was diluted with CHCl3and filtered through a PTFE syringe filter. The filtrate was concentrated under reduced pressure and the residue was used in the next step without any purification. LRMS (ESI) calcd from C17H23N2O9[M+H]+399.14, found 399.30. Synthesis of 3',6'-diacetoxy-4'-((methyl(prop-2-yn-1-yl)amino)methyl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid (compound 17)

[0131] N-methyl propargylamine (27 uL, 0.319 mmol, 1.2 eq.), a formaldehyde 37 wt% solution in water (0.008 g, 0266 mmol, 1.0 eq.) and CH3CN (3 mL) were added to a heavy-wall pressure vessel and the reaction mixture was stirred at 95 °C for 1 h. Upon cooling to rt, 5- carboxyfluorescein (0.100 g, 0.266 mmol, 1.0 eq.), CH3CN (3.2 mL) and H2O (2.4 mL) were added, and the reaction mixture was stirred at 95 °C for 3 h. Analysis by MS showed slow progression of the reaction, the crude mixture was then concentrated in vacuo and saved for further reaction. Fresh N-methyl propargylamine and formaldehyde solution were reacted as indicated above, then the residue saved before was added to the reaction mixture and stirring continued at 95 °C for 3 h. After cooling to rt, the crude mixture was concentrated under reduced pressure. The crude mixture contained 33% compound 15 as determined by LCMS. The residue was dispersed in dry DCM (10 mL) and then DIPEA (0.28 mL, 1.594 mmol, 6.0 eq.) and acetic anhydride (0.18 mL, 1.860 mmol, 7.0 eq) were added. Next, the resulting mixture was stirred at rt overnight. The crude mixture was concentrated in vacuo. The residue was dissolved in EtOAc and washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrate in vacuo. Compound 17 was isolated via reverse phase preparatory HPLC (5-95% CH3CN / H2O, linear gradient, with constant 0.1% v / v FA) as an off-white solid, 0.024 g (17% isolated yield over 2 steps). LRMS (ESI) calcd from C30H24NO9 [M+H]+542.14, found 542.40. Synthesis of 3',6'-diacetoxy-4'-((((1-(2-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-2- oxoethyl)-1H-1,2,3-triazol-4-yl)methyl)(methyl)amino)methyl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid (compound 18)

[0132] Compound 17 (0.024 g, 0.044 mmol, 1.0 eq.), compound 15 (0.027 g, 0.089 mmol, 2.0 eq.) and CuI (0.001 g, 0.004 mmol, 0.1 eq.) were added to a Schlenk flask. The flask was then placed under high vacuum, followed by backfilling with Ar. Next, THF (0.75 mL) and DIPEA (0.01 mL, 0.075 mmol, 1.7 eq.) were added and the mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with water. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was used in the next step without any purification. LRMS (ESI) calcd from C42H47ClN5O12[M+H]+848.29, found 848.60. Synthesis of bis(acetoxymethyl) 2,2'-((4-((3',6'-diacetoxy-4'-((((1-(2-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4- yl)methyl)(methyl)amino)methyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5- yl)carbamoyl)-2-methoxyphenyl)azanediyl)diacetate (compound 19)

[0133] Compound 18 (0.037 g, 0.044 mmol, 1.0 eq.), compound 16 (0.017 g, 0.044 mol, 1.0 eq.) and HATU (0.017 g, 0.044 mol, 1.0 eq.) were added to a Schlenk flask. The flask was first placed under high vacuum and then was back-filled with Ar using a balloon. Dry DMF (2.5 mL) and DIPEA were added (0.03 mL, 0.153 mmol, 3.5 eq.) and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed two times with brine. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. Compound 19 was isolated via reverse phase preparatory HPLC (10-95% CH3CN / H2O, linear gradient) as an off- white solid. LRMS (ESI) calcd from C59H67ClN7O20[M+H]+1228.41, found 1228.80. Synthesis of 3',6'-diacetoxy-4'-((((1-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)- 1H-1,2,3-triazol-4-yl)methyl)(methyl)amino)methyl)-3-oxo-3H-spiro[isobenzofuran-1,9'- xanthene]-5-carboxylic acid (compound 20)

[0134] Compound 17 (0.026 g, 0.048 mmol, 1.0 eq), compound 11 (0.038 g, 0.096 mmol, 2.0 eq.) and CuI (0.001 g, 0.005 mmol, 0.1 eq.) were added to a Schlenk flask. After being put under high vacuum, the flask was back-filled with Ar using a balloon. THF (0.75 mL) and DIPEA (0.02 mL, 0.082 mmol, 1.7 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was used in the next step without any purification. LRMS (ESI) calcd from C46H55ClN5O14[M+H]+936.34, found 936.70. Synthesis of bis(acetoxymethyl) 2,2'-((4-((3',6'-diacetoxy-4'-((((1-(21-chloro-8-oxo-3,6,12,15- tetraoxa-9-azahenicosyl)-1H-1,2,3-triazol-4-yl)methyl)(methyl)amino)methyl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthen]-5-yl)carbamoyl)-2-methoxyphenyl)azanediyl)diacetate (compound 21)

[0135] Compound 20 (0.037 mmol, 1.0 eq.), compound 18 (0.037 mmol, 1.0 mmol, 1.0 eq.) and HATU (0.014 g, 0.037 mmol, 1.0 eq.) were placed in a Schlenk flask, then the flask was placed under high vacuum. Next, the flask was back-filled with Ar using a balloon. DMF (2 mL) and DIPEA were added (0.02 mL, 0.129 mmol, 3.5 eq.) and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed twice with brine. The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to separation via reverse phase preparatory HPLC (10-95% CH3CN / H2O, linear gradient). Analysis of the isolated mass after lyophilization showed a 1:1 mixture compound 21 and mono deacetylated compound 21. The mixture was dissolved in DCM (0.4 mL) and subjected to reacetylation by adding acetic anhydride (3.5 eq.) and DIPEA (3 eq.). The reaction mixture was stirred at rt overnight. By LCMS, full conversion into compound 21 was observed, the crude mixture was concentrated in vacuo and further dried under high vacuum. LRMS (ESI) calcd from C63H75ClN7O22[M+H]+1316.46, found 1316.80. Synthesis of dimethyl 4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)isophthalate (compound 22)

[0136] To a heavy wall pressure vessel were added 5-carboxyfluorescein (0.100 g, 0.226 mmol, 1.0 eq.), MeOH (7 mL) and MSA (0.7 mL). The resulting mixture was stirred at reflux for 72 h. After cooling to rt, the crude mixture was poured into an addition funnel and added dropwise into a beaker containing ice cold water. While adding the crude mixture, the aqueous solution was basified by the portion wise addition of KOH (0.545 g, 0.9 eq. relative to MSA). The resulting orange precipitate was collected through vacuum filtration and air dried overnight. LRMS (ESI) calcd from C23H17O7[M+H]+405.10, found 405.20. Synthesis of 3-(((9-(2,4-bis(methoxycarbonyl)phenyl)-6-hydroxy-3-oxo-3H-xanthen-5- yl)methyl)(methyl)amino)propanoic acid (compound 23)

[0137] To a heavy wall pressure vessel were added 3-(methylamino)propanoic acid (0.047 g, 0.335 mmol, 1.2 eq.), K2CO3(0.046 g, 0.335 mmol, 1.2 eq.), a formaldehyde 37 wt% solution in water (0.008 g, 0.279 mmol, 1.0 eq.) and CH3CN (2.5 mL). The resulting mixture was stirred at 95 °C for 1 h. Upon cooling to rt, compound 22 (0.113 g, 0.279 mmol, 1.0 eq.) and CH3CN (2 mL) were added, and the reaction mixture was stirred at 95 °C for 3 h. The crude mixture was concentrated in vacuo and analyzed by MS. Incomplete reaction was observed and the crude mixture was reacted again. Fresh 3-(methylamino)propanoic acid, K2CO3 and formaldehyde solution were reacted as before, then the recovered mixture from the first reaction was added to the vessel and reacted for 3 h. The crude mixture was concentrated under reduced pressure and subjected to separation via reverse phase preparatory HPLC (10-95% CH3CN / H2O, lineargradient, with constant 0.1% v / v FA). An orange solid was recovered, 0.032 g (22% isolated yield). LRMS (ESI) calcd from C28H26NO9 [M+H]+520.16, found 520.30. Synthesis of dimethyl 4-(5-(18-chloro-2-methyl-5-oxo-9,12-dioxa-2,6-diazaoctadecyl)-6- hydroxy-3-oxo-3H-xanthen-9-yl)isophthalate (compound 24)

[0138] Compound 23 (0.030 g, 0.058 mmol, 1.0 eq.), 2-(2-((6- chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (0.015 g, 0.058 mmol, 1.0 eq.) and HATU (0.022 g, 0.058 mmol, 1.0 eq.) were added to a Schlenk flask. Next, the flask was placed under high vacuum followed by back-filling with Ar using a balloon. Then, dry DMF (2 mL) and DIPEA (0.034 g, 0.260 mmol, 4.5 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The desired compound was isolated via reverse phase preparatory HPLC (10-95% CH3CN / H2O, linear gradient, with constant 0.1% v / v FA) as an orange solid (0.029 g, 69% isolated yield). LRMS (ESI) calcd from C30H24NO9 [M+H]+725.28, found 725.50. Synthesis of 4-(5-(18-chloro-2-methyl-5-oxo-9,12-dioxa-2,6-diazaoctadecyl)-6-hydroxy-3- oxo-3H-xanthen-9-yl)isophthalic acid (compound 25)

[0139] A solution of compound 24 (0.026 g, 0.036 mmol, 1.0 eq.) in MeOH (0.25 mL) was treated with KOH (0.100 g, 1.793 mmol, 50.0 eq.) in H2O (0.3 mL). The reaction mixture was stirred at rt overnight. Completion of the reaction was verified by MS. Then, HCl 6 M (0.33 mL, 1.1 eq. relative to KOH) was added. The crude mixture was diluted with MeOH and the solution was carefully transferred to a vial, leaving behind the salts. The solution was concentrated in vacuo and further dried under high vacuum. The crude mixture was used in the next step without any purification. LRMS (ESI) calcd from C36H42ClN2O10[M+H]+697.25, found 697.60. Synthesis of 3',6'-diacetoxy-4'-(18-chloro-2-methyl-5-oxo-9,12-dioxa-2,6-diazaoctadecyl)-3- oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid (compound 26)

[0140] To a solution of compound 25 (0.022 g, 0.032 mmol, 1.0 eq.) in DCM (0.75 mL) were added DIPEA (0.03 mL, 0.189 mmol, 6.0 eq.) and acetic anhydride (0.02 mL, 0.221 mmol, 7.0 eq.). The reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The crude mixture was used in the next step without any purification. LRMS (ESI) calcd from C40H46ClN2O12 [M+H]+781.27, found 781.50.Synthesis of bis(acetoxymethyl) 2,2'-((4-((3',6'-diacetoxy-4'-(18-chloro-2-methyl-5-oxo-9,12- dioxa-2,6-diazaoctadecyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)carbamoyl)-2- methoxyphenyl)azanediyl)diacetate (compound 27)

[0141] The crude mixture containing compound 26 (0.032 mmol, 1.0 eq.), compound 16 (0.013 g, 0.032 mmol, 1.0 eq.) and HATU (0.012 g, 0.032 mmol, 1.0 eq.) were placed in a Schlenk flask. The flask was put under high vacuum and then back-filled with Ar using a balloon. DMF (1.75 mL) and DIPEA (0.02 mL, 0.110 mmol, 3.5 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude mixture was subjected to separation via reverse phase preparatory HPLC (10-95% CH3CN / H2O, linear gradient), compound 27 was recovered as a light orange solid (0.002 g, 5% isolated yield over 2 steps). LRMS (ESI) calcd from C57H66ClN4O20[M+H]+1161.40, found 1161.70. Synthesis of 5-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl diacetate (compound 28)

[0142] To a Schlenk flask were added 5-FAM diacetate (0.100 g, 0.217 mmol, 1.0 eq.), 2-(2-((6- chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (0.057 g, 0.217 mmol, 1.0 eq.) and HATU (0.083 g, 0.217 mmol, 1.0 eq.). The flask was placed under high vacuum and then back-filled with Ar using a balloon. Dry DMF (6 mL) and DIPEA (0.17 mL, 0.978 mmol, 4.5 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine two times. The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The crude mixture was used in the next step without any purification. LRMS (ESI) calcd from C35H37ClNO10[M+H]+666.21, found 666.50. Synthesis of 5-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(6-hydroxy-3-oxo-3H- xanthen-9-yl)benzoic acid (compound 29)

[0143] A solution of the crude mixture containing compound 28 (0.217 mmol, 1.0 eq.) in MeOH (0.3 mL) was treated with KOH (0.030 g, 0.543 mmol.2.5 eq.) in H2O (0.2 mL). The reaction mixture was stirred at rt for 30 min. Then, the crude solution was acidified to pH 1-2 by adding HCl 1 M. Once acidified, MeOH was removed under reduced pressure. Water was added to the residue to precipitate an orange solid. The solid was recovered via vacuum filtration and air dried (0.134 g). LRMS (ESI) calcd from C31H33ClNO8 [M+H]+582.19, found 582.50. Synthesis of 2-(5-(((2-carboxyethyl)(methyl)amino)methyl)-6-hydroxy-3-oxo-3H-xanthen-9- yl)-5-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoic acid (compound 30)

[0144] To a heavy wall pressure vessel were added 3-(methylamino)propanoic acid (0.037 g, 0.268 mmol, 1.2 eq.), K2CO3 (0.037 g, 0.268 mmol, 1.2 eq.), a formaldehyde 37 wt% solution in water (0.007 g, 0.223 mmol, 1.0 eq.) and CH3CN (2.5 mL). The resulting mixture was stirred at 95 °C for 1 h. Upon cooling to rt, compound 29 (0.130 g, 0.223 mmol, 1.0 eq.), CH3CN (1.25 mL) and H2O (0.6 mL) were added, and the reaction mixture was stirred at 95 °C for 3 h. After cooling to rt, the crude mixture was concentrated under reduced pressure. The residue was subjected to separation via preparatory reverse phase preparatory HPLC (5-95% CH3CN / H2O, linear gradient, with constant 0.1% v / v FA). Compound 30 was recovered as an orange solid (0.037 g, 24% isolated yield). LRMS (ESI) calcd from C36H42ClN2O10[M+H]+697.25, found 697.50. Synthesis of 2-(5-(((3-((4-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)-3- methoxyphenyl)amino)-3-oxopropyl)(methyl)amino)methyl)-6-hydroxy-3-oxo-3H-xanthen- 9-yl)-5-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoic acid (compound 31)

[0145] Compound 30 (0.037 g, 0.053 mmol.1.0 eq.), compound 18 (0.021 g, 0.053 mmol, 1.0 eq.) and HATU (0.020 g, 0.053 mmol) were placed in a Schlenk flask. The flask was put under high vacuum and then back-filled with Ar using a balloon. Dry DMF (1.5 mL) and DIPEA were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The crude mixture was subjected to separation via preparatory reverse phase preparatory HPLC (5-95% CH3CN / H2O, linear gradient, with constant 0.1% v / v FA). The desired compound was obtained as an orange solid (0.008 g, 14% isolated yield). LRMS (ESI) calcd from C53H62ClN4O18 [M+H]+1077.37, found 1077.70. Synthesis of bis(acetoxymethyl) 2,2'-((4-(3-(((3',6'-diacetoxy-5-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-4'- yl)methyl)(methyl)amino)propanamido)-2-methoxyphenyl)azanediyl)diacetate (compound 32)

[0146] To a solution of compound 31 (0.008 g, 0.007 mmol, 1.0 eq.) in DCM (0.75 mL) were added DIPEA (7.8 uL, 0.045 mmol, 6.0 eq.) and acetic anhydride (4.9 uL, 0.052 mmol, 7.0 eq.). The reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic layer was dried over Na2SO4, filtered and concentration in vacuo. The desired product was isolated via reverse phase preparatory HPLC (5-95% CH3CN / H2O, linear gradient, with constant 0.1% v / v FA). Compound 32 was obtained as a white solid (0.005 g, 59% isolated yield). LRMS (ESI) calcd from C57H66ClN4O20[M+H]+1161.40, found 1161.80.Synthesis of 3-(N-((6-acetoxy-9-(4-(bis(2-ethoxy-2-oxoethyl)amino)-3-(2-(2-(bis(2-ethoxy-2- oxoethyl)amino)-5-methylphenoxy)ethoxy)phenyl)-2,7-difluoro-3-oxo-3H-xanthen-4- yl)methyl)acetamido)propanoic acid (compound 33)

[0147] To a heavy-wall pressure vessel were added 3-aminopropanoic acid (0.010 g, 0.106 mmol, 1.2 eq.), a formaldehyde 37 wt% solution in water (0.003 g, 0.088 mmol, 1.0 eq.) and CH3CN (1.5 mL). The resulting mixture was stirred at 95 °C for 1 h. Upon cooling to rt, Fluo-4 ethyl ester (0.075 g, 0.088 mmol, 1.0 eq.) and CH3CN (1.8 mL) were added. The reaction FLuo- 4mixture was stirred at 95 °C for 3 h. The crude mixture was concentrated in vacuo. The residue was dissolved in DCM (5 mL) and DIPEA (0.09 mL, 0.530 mmol, 6.0 eq.) and acetic anhydride (0.06 mL, 0.619 mmol, 7.0 eq.) were added. The reaction mixture was then stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The crude mixture was subjected to separation via reverse phase preparatory HPLC (10-95% CH3CN / H2O, linear gradient, with constant 0.1% v / v FA). Compound 33 was isolated as an orange solid (0.035 g, 38% isolated yield). LRMS (ESI) calcd from C52H58F2N3O17 [M+H]+1034.37, found 1034.70. Synthesis of diethyl 2,2'-((4-(4-(2-acetyl-18-chloro-5-oxo-9,12-dioxa-2,6-diazaoctadecyl)-2,7- difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-2-(2-(2-(bis(2-ethoxy-2-oxoethyl)amino)-5- methylphenoxy)ethoxy)phenyl)azanediyl)diacetate (compound 34)

[0148] Compound 33 (0.035 g, 0.034 mmol, 1.0 eq.), 2-(2-((6- chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (0.009 g, 0.034 mmol, 1.0 eq.) and HATU (0.013 g, 0.034 mmol, 1.0 eq.) were added to a Schlenk flask. The flask was put under high vacuum and then back-filled with Ar using a balloon. Dry DMF (2 mL) and DIPEA (0.03 mL, 0.152 mmol, 4.5 eq.) were added and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was dissolved with CHCl3and applied to a preparatory TLC plate, resolving with 6% MeOH in CHCl3. Compound 34 was obtained as an orange solid (0.020 g, 49% isolated yield). LRMS (ESI) calcd from C60H76ClF2N4O17 [M+H]+1197.49, found 1197.80. Synthesis of 2,2'-((4-(4-(2-acetyl-18-chloro-5-oxo-9,12-dioxa-2,6-diazaoctadecyl)-2,7- difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-2-(2-(2-(bis(carboxymethyl)amino)-5- methylphenoxy)ethoxy)phenyl)azanediyl)diacetic acid (compound 35)

[0149] To a solution of compound 34 (0.020 g, 0.017 mmol, 1.0 eq.) in MeOH (0.5 mL) was added KOH (0.019 g, 0.334 mmol, 20.0 eq.) in H2O (0.5 mL). The reaction mixture was stirred at rt for 5 h. Full conversion was verified by MS and HCl 1 M (0.83 mL, 2.5 eq. relative toKOH) was added to the crude mixture. The crude mixture was concentrated in vacuo, further dried under high vacuum and used in the next step without any purification. LRMS (ESI) calcd from C52H60ClF2N4O17[M+H]+1085.36, found 1085.70. Synthesis of bis(acetoxymethyl) 2,2'-((4-(6-(acetoxymethoxy)-5-(2-acetyl-18-chloro-5-oxo- 9,12-dioxa-2,6-diazaoctadecyl)-2,7-difluoro-3-oxo-3H-xanthen-9-yl)-2-(2-(2-(bis(2- (acetoxymethoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)phenyl)azanediyl)diacetate and bis(acetoxymethyl) 2,2'-((4-(6-(acetoxymethoxy)-4-(2-acetyl-18-chloro-5-oxo-9,12- dioxa-2,6-diazaoctadecyl)-2,7-difluoro-3-oxo-3H-xanthen-9-yl)-2-(2-(2-(bis(2- (acetoxymethoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)phenyl)azanediyl)diacetate (compounds 36-1 and 36-2)

[0150] To a solution of the crude mixture containing compound 35 (0.017 mmol, 1.0 eq.) in dry DMF (1.5 mL) was added DIPEA (0.06 mL, 0.309 mmol, 18.5 eq.). The solution was cooled in an ice bath before bromomethyl acetate (0.02 mL, 0.184 mmol, 11.0 eq.) was added dropwise. The ice bath was removed, and the reaction mixture was stirred at rt overnight. The crude mixture was diluted with CHCl3and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude mixture was subjected to separation via reverse phase preparatory HPLC (10-95% CH3CN / H2O, linear gradient, with constant 0.1% v / v FA). The two possible regioisomers were recovered and arbitrarily assigned as 36-1 (0.003 g) and 36-2 (0.003 g). LRMS (ESI) calcd from C67H80ClF2N4O27[M+H]+1445.47, found 1445.80.80 (36-1) and 1445.90 (36-2). Synthesis of acetoxymethyl 3-(6-(acetoxymethoxy)-4,5-dichloro-2-(25-chloro-3-oxo- 7,10,13,16,19-pentaoxa-4-azapentacosyl)-9-(3-methoxy-4-(13-(2-methoxyphenyl)-1,4,10- trioxa-7,13-diazacyclopentadecan-7-yl)phenyl)-3-oxo-3H-xanthen-7-yl)propanoate (compound 39)

[0151] To a solution of 21 mg (25 umol, 9 eq) of 3,3'-(4,5-dichloro-6-hydroxy-9-(3-methoxy-4- (13-(2-methoxyphenyl)-1,4,10-trioxa-7,13-diazacyclopentadecan-7-yl)phenyl)-3-oxo-3H- xanthene-2,7-diyl)dipropionic acid in 300 uL DMF was added 40 uL (230 umol) DIPEA, followed by 2.3 uL (25 umol, 1 eq) acetic anhydride. After the reactions stirred at room temperature for 30 minutes, LCMS showed ~70% (at 254 nm absorbance) to the desired lactone intermediate (LRMS M+H 835.5 m / z, calculated 835.2 m / z): 3-(10,12-dichloro-6-(3-methoxy-4- (13-(2-methoxyphenyl)-1,4,10-trioxa-7,13-diazacyclopentadecan-7-yl)phenyl)-2,9-dioxo-3,4- dihydro-2H,9H-pyrano[3,2-b]xanthen-8-yl)propanoic acid (compound 37). 12 mg (31 umol, 1.2 eq) of the halo tag ligand were then added to the reaction mixture, and it stirred at room temperature for another 30 minutes. LCMS of the reaction mixture showed full conversion ofthe lactone intermediate to the desired amide (LRMS M+H 1190.8 m / z, calculated 1190.5 m / z): 3-(4,5-dichloro-7-(25-chloro-3-oxo-7,10,13,16,19-pentaoxa-4-azapentacosyl)-6-hydroxy-9-(3- methoxy-4-(13-(2-methoxyphenyl)-1,4,10-trioxa-7,13-diazacyclopentadecan-7-yl)phenyl)-3- oxo-3H-xanthen-2-yl)propanoic acid (compound 38). 50 uL (eq) bromomethyl acetate were then added to the reaction mixture, and the reaction stirred at room temperature for three hours. LCMS of the reaction mixture showed ~ 60% conversion at 254 nm to the desired AM product (compound 39, LRMS M+H 1334.9 m / z, calculated 1334.5 m / z). The crude reaction mixture was diluted in 5:3 acetonitrile / water and purified by preparative HPLC (acetonitrile / water 0.1% formic acid gradient on C18). The combined pure fractions were lyophilized at -105 C to yield 5 mg.

[0152] The shorter halo tag ligand variant acetoxymethyl 3-(6-(acetoxymethoxy)-4,5-dichloro-2- (3-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-3-oxopropyl)-9-(3-methoxy-4-(13-(2- methoxyphenyl)-1,4,10-trioxa-7,13-diazacyclopentadecan-7-yl)phenyl)-3-oxo-3H-xanthen-7- yl)propanoate was similarly synthesized. (LRMS M+H 1202.7 m / z, calculated 1202.4 m / z)

[0153] The following scheme outlines a synthesis for the azido or alkynyl-terminating general formula VII.

[0154] The following scheme outlines a synthesis for the azido or alkynyl-terminating general formula VIII.In vitro experiments (1) Preparation of compounds for fluorescence polarization data acquisition.

[0155] First, made the buffer solution in which to dissolve / test the desired compounds which is composed of TMR ligand (Promega) as a positive control, HALOTAG®Standard Protein (Promega), and Thallos HALOTAG®Ligand (ION). Combined 1X PBS (Gibco) with 0.01% w / v CHAPS hydrate (Sigma). Added adequate amount of TMR (Promega) solution to the buffer solution to have a working concentration of 200nM. Hydrolyzed Thallos HALOTAG® Ligand (ION) using KOH. Added adequate amount of this solution to the buffer solution to have a working concentration of 200nM. Added adequate amount of HALOTAG® Standard Protein (Promega) to the buffer solution to have a working concentration of 800 nM. (2) Fluorescence polarization data acquisition.

[0156] Added two sets of 20µL of PBS + CHAPS solution, 20µL of TMR in PBS + CHAPS solution, and 20 µL of Thallos HALOTAG® Ligand in PBS + CHAPS solution to a 384-well plate (Falcon). Used a FlexStation (Molecular Devices) to collect data. Set the excitation wavelength to 550 nm and the emission wavelength to 590 nm when reading TMR data. For the Thallos data, set excitation to 485 nm and emission to 515 nm. Collected baseline fluorescence polarization data for 1 minute. After adding the compound being tested to each well, added 20ul of PBS + CHAPS solution to one set of wells and 20ul of HALOTAG® Standard protein in PBS + CHAPS solution to the other set of wells. Collect fluorescence polarization data for 10 minutes.

[0157] FIG.1 shows the fluorescence polarization of compound I-2 and compound II-2 vs. control experiments. The polarization increased in the combinations of compound I-2 orcompound II-2 with HALOTAG®, indicating that substrate-ligand binding occurred. When HALOTAG® protein was not added (solid line), the polarization decreased or remained the same.

[0158] FIG.2 shows the fluorescence polarization of compound IVA-7 and compound VIA-1 vs. control experiments. The polarization increased in the combinations of compound IVA-7 or compound VIA-1 with HALOTAG®, indicating that substrate-ligand binding occurred (upper lines). When HALOTAG® protein was not added (lower lines), the polarization decreased or remained the same. (3) Thallium Sensitivity

[0159] Thallos HALOTAG® was hydrolyzed using KOH. Two chloride free buffer solutions were made. The first solution (low Tl+) was made with 1 mM Tl2SO4, and the second solution (high Tl+) was made with 45 mM Tl2SO4. Enough of the hydrolyzed indicator was added to have a final concentration of 3 µM of the indicator.

[0160] An amount of 100 µL of the 1 mM Tl2SO4solution was added to each of the last 11 wells of a row in a 96-well plate. An amount of 200 µL of the 45 mM Tl2SO4 solution was added to the first well of the row. An amount of 100 µL of the high Tl+solution was taken and diluted in half by adding it to the well containing 100 µL of the low Tl+solution. An amount of 100 µL of this solution was removed and diluted by half again when adding it to next well in the row with 100 µL of the low Tl+solution. This procedure was continued down the row until reaching the well in column 11, from which 100 µL of the solution was taken and discarded instead of adding it to column 12. The well in column 12 had 100 µL of 1 mM Tl2SO4solution. A bottom read fluorescence of each well was taken using a Cytation5 instrument (BioTek). FIG.3 shows Tl+ sensitivity of compound I-2 and compound II-2.

[0161] A similar experiment was performed using compound IVA-7 and compound VIA-1 and a CaSO4 solution. FIGS.4A and 4B show the Ca sensitivity of compounds IVA-7 and compound VIA-1.

[0162] A similar experiment was performed using compound VII-2 and a Na2SO4 solution. FIG. 5 shows the Na sensitivity of compound VII-12. (4) Preparation of cells and In Situ Experiments Cell Transfection

[0163] Seeded CHO K1 cells (FISHER) into a 6-well plate for transfection. Used pHTC Halotag(registered trademark) CMV-neo Vector and Lipofectamine 2000 (THERMO) to transfect cells. Added 600ug / ml G418 in media solution 2 days after transfection. Conductedlimited clone dilution in a 384 well plate. Confirmed HALOTAG® expression by adding TMR. Expanded cells that expressed HALOTAG®.

[0164] Seeded HEK 293 cells (ATCC) into a 6 well plate. Used pHTC HALOTAG® CMV-neo Vector and Lipofectamine 2000 (THERMO) to transfect cells. Allowed cells to grow and look healthy before adding 300 µg / ml G418 solution. Conducted limited dilution cloning in 96-well plate. Confirmed HALOTAG® expression by adding TMR. Expanded cells that expressed HALOTAG®. Cell Based Assays

[0165] Prepared dye load buffer with the desired indicator, dye load may include probenecid, pluronic, TRS. Add the dye load to cells (both wild type (WT) and HALOTAG®expressing (HT+) cells that had been allowed to grow in a 96-well plate overnight. Collect bottom read fluorescence and images on a Cytation5 (BioTek). In some cases after loading, cells were washed to allow unbound dye to efflux from cells over a period of time. Wash solution may be dye load or media, may include TRS, may not include probenecid. Collect unloading data on the Cytation5 as well. Then added stimulus solution to record fluorescence change and differences between WT cells and HT+ cells. Use both Cytation5 and FlexStation.

[0166] Compound IVA-7 was allowed to unload from both CHO wild-type (WT) cells and CHO HALOTAG® (+) cells, 100nM ionomycin, a calcium ionophore that increases intracellular calcium concentrations, was added on the FlexStation. Data acquisition shows that there is an increase in the fluorescence of CHO HT(+) cells while there is no change in the wild type cells. Data suggests that there was enough dye that remained in CHO HT(+) cells because the dye was covalently bound to HT-protein expressed in the cells and therefore was not effluxed as in the WT cells. This demonstrates signal discrimination between HT-expressing cells and WT cells. FIG.6 shows a comparison of relative fluorescence (F / F0) over time for HT and WT cells in the presence of compound IVA-7.

[0167] HEK WT and HEK HT(+) cells were loaded with compound I-2. After loading for 30 minutes (pre-stimulus images), a stimulus solution of 1 mM Tl+was added. A large increase in fluorescence is observed in HEK HT(+) cells in response to analyte addition, whereas a negligible increase is observed in WT cells that do not retain significant concentrations of the indicator. (5) Streptavidin Coated Slides

[0168] A glass slide was coated with streptavidin. Compound VII-11, having a biotin coupling agent, was added to the slide. The slide was treated with increasing concentrations of potassium ions (K2SO4). FIG.7 shows the K sensitivity of compound VII-11 vs a control with slidestreated with compound VII-11 which was not biotinylated. These results show that the potassium indicator retains its sensitivity to potassium when immobilized on a surface.

Claims

CLAIMS What is claimed is:

1. A compound with the chemical structure (IA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; O U represents or ; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (I) can exist in the following tautomeric forms:.

2. The compound of claim 1, wherein Linker is: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, Het;group, or Ac; and Het represents a heterocyclic group.

3. The compound of claim 1, wherein Linker is: –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s–, where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; and where Y1is Het and Y2is –C(O)–, –NH–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, or –C(O)–NR11–.

4. The compound of claim 2 or 3, wherein Het is a 1,2,3-triazole heterocycle.

5. The compound of claim 4, wherein the 1,2,3-triazole heterocycle is:.

6. The compound of claim 2 or 3, wherein Het is a pyridazine or dihydropyridazine heterocycle.

7. The compound of claim 6, wherein the pyridazine or dihydropyridazine heterocycle is:where R10is H or Me.

8. The compound of any one of claims 1-7, where L is a ligand for a self-labeling enzyme.

9. The compound of claim 8, wherein L is:, where t is 2-8 and u is 2 to 10.

10. The compound of claim 9, wherein L is :.

11. The compound of claim 8, wherein L comprises benzylguanine or chloropyrimidine.

12. The compound of claim 11, wherein L is:.

13. The compound of claim 8, wherein L comprises benzylcytosine.

14. The compound of claim 13, wherein L is:.

15. The compound of any one of claims 1-7, wherein L comprises a maleimide group.

16. The compound of claim 15, wherein L is:.

17. The compound of any one of claims 1-7, wherein L comprises a N-hydroxysuccinimide (NHS) group.

18. The compound of claim 17, wherein L is:.

19. The compound of any one of claims 1-7, wherein L comprises a biotin group.

20. The compound of claim 19, wherein L is:.

21. The compound of any one of claims 1-7, wherein L comprises a chemical handle.

22. The compound of claim 21, wherein L is:

23. The compound of any one of claims 1-7, wherein L is: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

24. The compound of any one of claims 1-20, wherein the compound has the structure (IA):wherein each X1and X2independently represent: a bond, –(CH2)m–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–O–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–NH–, –NH–(CH2)m–(CH2–CH2–O)p–(CH2)n–, –O–(CH2)m–(CH2–CH2–O)p–(CH2)n–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–O–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–NH–, –NH–(CH2)m–(O–CH2–CH2)p–(CH2)n–, or –O–(CH2)m–(O–CH2–CH2)p–(CH2)n–; and where m is 0-4; n is 0-4; and p is 0-4; wherein, when R1is CO2R’, the compound (I) can exist in the following tautomeric forms:.

25. The compound of claim 24, wherein the compound has the formula: (I-1), (I-2), (I-3), (I- 4), (I-5), (I-6), (I-7), (I-8), (I-9), (I-10), (I-11), (I-12), (I-13), (I-14), (I-15), (I-16), (I-17), (I-18), or (I-19):;;;W.

26. The compound of any one of claims 1-20, wherein the compound has the formula: (I-20), (I-21), or (I-22):.

27. The compound of claim 21 or 22 , wherein the compound has the formula: (I-23), (I-24), (I-25), or (I-26):

28. A compound with the chemical structure (I) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–,where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:; where R10is H or Me; L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (I) can exist in the following tautomeric forms:.

29. A fluorescent probe comprising a compound as described in any one of claims 1 to 28 coupled to a biological macromolecule or solid substrate.

30. A compound with the chemical structure (II) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; where R11is H, an alkyl group, or Ac; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; U representsLinker is a linking group; and L represents a coupling agent; wherein, when R1is CO2R’, the compound (II) can exist in the following tautomeric forms:.

31. The compound of claim 30, wherein Linker is:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group.

32. The compound of claim 30, wherein Linker is: –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s–, where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; and where Y1is Het and Y2is –C(O)–, –NH–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, or –C(O)–NR11–.

33. The compound of claim 31 or 32, wherein Het is a 1,2,3-triazole heterocycle.

34. The compound of claim 33, wherein the 1,2,3-triazole heterocycle is:.

35. The compound of claim 31 or 32, wherein Het is a pyridazine or dihydropyridazine heterocycle.

36. The compound of claim 35, wherein the pyridazine or dihydropyridazine heterocycle is:where R10is H or Me.

37. The compound of any one of claims 30-36, where L is a ligand for a self-labeling enzyme.

38. The compound of claim 37, wherein L is:, where t is 2-8 and u is 2 to 10.

39. The compound of claim 38, wherein L is :.

40. The compound of claim 37, wherein L comprises benzylguanine or chloropyrimidine.

41. The compound of claim 40, wherein L is:.

42. The compound of claim 37, wherein L comprises benzylcytosine.

43. The compound of claim 42, wherein L is:.

44. The compound of any one of claims 30-36, wherein L comprises a maleimide group.

45. The compound of claim 44, wherein L is:.

46. The compound of any one of claims 30-36, wherein L comprises a N- hydroxysuccinimide (NHS) group.

47. The compound of claim 46, wherein L is:

48. The compound of any one of claims 30-36, wherein L comprises a biotin group.

49. The compound of claim 48, wherein L is:.

50. The compound of any one of claims 30-36, wherein L comprises a chemical handle.

51. The compound of claim 50, wherein L is:

52. The compound of any one of claims 30-36, wherein L is: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

53. The compound of any one of claims 30-52, wherein the compound has the structure (IIA):wherein each X1and X2independently represent: a bond, –(CH2)m–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–O–, –(CH2)m–(CH2–CH2–O)p–(CH2)n–NH–, –NH–(CH2)m–(CH2–CH2–O)p–(CH2)n–, –O–(CH2)m–(CH2–CH2–O)p–(CH2)n–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–O–, –(CH2)m–(O–CH2–CH2)p–(CH2)n–NH–, –NH–(CH2)m–(O–CH2–CH2)p–(CH2)n–, or –O–(CH2)m–(O–CH2–CH2)p–(CH2)n–; and where m is 0-4; n is 0-4; and p is 0-4 wherein, when R1is CO2R’, the compound (IIA) can exist in the following tautomeric forms:.

54. The compound of claim 53, wherein the compound has the formula: (II-1), (II-2), (II-3), (II-4):

55. The compound of any one of claims 30-52, wherein the compound has the structure (IIB):

56. The compound of claim 55, wherein the compound has the structure (II-5):

57. A compound with the chemical structure (II), or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl;where R11is H, an alkyl group, or Ac; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; O U represents or ; Linker represents: –(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; and Het represents a heterocyclic group selected from the group consisting of:where R10is H or Me; L represents a ligand for coupling to a protein selected from the group consisting of:,where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (II) can exist in the following tautomeric forms:.

58. A fluorescent probe comprising a compound as described in any one of claims 30 to 57 coupled to a biological macromolecule or material.

59. A compound with the chemical structure (III), or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; R11 is H, an alkyl group, or Ac; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (III) can exist in the following tautomeric forms:.

60. The compound of claim 59, wherein Linker is:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group.

61. The compound of claim 59, wherein Linker is:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; and where Y1is Het and Y2is –C(O)–, –NH–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, or –C(O)–NR11–.

62. The compound of claim 60 or 61, wherein Het is a 1,2,3-triazole heterocycle.

63. The compound of claim 62, wherein the 1,2,3-triazole heterocycle is:.

64. The compound of claim 60 or 61, wherein Het is a pyridazine or dihydropyridazine heterocycle.

65. The compound of claim 64, wherein the pyridazine or dihydropyridazine heterocycle is:where R10is H or Me.

66. The compound of any one of claims 59-65, where L is a ligand for a self-labeling enzyme.

67. The compound of claim 66, wherein L is:, where t is 2-8 and u is 2 to 10.

68. The compound of claim 67, wherein L is :.

69. The compound of claim 66, wherein L comprises benzylguanine or chloropyrimidine.

70. The compound of claim 69, wherein L is:.

71. The compound of claim 66, wherein L comprises benzylcytosine.

72. The compound of claim 71, wherein L is:.

73. The compound of any one of claims 59-65, wherein L comprises a maleimide group.

74. The compound of claim 73, wherein L is:.

75. The compound of any one of claims 59-65, wherein L comprises a N- hydroxysuccinimide (NHS) group.

76. The compound of claim 75, wherein L is:.

77. The compound of any one of claims 59-65, wherein L comprises a biotin group.

78. The compound of claim 77, wherein L is:.

79. The compound of any one of claims 59-65, wherein L comprises a chemical handle.

80. The compound of claim 79, wherein L is:

81. The compound of any one of claims 59-65, wherein L is: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

82. The compound of any one of claims 59-81, wherein the compound has the formula: (III- 1):

83. A compound with the chemical structure (III), or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; where Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; R11is H, an alkyl group, or Ac; Linker represents:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: l ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (III) can exist in the following tautomeric forms:.

84. A fluorescent probe comprising a compound as described in any one of claims 59 to 83 coupled to a biological macromolecule or material.

85. A compound with the chemical structure (IVA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; each R12independently represents H, Me, halogen, or NO2; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (IVA) can exist in the following tautomeric forms:.

86. A compound with the chemical structure (IVB) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (IVB) can exist in the following tautomeric forms:.

87. The compound of claim 85 or 86, wherein Linker is:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group.

88. The compound of claim 85 or 86, wherein Linker is:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; and where Y1is Het and Y2is –C(O)–, –NH–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, or –C(O)–NR11–.

89. The compound of claim 87 or 88, wherein Het is a 1,2,3-triazole heterocycle.

90. The compound of claim 89, wherein the 1,2,3-triazole heterocycle is:.

91. The compound of claim 86 or 87, wherein Het is a pyridazine or dihydropyridazine heterocycle.

92. The compound of claim 91, wherein the pyridazine or dihydropyridazine heterocycle is:where R10is H or Me.

93. The compound of any one of claims 85-92, where L is a ligand for a self-labeling enzyme.

94. The compound of claim 93, wherein L is:, where t is 2-8 and u is 2 to 10.

95. The compound of claim 93, wherein L is :.

96. The compound of claim 93, wherein L comprises benzylguanine or chloropyrimidine.

97. The compound of claim 96, wherein L is:.

98. The compound of claim 93, wherein L comprises benzylcytosine.

99. The compound of claim 98, wherein L is:.

100. The compound of any one of claims 85-92, wherein L comprises a maleimide group.

101. The compound of claim 100, wherein L is:.

102. The compound of any one of claims 85-92, wherein L comprises a N- hydroxysuccinimide (NHS) group.

103. The compound of claim 102, wherein L is:

104. The compound of any one of claims 85-92, wherein L comprises a biotin group.

105. The compound of claim 104, wherein L is:.

106. The compound of any one of claims 85-92, wherein L comprises a chemical handle.

107. The compound of claim 106, wherein L is:

108. The compound of any one of claims 85-92, wherein L is: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

109. The compound of any one of claims 85-108, wherein the compound has the formula (IVA-1), (IVA-2), (IVA-3), (IVA-4), (IVA-5), (IVA-6), (IVA-7), (IVA-8), (IVA-9), or (IVA- 10):AA.

110. A compound with the chemical structure (IVA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; R12represents H, Me, halogen, or NO2; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl;where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4;each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (IVA) can exist in the following tautomeric forms:.

111. A compound with the chemical structure (IVB) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: l ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (IVB) can exist in the following tautomeric forms:.

112. A fluorescent probe comprising a compound as described in any one of claims 85 to 111 coupled to a biological macromolecule or material.

113. A compound with the chemical structure (VA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; R12represents H, Me, halogen, or NO2; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (VA) can exist in the following tautomeric forms:.

114. A compound with the chemical structure (VB), or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; and L represents a coupling agentwherein, when R1is CO2R’, the compound (VB) can exist in the following tautomeric forms:.

115. The compound of claim 113 or 114, wherein Linker is:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group.

116. The compound of claim 113 or 114, wherein Linker is: –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s–, where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; and where Y1is Het and Y2is –C(O)–, –NH–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, or –C(O)–NR11–.

117. The compound of claim 115 or 116, wherein Het is a 1,2,3-triazole heterocycle.

118. The compound of claim 117, wherein the 1,2,3-triazole heterocycle is:.

119. The compound of claim 115 or 116, wherein Het is a pyridazine or dihydropyridazine heterocycle.

120. The compound of claim 119, wherein the pyridazine or dihydropyridazine heterocycle is:where R10is H or Me.

121. The compound of any one of claims 113-120, where L is a ligand for a self-labeling enzyme.

122. The compound of claim 121, wherein L is: , where t is 2-8 and u is 2 to 10.

123. The compound of claim 122, wherein L is :.

124. The compound of claim 121, wherein L comprises benzylguanine or chloropyrimidine.

125. The compound of claim 124, wherein L is:.

126. The compound of claim 121, wherein L comprises benzylcytosine.

127. The compound of claim 126, wherein L is:.

128. The compound of any one of claims 113-120, wherein L comprises a maleimide group.

129. The compound of claim 128, wherein L is:.

130. The compound of any one of claims 113-120, wherein L comprises a N- hydroxysuccinimide (NHS) group.

131. The compound of claim 130, wherein L is:.

132. The compound of any one of claims 113-120, wherein L comprises a biotin group.

133. The compound of claim 132, wherein L is:.

134. The compound of any one of claims 113-120, wherein L comprises a chemical handle.

135. The compound of claim 134, wherein L is:

136. The compound of any one of claims 113-120, wherein L is: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

137. The compound of any one of claims 113-136, wherein the compound has the formula: (VA-1), (VA-2), or (VA-3):

138. A compound with the chemical structure (VA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; R12represents H, Me, halogen, or NO2; Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents:–(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; orL comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (VA) can exist in the following tautomeric forms:.

139. A compound with the chemical structure (VB) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R11is H, an alkyl group, or Ac; Y3is O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: l ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (VB) can exist in the following tautomeric forms:.

140. A fluorescent probe comprising a compound as described in any one of claims 59 to 139 coupled to a biological macromolecule or material.

141. A compound with the chemical structure (VIA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R12represents H, Me, halogen, or NO2; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; and L represents a coupling agent wherein, when R1is CO2R’, the compound (VIA) can exist in the following tautomeric forms:.

142. A compound with the chemical structure (VIB) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker is a linking group; andL represents a coupling agent wherein, when R1is CO2R’, the compound (VIB) can exist in the following tautomeric forms: L.

143. The compound of claim 141 or 142, wherein Linker is:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group.

144. The compound of claim 141 or 142, wherein Linker is:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; andwhere Y1is Het and Y2is –C(O)–, –NH–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, or –C(O)–NR11–.

145. The compound of claim 142 or 144, wherein Het is a 1,2,3-triazole heterocycle.

146. The compound of claim 145, wherein the 1,2,3-triazole heterocycle is:.

147. The compound of claim 142 or 144, wherein Het is a pyridazine or dihydropyridazine heterocycle.

148. The compound of claim 147, wherein the pyridazine or dihydropyridazine heterocycle is:where R10is H or Me.

149. The compound of any one of claims 141-148, where L is a ligand for a self-labeling enzyme.

150. The compound of claim 149, wherein L is:, where t is 2-8 and u is 2 to 10.

151. The compound of claim 149, wherein L is :.

152. The compound of claim 149, wherein L comprises benzylguanine or chloropyrimidine.

153. The compound of claim 152, wherein L is:.

154. The compound of claim 149, wherein L comprises benzylcytosine.

155. The compound of claim 154, wherein L is:.

156. The compound of any one of claims 141-148, wherein L comprises a maleimide group.

157. The compound of claim 156, wherein L is:.

158. The compound of any one of claims 141-148, wherein L comprises a N- hydroxysuccinimide (NHS) group.

159. The compound of claim 158, wherein L is:

160. The compound of any one of claims 141-148, wherein L comprises a biotin group.

161. The compound of claim 160, wherein L is:.

162. The compound of any one of claims 141-148, wherein L comprises a chemical handle.

163. The compound of claim 162, wherein L is:

164. The compound of any one of claims 141-148, wherein L is: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

165. The compound of any one of claims 141-164, wherein the compound has the formula (VIA-1), (VIA-2); (VIA-3); (VIA-4); or (VIA-5):.

166. A compound with the chemical structure (VIA) or a salt thereof:where: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; R12represents H, Me, halogen, or NO2; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (VIA) can exist in the following tautomeric forms:.

167. A compound with the chemical structure (VIB) or a salt thereof: Lwhere: each R independently represent H, Me, Et,–CH2OAc, or a counter cation; R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R2and R3independently represent H, F, or Cl; each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; Linker represents:–(CH2)m–, –(CH2)m–Y1–(CH2)n–, –(CH2)m–Y1–(CH2)n–Y2–(CH2)o–, –(CH2)m–(CH2CH2O)p–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–, –(CH2)m–(CH2CH2O)p–(CH2)n–Y1–(CH2)o–(CH2CH2O)q–(CH2)r–Y2–(CH2)s–, –(CH2)m–(OCH2CH2)p–, –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–, or –(CH2)m–(OCH2CH2)p–(CH2)n–Y1–(CH2)o–(OCH2CH2)q–(CH2)r–Y2–(CH2)s– where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group selected from the group consisting of:L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; orL comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac; wherein, when R1is CO2R’, the compound (VIB) can exist in the following tautomeric forms:L.

168. A fluorescent probe comprising a compound as described in any one of claims 141 to 167 coupled to a biological macromolecule or material.

169. A compound with the chemical structure (VII) or a salt thereof: Lwhere: R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R3independently represent H, F, or Cl; R4represents H, a counter cation, an alkyl, or an acyloxyalkyl group R6represents H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; each R independently represent H, Me, Et,–CH2OAc, or a counter cation; Linker1and Linker2are linking groups; “Na / K Chelator” is a chelator selective for sodium or potassium ions; and L represents a coupling agent.

170. The compound of claim 169, wherein the Na / K Chelator is a cryptand selective for sodium or potassium ions.

171. The compound of claim 170, wherein the cryptand selective for sodium or potassium ions c172. The compound of claim 169, where the Na / K Chelator is a crown ether or aza-crown ether selective for sodium or potassium ions.

173. The compound of claim 172, wherein the Na / K Chelator is a monoaza-15-crown-5, monoaza-18-crown-6, diaza-15-crown-5, diaza-18-crown-6.

174. The compound of claim 173, wherein the Na / K Chelator is (C-5), (C-6), or (C-7):; where:R6is H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4; and R7is H or Me.

175. The compound of any one of claims 169-174, wherein Linker1and Linker2are each independently:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; and Het represents a heterocyclic group.

176. The compound of any one of claims 169-174, wherein Linker1and Linker2are each independently:where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; and where Y1is Het and Y2is –C(O)–, –NH–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, or –C(O)–NR11–.

177. The compound of claim 175 or 176, wherein Het is a 1,2,3-triazole heterocycle.

178. The compound of claim 177, wherein the 1,2,3-triazole heterocycle is:.

179. The compound of claim 175 or 176, wherein Het is a pyridazine or dihydropyridazine heterocycle.

180. The compound of claim 179, wherein the pyridazine or dihydropyridazine heterocycle is:where R10is H or Me.

181. The compound of any one of claims 169-180, where L is a ligand for a self-labeling enzyme.

182. The compound of claim 181, wherein L is:, where t is 2-8 and u is 2 to 10.

183. The compound of claim 181, wherein L is :.

184. The compound of claim 181, wherein L comprises benzylguanine or chloropyrimidine.

185. The compound of claim 184, wherein L is:.

186. The compound of claim 181, wherein L comprises benzylcytosine.

187. The compound of claim 186, wherein L is:.

188. The compound of any one of claims 169-180, wherein L comprises a maleimide group.

189. The compound of claim 188, wherein L is:.

190. The compound of any one of claims 169-180, wherein L comprises a N- hydroxysuccinimide (NHS) group.

191. The compound of claim 190, wherein L is:

192. The compound of any one of claims 169-180, wherein L comprises a biotin group.

193. The compound of claim 192, wherein L is:.

194. The compound of any one of claims 169-180, wherein L comprises a chemical handle.

195. The compound of claim 194, wherein L is:

196. The compound of any one of claims 169-180, wherein L is: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

197. The compound of any one of claims 169-196, wherein the compound has the formula: (VII-1), (VII-2), (VII-3), (VII-4), (VII-5), (VII-6), (VII-7), (VII-8), (VII-9), (VII-10), (VII-11), or (VII-12):(VII-1);where R4and R8is H, a counter cation, an alkyl, or an acyloxyalkyl group and R7is H or Me; each R3independently represent H, F, or Cl; W1 is NH2, N(Me)2, N(Et)2, OH, OAc, or OAlkyl; andR6is H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4; and R7is H or Me.

198. A compound with the chemical structure (VII) or a salt thereof: Lwhere: R1is H, Me, OR, CO2R’, SO3R’, NR2, SO3NR2, OC(O)R; R’ is H or a counter cation each R3independently represent H, F, or Cl; R4represents H, a counter cation, an alkyl, or an acyloxyalkyl group R6represents H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4 each W1and W2independently represent NH2, N(Me)2, N(Et)2, O, OH, OAc, or OAlkyl; each R independently represent H, Me, Et,–CH2OAc, or a counter cation; where “Na / K Chelator” is a chelator selective for sodium or potassium ions, wherein the chelator is selected from the group consisting of ;R6is H, alkyl, or –(CH2CH2O)p-CH3; p = 1-4; and R7is H or Me; Linker represents: ,where m is 0-4; n is 0-4; o is 0-4; p is 0-4; q is 0-4; r is 0-4; s is 0-4; each Y1and Y2independently represent O, NR11, –C(O)–, –O–C(O)–, –C(O)–O–, –NR11–C(O)–, –C(O)–NR11–, or Het; R11is H, an alkyl group, or Ac; Het represents a heterocyclic group selected from the group consisting of:; where R10is H or Me; L represents a ligand for coupling to a protein selected from the group consisting of: ,, , , , where t is 2-8 and u is 2 to 10; or L comprises a functional group selected from the group consisting of:L represents a functional group selected from the group consisting of: –OH, –NHR11, –O–C(O)–OR9, –O–C(O)–NHR11, –C(O)–OR9, –C(O)–NHR11, –NR11–C(O)– OR9, or –NR11–C(O)–NHR11, where R9is H, alkyl, counter cation, or acyloxyalkyl and R11is H, an alkyl group, or Ac.

199. A fluorescent probe comprising a compound as described in any one of claims 169 to 198 coupled to a biological macromolecule or material.

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