Bioorthogonal Turn-on Probes

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application Serial No. 61/81.9,913, filed on May 6, 2013, which is incorporated by reference in its entirety.

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TECHNICAL FIELD

This present application relates to fluorescent tetrazine-containirig compounds consisting of a single pi-system. Also provided herein are methods of using the compounds provided herein for biomedical imaging. BACKGROUND

Visualizing biomolecular processes has been enhanced by combining fluorophores with bioorthogonal chemistry, resulting in new tools to study the complex biochemical milieu of living cells and organisms. (N, K. Devaraj, et al., Angew. Chem. 2009, 121, 7147-7150; M.

Boyce and C. R. Bertozzi, Nat. Methods 2011, 8, 638; and C. L. Droumaguet and C. Wang, Q. Wang, Chem. Soc. Rev. 2010, 39, 1233.) The resulting probes have been applied to image glycosylation and phospholipid uptake, cellular proteins, and intracellular drug distribution. (P.

Shieh, et aL J Am. Chem. Soc. 2012, 134, 17428; S. T. Laughlin, et ai., Science 2008, 320, 664;

M. J. Ha.nga.uer and C, R. Bertozzi, Angew. Chem. 2008, 120, 2353; J. Yang, et. al, Angew, Chem.

2012, 124, 7594-7597: K. Lang, et al, Nat. Chem. 2012, 4, 298; D. S. Liu, et al., J Am. Chem. Soc. 20.12, 1.34, 792; N. . Devaraj, et al, Bioconjug. Chem. 2008, 19, 2297; i. Z. Yao, et al, J.

Am. Chem. Soc. 2012, 134, 3720; K. S. Yang, et Angew. Chem. 2012, 124, 6702-6707; G.

Budin, et al, Angew. Chem. 201 1 , 123, 9550-9553; T. Reiner, et al, Chemhiochem 2010, 1.1,

2374.)

Bioorthogonal "click." chemistries are widely used in chemical biology for a myriad of applications such as activity based protein profilmg, crosslinkmg of proteins, monitoring cell proliferation , generation of novel enzyme inhibitors, monitoring the synthesis of newly formed proteins, protein target identification, and studying glycan processing. Perhaps the most fascinating applications involve using these bioorthogonal chemistries to assemble molecules in the presence of living systems such as live cells or even whole organisms (Baskin et al, 2007, Proc Natl Acad Sci U S A, 104, 16793-7; Laughlin et ah, 2008, Science, 320, 664-7; Prescher and Bertozzi, 2005, Nat Chem Biol, 1, 13-21; Neef and Schultz, 2009, Angew Chem hit Ed Engl, 48, 1498-500; Ning et al., 2008, Angewandte Chemie-Intemational Edition, 47, 2253- 2255). These latter applications require that the chemistry' be non-toxic and possess kinetics that allow fast reaction to occur with micronioiar concentrations of reagents in a time span of minutes to hours.

To fulfill these criteria, various "copper-free" click chemistries have been reported, such as the strain-promoted azide-alkyne cycloaddition and the Staudinger ligation, to react with azides on the surface of live cells both in culture and in. in vivo systems such as mice and zebrafish (Prescher and Bertozzi, 2005, Nat Chem Biol, 1, 13-21). However, to date, the application of "click" chemistry in living systems, has been largely limited to extracellular targets and no technique has shown reliable ability to specifically label and image intracellular targets (Baskin and Bertozzi, 2007, QSAR Comb. Sci, 26, 1211-1219). The reasons for this are likely several. In addition to fulfilling the stability, toxicity, and diemoseleetivity requirements of "click" chemistry, intracellular live cell labeling requires reagents that can easily pass through biological membranes and kinetics that enable rapid labeling even with the low concentrations of agent that make it across the cell membrane. Additionally, a practical intracellular bioorthogonal coupling scheme would need to incorporate a mechanism by which the fluorescent tag increases in fluorescence upon covalent reaction to avoid visualizing accumulated but unreacted imaging probes (i.e. background). This "turn-on" would significantl increase die signal-to- background ratio, which is particularly relevant to imaging targets inside living cells since a stringent w ⁷ ashout of unreacted probe is not possible.

In previous years a number of elegant probes have been introduced whose fluorescence increases after azide-alkyne cycloaddition or staudinger ligation coupling reactions (Sivakumar et al, 2004, Org Lett, 6, 4603-6; Zhou and Fahrni, 2004, J Am Chem Soc, 126, 8862-3;

Hangauer and Bertozzi, 2008, Angew Chem Int Ed Engl, 47, 2394-7: Lemieux et ah, 2003, J Am Chem Soc, 125, 4708-9). Most of these strategies either require a reactive moiety intimately attached to the fmorophore thus requiring synthesis of new fluorophore scaffolds or take advantage of a F RET based activation requiring appendage of an additional molecule that can act as an energy transfer agent. Furthermore, most probes utilizing these popular- coupling schemes have to date been unable to label intracellular targets in live cells.

The bioorthogonal Diels- Alder reaction is compatible with aqueous environments and has second order rate constants that are kno wn to be enhanced up to several hundred-fold in aqueous media in comparison to organic solvents. (Rideout DC et al., 1980, J Am Chern Soc 102:7816- 7817; Graziano G, 2004, J Phys Org Chem 17: 100-101). Many Dieis-Alder reactions are reversible, therefore, they may not be suitable for biological labeling. (Kwart et a,L, 1968, Chem Rev 68:415-447), however, the inverse electron demand Dieis-Alder cycloaddition of olefins with tetrazines results in irreversible coupling giving dihydropyridazine products. During this reaction, dinitrogen is released in a retro Dieis-Alder step. (Sauer J et al., 1965, Chem Ber

998:1435-1445). A variety of tetrazines and dienophiles including cyclic and linear alkenes or alkynes have been studied in this reaction.. Selection, of the appropriate reaction partners, allows for tuning of the coupling rate by several orders of magnitude. (Balcar J et al., 1983, Tel Lett 24:1481 -1484; Thalhammer F et al., 1990, Tet Lett 47:6851-6854). See also US 2006/0269942, WO 2.007/144200, and US 2008/0181847.

In some previously described fiuorophores, the capacity of in situ chemical conjugation, has been paired with fluorogenic turn-on, whereby fluorophore emission increases upon reaction, with its bioorthogonal counterpart ("turn-on" probes). See, e.g., N. K. Devaraj, et al., Angew. Chem 2009, 121, 7147-7150. This has the very attractive feature of reducing background fluorescence when doing in vivo imaging, potentially allowing real time imaging, without washing or clearance steps. Existing methods that exploit azide-pliosphme, azide-alkyne, or inverse electron demand Dieis-Alder tetrazine cyeloadditions have not provided probes with high fluorescence turn-on ratios and fast kinetics without using a catalyst.

Provided herein are compounds of Formula (I) or (II):

or a salt thereof,

wherein: F is a fluorophore;

L is a conjugated linker; and

Tz is a substituted or imsubstituted tetrazine;

wherein the linker bridges the Tz and F moieties in a single conjugated pi-system.

Compound of Formula (I) or (II) can be reacted with a dienophile to prepare compounds of Formula (III) or (IV):

p L Z

F Z (IV) or a salt thereof,

wherein:

F is a fluorophore;

I. is a conjugated linker; and

Z is a moiety comprising the reaction product of a diene and a dienophile, wherein the diene is a tetrazine or a derivative thereof;

wherein the linker bridges the Z and F moieties in a single conjugated pi~system.

Unless otherwise defined, all technical and scientific tenns used herein have the same meaning as commonly understood by one of ordinary skill in the art to which th s invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety, in case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS FIG 1 is a. fluorescence emission spectra for compound 4b in acetonitrile at baseline (black) and after addition of TCO (green); excitation at 490nm. FIG, 2 illustrates normalized fluorescence turn-on after addition of TCO (240 μΜ) to a solution of the indicated fluorophore (1 μΜ) in aeetonitrile; initial intensity 1,0 a.u.

FIG. 3 is a photograph showing equimolar solutions of compound 2a (at left) and 2a plus TCO (at right) under excitation by a handheld UV lamp.

FIG. 4 is a 3D model of compound 4a, illustrating a twisted phenyl linker between the

BO DIP Y and Tz chromophores and the orientation of the donor and acceptor transition dipoles (gray arrows).

Fid 5 shows the absorption and fluorescence emission spectra of 4b in toluene and aeetonitrile.

FIG, 6 illustrates the fluorogenic activation of 4b in water.

FIG, 7 illustrates the biological application of the compounds provided herein. FIG. 7A shows fluorogenic imaging of EGFR expression on both fixed and live A431 cells. FIG. 7B are fluorogenic live-cell images of intracellular nanopartieles internalized by RAW 264.7 cells.

FIG, 8 illustrates a tetrazine transition, dipoie as collinear with or parallel to the transition dipoie of the fluorophore.

FIG. 9 shows the fluorogenic activation of HELIOS probes upon reaction with TCOc (H - HELIOS).

FIG. 10 shows the fluorescence emission, spectra of HELIOS 400Me in PBS (pH 7.4), aeetonitrile (dielectric constant 37.5), and toluene (dielectric constant 2.4).

Fid II A shows no wash fluorogenic imaging of EGFR. expression on A431 cells using

HELIOS 370H. Bright, membrane specific staining is visible within seconds, peaks within 3 minutes and is stable thereafter. FIG. 11B shows cells imaged with HELIOS 370H (left) and control cells (right) exhibiting autofluorescence at baseline, prior to addition of dye.

FIG. 1.2 illustrates no-wash fluorogenic imaging of intracellular targets. FIG, 12 A shows mitochondrial imaging of OVCA-429 cells with RFP-tagged mitochondria were incubated with an anti-mitochondria-TCO antibody, rinsed briefly, and then imaged after addition of 100 nrn HELIOS-388H in PBS. FIG. I2B shows actin imaging of COS-1 cells incubated with phalloidin-TCO (1 §/πιΙ,) and DRAQ5 nuclear counterstain (ΙμΜ, BioStatus), rinsed briefly, and then imaged, upon addition of the indicated HELIOS probe at lOOnM. Control images were collected at matched dye concentrations in- the absence of phalloidin-TCO. FIG. 13 illustrates in vivo imaging of a HELIOS probe as described herein.

DETAILED DESCRIPTION

Definitions

For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise. As used herein, the term "about" is meant to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term "about", whether or not the term is explicitly used, unless explicitly stated otherwise. As used herein, the singular- forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The term "alkyl" includes straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyi, heptyl, octyi, nonyl, decyl, etc.) and branched-chain alkyl groups (e.g., isopropyl. tert-butyl, isobiityl, etc.). In certain embodiments, a straight chain or branched chain alkyl has twelve or fewer carbon atoms in its backbone (e.g., Ci-C \z for straight chain; C3-C 12 for branched chain). The term Ci-C 12 includes alkyl groups containing 1 to 12 carbon atoms.

The term "aikenyl" includes aliphatic groups that may or may not be substituted, as described above for alkyls, containing at least one double bond and at least two carbon atoms. For example, the term "aikenyl" includes straight-chain aikenyl groups (e.g., ethenyl, propenyl, butenvl, pentenyl, bexenyl, heptenyl, octenyl, nonenyl, and decenyl) and branched-chain aikenyl groups. In certain, embodiments, a straight chain or branched chain aikenyl group has twelve or fewer carbon atoms in its backbone (e.g., C2-C12 for straight chain; C3-C 12 for branched chain). The term C2-C ₁₂ includes aikenyl groups containing 2 to 12 carbon atoms.

The term "alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond and two carbon atoms. For example, the term "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, and decynyl) and branched-chain alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has twelve or fewer carbon atoms in its backbone (e.g., C2-C 12 for straight chain; C3-C 12 for branched chain). The term C ₂ -C 12 includes alkynyl groups containing 2 to 12 carbon atoms. ^' The term "alkoxy" is used in its conventional sense, and refers to alky! groups linked to molecules via an oxygen atom. In some embodiments, an alkoxy has twelve or fewer carbon atoms in its backbone (e.g., a Ci-C n alkoxy). For example, Ci-C 10, O-C , Ci-C 0, C1-C 4, Ci-C 3, or C1-C 2. Non-limiting examples of an alkoxy group include metlioxy, ethoxy, propoxy, butoxy, and hexoxy.

The terms "halo" or "halogen", used alone or in combination with other terms, refers to fluoro, chloro, bromo and iodo.

The term "haloalkyl" refers to an alky! group in which one or more of the hydrogen atoms has been replaced by a halogen atom. The term "C _n -C _m haloalkyl" refers to a C _a -C m alkyl group having n to m carbon atoms, and from at least one up to {2(n to m)+l } halogen atoms, which may either be the same or different. In some embodiments, the halogen atoms are fluoro atoms. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Eixample haloalkyl groups include CF3, C2.F5, CHF ^' 2, CCT3, CHCb, C2CI5 and the like, in some

embodiments, the haloalkyl group is a fmoroalkyl group.

The term "haloalkoxy", employed alone or in combination with, other terms, refers to a group of formula -O-haloalkyl, wherein the haloalkyl group is as defined above. The term "C _n -C m haloalkoxy" refers to a haloalkoxy group, the haloalkyl group of which has n to m carbons. Example haloalkoxy groups include trifiuoroniethoxy and die like. In some embodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms,

The term "amino" refers to a group of formula. -NH2.

The term "carbamyl" refers to a group of formula -C(0)NH ₂ .

The term "carbonyl", employed alone or in combination with other terms, refers to a -C(=0)- group, which also may be written as C(O).

The term "oxo" refers to oxygen as a divalent, substituent, forming a carbonyl group, or attached to a heteroatom forming a sulfoxide or sulfone group, or an N-oxide group.

^' The term "aromatic" refers to a carbocycle or heterocycle having one or more

polyunsaturated rings having aromatic character (i.e., having (4n + 2) delocalized π (pi) electrons where n is an integer).

The term "carbocyciyl" includes a cyclic aliphatic group which may be saturated or unsaturated. For example, carbocyciyl groups include cyciopropyl, cyclopentyl, cyclohexyl. cycloheptyl, and cyelooctyl. In some embodiments, carbocyclyls have from 3-8 carbon atoms in their ring structure, for example, they can have 3, 4, 5 or 6 carbons in the ring structure.

In general, the term "aryl" includes groups, including 5- and 6-membered single-ring aromatic groups, such as benzene and phenyl. Furthermore, the term "aryi" includes multi cyclic aryl groups, e.g., tricyclic, Bicyclic, such as naphthalene and anthracene.

The term "heteroaryl" includes groups, including 5- and 6- membered single-ring aromatic groups, that have from one to four heteroatoms, for example, pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazoie, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, the term "heteroaryl" includes multicyclic heteroaryl groups, e.g., tricyclic, bicyclic, such as benzoxazole, benzodioxazole, benzothiazole, benzoimidazoie, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthyridme, indole, benzofuran, purine, benzofuran, quinazoline, deazapurine, indazole, or indolizine.

The term "heterocyclyl" includes non-aromatic groups, including but not limited to, 3- to 10-membered single or multiple non-aromatic rings having one to five heteroatoms, for example, oxetane, piperazine, pyrrolidine, piperidine, or homopiperazine.

The term "substituted" means that an. atom, or group of atoms replaces hydrogen as a "substituent" attached to another group. For aryl and. heteroaryl groups, the term "substituted", unless otherwise indicated, refers to any level of substitution, namely mono, di, tri, tetra, or penta substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In some cases, two sites of substitution may come together to form a 3-10 membered carbocyclyl or heterocyclyl ring.

Substituents include, but are not limited to, Cy ¹ , Ci-6 aikyl, C2-0 alkenyl, C2-6 alkynyl, halo, Ci-6 haloalkyl, CN, NC% OR ^al , SR ^al , C(0)R ^bl , C(0)NR ^cl R ^dl , C(0)OR ^al , OC(0)R ^bf , OC(G)NR ^ci R ^dl , C(= R ^el )NR ^cl R ^dl , NR ^cl C(=NR ^el )NR ^cl R ^dl , NR ^cl R ^di , NR ^cl C(0)R ^bl ,

NR ^cl C(0)OR ^al , NR ^ci C(G)NR ^cl R ^dl , NR ^cl S(0)R ^bl , NR ^ci S(0) ₂ R ^bl , NR ^cl S(0) ₂ NR ^c! R ^di , S(0)R ^l , S(O)NR ^0t R ^di , S(0) ₂ R ^bl and S(0)2NR ^cl R ^di ;

each Cy ³ is independently Ce-io aryl, C3-10 carbocyclyl, 5-10 membered heteroaryl or 4-10 membered heterocyclyl, each, of which is imsubstituted or substituted by 1, 2, 3, 4 or 5 substituents independently selected from Cw alkyl, C2-0 alkenyl, C2-6 alkynyl, halo, CN, NC¾, OR ^al , SR ^al , C(0)R ^bl , C(0)NR ^cl R ^dl , C(0)OR ^al , OC(())R ^bi , OC(0)NR ^cl R ^dl , C( ^:::: NR ^cl )NR ^ci R ^dl , NR ^c¾ C(-NR ^el )NR ^0¾ R ^dl , NR ^cl R ^d3 , NR ^c, C(0)R ^bl , NR ^ci C(0)OR ^a \ NR ^0l C(O)NR ^cl R ^dl ,

NR ^ci S(0)R ^bl , NR ^cl S(0) ₂ R ^bi , NR ^cl S(0) ₂ NR ^ol R ^dl , S(0)R ^bl , S(0)NR ^cl R ^di , S(0) ₂ R ^bl ,

S(0) ₂ NR ^c, R ^di and oxo; - each R ^al , R ^l , R ^cl , R ^dl , is independently selected from H, Cw alkyl C1-4 haloalkyi, C2-5 alkenyl, C2-6 alkynyl, Ce-Hi aryl, Cs-io carbocyclyl, 5-10 membered heteroaryl, 4-10 membered heterocyclyi, C6-10 aryl-Ci-4 alkyl, Cs-io carbocyclyl-Ci-4 alkyl, (5-10 membered heteroaryl)-C _1-4 alkyl or (4-10 membered heterocyclyi)-Ci-4 alkyl, wherein said Ct-e alkyl, C2-6 alkenyl, C2-6 alkynyl, Ce-ιο and, Cs-i o carbocyclyl, 5-10 membered heteroaryl, 4-10 membered heterocyclyi, Ce-io aryl-d-4 alkyl, C3-10 carbocyelyl-Ci-4 alkyl, (5-10 membered heteroaryl)-Ci-4 alkyl and (4-10 membered heterocyclyl)-Ci-4 alkyl is optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from CM alkyl, CM haloalkyi, halo, CN, OR ⁸⁴ , SR* ⁴ , C(0)R ^M ,

C(())NR ^c4 R ^d4 , C(0)OR ^a4 , OC(0)R , OC(0)NR ^c R ^d4 , N R ^{O I} R ^L . NR ^c4 C(0)R ^b4 ,

NR ^c C (0)NR ^c4 R ^d4 ₅ NR ^c4 C(Q)OR ^a4 , C(= R ^e4 )NR ^o4 R ^d4 , NR ^o4 C(=NR ^e4 ) R ^c4 R ^d4 , S(0)R ^M , 8(0)NR ^c4 R ⁴ , S(()) ₂ R ⁴ , NR ^c 8(G) ₂ R ^b4 , N:R ^c4 8(0) ₂ NR ^c4 R ^d4 and S(0) ₂ NR ^c4 R ^d4 ;

each R ^a4 , R ^b4 , R ^c4 and. R ^d4 is independently selected from H, CM alkyl. CM haloalkyi, C ₂ - 4 alkenyl. and C2-4 alkynyl, wherein said CM alkyl, C2-4 alkenyl, and C2-4 alkynyl, is optionally substituted with 1, 2, or 3 substituents independently selected from OH, CN, amino, halo, CM alkyl. C alkoxy, C1-4 alkyl.th.io, CM aikylamino, di(C alkyl)amino, C M haloalkyi, and CM haloalkoxy:

Where substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, for example,— CH2O— is equivalent to— OCH2— . in some embodiments, one or more substituents can be a group reactive with a biologically active molecule or a detectable agent.

The abbreviation "PEG" as used herein refers to a polyethylene glycol polymer.

A "reactive moiety" as used herein refers to a reactive moiety for conjugation between the fluorophore and/or the dienophile and a biologically active compound (e.g., an antibody). Non-limiting examples of reactive moietys include hydroxyl, amine, thiol, carboxvl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, d

wherein n is an integer

As used herein, chemical structures which contain one or more stereocenters depicted with dashed and bold bonds (i.e., ^" are meant to indicate absolute stereochemistry of the .stereocenter(s) present in the chemical structure. As used herein, bonds symbolized by a simple line do not indicate a stereo-preference. Unless otherwise indicated, chemical structures, which include one or more stereocenters, illustrated herein without indicating absolute or relative stereochemistry encompass all possible stereoisomeric forms of the compound (e.g.,

diastereorners, enantiomers) and mixtures thereof (e.g., racemic mixtures). Structures with a single bold or dashed line, and at least one additional simple line, encompass a single

enantiomeric series of ail possible diastereorners.

Resolution of racemic mixtures of compounds can be carried out by any of numerous methods known in the art. An exemplary method includes fractional recrystailization using a chiral resolving acid which is an optically active, salt-forming organic acid. Suitable resol ving agents for fractional recrystailization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, maiidelic acid, malic acid, lactic acid, or the various optically active camphor sulfonic acids such as camphor sulfonic acid. Other resolving agents suitable for fractional crystallization methods include

stereoisomerically pure forms of methylbenzylamine (e.g., S and R forms, or diastereomerieally pure forms), 2-phenylgiycinol, norephedrine, ephedrme, N-methyiephedrine,

cyclohexylemyiamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by one skilled in the art. The term "counter anion" as used herein is intended to include any counter anions of inorganic and organic acids. Exemplary anions include, but are not limited to: chloride, bromide, iodide, nitrate, sulfate, bisulfate, sulfite, bisulfite, phosphate, acid phosphate, perchlorate, chlorate, chlorite, hypochlorite, periodate, iodate, iodite, hypoiodite, carbonate, bicarbonate, isonicotinate, acetate, trichloroacetate, trifluoroacetate, lactate, salicylate, citrate, taitrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate. formate, benzoate, glutamate, methanesuifonate.

trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, ρ-toluenesulfonate, p- triiluoromethyibenzenesulfonate, hydroxide, aluminates, and borates.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "salt" includes any ionic form of a compound and one or more counter-ionic species (cations and/or anions). Salts also include zwitterionic compounds (i.e., a molecule containing one more cationic and anionic species, e.g., zwitterionic amino acids). Counter ions present in a salt can include any cationic, anionic, or zwitterionic species. Exemplar} _' anions include, but are not limited to: chloride, bromide, iodide, nitrate, sulfate, bisulfate, sulfite, bisulfite, phosphate, acid phosphate, perchlorate. chlorate, chlorite, hypochlorite, periodate, iodate, iodite, hypoiodite, carbonate, bicarbonate, isonicotinate, acetate, trichloroacetate, trifluoroacetate, lactate, salicylate, citrate, taitrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesuifonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, p-trifluoromethyibenzenesulibnate, hydroxide, aluminates, and borates.

Exemplar}-' cations include, but are not limited to: monovalent alkali metal cations, such as lithium, sodium, potassium, and cesium, and divalent alkaline earth metals, such as beryllium, magnesium, calcium, strontium, and barium. Also included are transition metal cations, such as gold, silver, copper and zinc, as well as non-metal cations, such as ammonium salts.

1.1 Also provided herein are pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organi c acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the compounds provided herein include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the compounds provided herein can be synthesized from the paren t compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the -appropriate base or acid in water or in an organic sol vent, or in a mixture of the two; in some embodiments, a non-aqueous media like ether, ethyl acetate, aicohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (ACN) can he used. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed.. Mack Publishing Company, Easton, Pa., 1985, p. 1418 ^' and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salts are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

The term "essentially pure" refers to chemical purity of a compound provided herein that may be substantially or essentially free of other components which normally accompany or interact with the compound prior to purification. By way of example only, a compound may be "essentially pure" when, the preparation of the compo und, contains less than about 30%, less than about 25%, less than about 20%. less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, an "essentially pure" compound may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%t, about 98%, about 99% or greater. For the purposes of this document, preparations of mnctionalized polymers or conjugates differing only in the length of their polymer chain are considered to be essentially pure. An essentially pure compound may be obtained using chromatographic purification methods. As used herein, a "turn-on ratio" is a measure of the increase in fluorescence associated with probe activation and is the ratio of the fluorescence after and before activation of a compound provided herein. For example, a turn-on ratio for a compound of Formula (Hi) can be calculated by taking the ratio of the fluorescence intensity of a compound of Formula (III) over the fluorescence intensity of the corresponding compound of Formula (I), See, e.g., Thompson, M.A. et al., Methods Enzymol 2010 475: 27-59.

As used herein, "conjugated" refers to a compound having alternating single and multiple bonds and containing a system of connected p-orbitals with delocalized electrons.

Compounds

A fluorogenic probe enables the intrinsic fluorescence of a chromophore to be stably, but reversibly, suppressed. This can be accomplished by making customized modifications that directly perturb the intrinsic fiuorophore, or, more generally, by linking a fluorophore to moieties that can quench its fluorescence via photoinduced electron transfer (PET) or via energy transfer, e.g. Forster resonance energy transfer (FRET). Subsequent disruption of the quenching group restores the light emission. An optimal probe of this type thus requires both highly efficient quenching and a facile, selective mechanism to modulate the quencher— -either by cleavage of the linker or chemical reaction of the quencher itself.

Tetrazine-based probes achieve their fluorogenic turn-on by the latter route, as the tetrazine (Tz) chromophore can be characterized as both a quencher (via energy transfer) and a bioorthogonal reactant. First generation Tz-fiuorophore probes relied on FRET, a through-space transfer mechanism that depends on distance, molecular alignment, as well as the intensity and alignment of emission-absorption bands, all of which can limit energy transfer efficiency. As a result, although they possess many desirable characteristics, including very fast, catalyst free, chemical reactivity and high brightness after turn-on, these probes were limited by fluorescence turn-on ratios of just 10-20 fold (i.e. 90-95% baseline quenching). Similar tradeoffs limit most PET-based probes in the literature, as many highly efficient quenchers have no intrinsic bioorthogonal reactivity, and reactive moietys, such as azi de-modified fluorophores, have modest quenching ability and comparatively slow reaction kinetics. In contrast, through-bond energy transfer (TBET) enables the Tz-based probes provided herein to display both exceptional turn-on and chemical reactivity. In the present system, TBET is markedly more efficient than FRET, achieving turn-on ratios of greater than 10 and in some cases greater than 10 ³ (>99.9% baseline quenching). Several practical features can enhance TBET relative to FRET, including insensitivity to spectral overlap (allowing donor

chroniophores to excite significantly red-sbifted acceptors), decreased dependence on donor acceptor dipole alignment, and accelerated energy transfer kinetics (due to the lack of a strict orientational requir em ent) .

Many of the previously described tetrazine-based probes achieve their fluorogenic turn- on by a unique mechanism, in which the tetrazine (Tz) chrornophore is both quencher and bioorthogonal reactant. In published studies, flexibly-linked. Tz-fluorophore pairs-— chosen for their ready synthetic accessibility are quenched with moderate efficiency, yielding turn-on ratios on the order of 10-20 fold after reaction with dienophile targets. Although intriguing applications have been demonstrated, the limited turn-on ratios almost always result in native background during imaging applications. In some cases, a larger turn-on ratio is required for useful imaging. For example, a turn-on ratio of 10"' would be preferable for robust utility in cellular imaging applications, and a ratio of 10 ^J ma be necessary for low abundance targets and. super resolution imaging. Mechanistic observations have suggested that quenching in

bicnromophoric iluorophore-tetrazines occurs via Forster resonance energy transfer (FRET), offering a starting point for efforts to optimize turn-on. Although its relatively weak visible light absorbance inherently limits the range of tetrazine as a FRET acceptor, Forster theory dictates that energy transfer efficiency will be crucially dependent upon inter-chromophore distance (varying as r ⁶ ) and. upon transition dipole alignment, which are both optimizable parameters. Without being bound by theory, an alternative way of designing more efficient turn-on probes, involves adapting through-bond energy transfer (TBET) for fluorescence quenching as is illustrated by the compounds provided herein.

Accordingly, provided herein are compounds of Formula (I):

F—— L—— Tz

or a salt thereof,

wherein: F is a fluorophore;

L is a conjugated linker; and

Tz is a substituted or unsubstituted tetrazine;

wherein the linker bridges the Tz and F moieties in a single conjugated pi-system.

The single, conjugated pi-system may, in some embodiments, be non-coplanar due to steric factors within the compound that enforce a twist in the mterchromophore linkage between the F and L-Tz moieties. Such steric factors can originate from substituents on either the linker or on the fluorophore. Without being bound by theory, fluorescence can be increased when the L-Tz moiety is oriented with respect to the F moiety such that the tetrazine transition dipole is either collinear with or parallel to the transition dipole of the fluorophore (see, e.g., FIG. 8). In some embodiments, the pi-system is topologica!ly conjugation (i.e., a single systems of alternating single and double bonds). In other embodiments, for example, when the pi-system is coplanar, the conjugation of the pi-system is both topologically and functionally (i.e., wherein the alternating single and double bonds are coplanar) conjugated.

In some embodiments, the compounds provided herein enhance spatial donor-acceptor proximity, provide predictable donor-acceptor transition dipole orientation, and/or afford, the possibility of accessing alternate modes of fluorescence quenching.

A "fluorophore", as described herein, can be any small molecule that can re-emit light upon light excitation (e.g., light in the visible spectrum). For example, fluorophores can include rhodamines, fluoresceins, boron-dypyrromethanes, coumarins, pyrenes, cyanines, oxazines, acridines, auramine Os, and derivatives thereof. In some cases, derivatives include sulfonated derivatives such as sulfonated pyrenes, sulfonated coumarins, sulfonated rhodamines, and sulfonated cyanines (e.g., ALEXAFLUOR® dyes),

In particular-, boron-dypyrromethanes fluorophores may include boron-dipyrromethene (BODIPY®); 4,4-difluoro-5,7-dimemyl-4-bora-3a,4a-diaza-s-mdacene-3-prop ionic acid

(BODIPY® FL); 6-((4,4-difluoro-l,3-dimethyl-5-(4-m.ethoxyphen.yi)-4-bora-3 a,4a-diaz.a-s- indacene-2-propionyi)amino)liexanoic acid, succinimidyl ester (BODIPY® TRM-X); 6-(((4,4- dMuoro-5-(2-pyfrolyl)-4-bora-3a,4a-diaza-s-mdacene-3-yi)s1y acid, succinimidyl ester (BODIPY® 650/665-X); 5,5-difluoro-l, 3,7,9, 10-pentamethyl-5H- dipyrrolo[l ,2-c:2', Γ-f] [1 ,3,2]diazaboiiDin-4-ium-5-uide; 4,4~Difluoro-l,3,5,7-Tetramemyl-4- Bora-3a,4a-Diaza-s-Indacene-8-Propionic Acid; 4,4-Difluoro-5,7-Dimet yl-4-Bora-3a _> 4a-Diaza- s-Indacene-3 -Propionic Acid; 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-5-jjidacene-3- Pentanoic Acid; 6-((4,4-Difluoro-5,7-DiiBethyl-4-Bora-3a,4a-Diaza-s-Indacene -3- Propionyl)amino)hexa.noic Acid; 4,4-Difluoro-5-PhenyI-4~Bora~3a ₅ 4a-Diaza-s-Indacene~3~ Propionic Acid; 4,4-Difluoro-5,7-Dipheiiyl~4-Bora-3a,4a-Diaza-s-indacene-3-P ropionic Acid; 6- ((4,4-Difluoro- 1 ,3-Dinietliyl-5-(4-Methoxyphenyl)-4~Bora~3a ₅ 4a-Diaza-s-Indacene-2- Propionyl)amino)hexanoic Acid; 4,4-Difl oro-5-(2-TWenyl)-4-Bora-3a,4a-Diaza-s-JjQdacene-3- Propionic Acid; 4 ₅ 4-Difl oro-5-St. Tyl-4-Bora-3a,4a-Diaza-s~Indacene-3-Propionic Acid; 4,4- Ditluoro-5-(2-Pyrrolyl)^-Bora-3a,4a-Diaza-s-Indacene-3-Propi omc Acid; 4,4-Difiuoro-5-(4- Phenyl- 1 ,3 -Butadien l)-4-Bora-3 a,4a-Diaza~s-Indacene-3 -Propionic Acid; 6~(((4-(4,4-Difluoro- 5~(2-TWeriyl)-4 Bora-3a,4a-Oiaza-s ^ Acid; 6-

(((4,4-Difluoro-5-(2-Thienyi)-4~Bora~3a,4a-Diaza-s-Indace ne-3- yi)styryloxy)acetyi)arainohexaiioic Acid; 6-(((4,4-Difluoro-5-(2-PyrrolyI)-4-Bora-3a,4a-Diaza-s- Indacene-3-yl)Styryloxy)Acetyi)Aminohexanoic Acid; 5- utyi-4,4-difluoro-4- ora-3a,4a~d.iaza~ s-indacene-3-nonanoic acid; 5-decyi-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propion ic acid; 4,4-difluoro-5,7-dimeth.yl-4-bora-3a,4a-diaza-s-indacene-3-d od acid; 4,4-difluoro-

5,7-dimethyl-4-bora-3 a,4a-diaza-s-indacene-3 -hexadecanoic acid; 4,4-difluoro-5 , 7-dimethy! -4- bora-3a,4a-diaza-s-i]idacene-3-pentanoic acid; 4,4-difl oro-5,7-diraethyl-4-bora-3a,4a-diaza-s- indacene-3-undecanoic acid; 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3- dodecanoic acid; 4,4~diiluoro-5-methyi-4-bora-3a ₃ 4a-diaza-s-indacen.e-3-dodecanoic acid; 4,4- difliioro-5-ociyl-4-bora-3a,4a-diaza-s-indacene-3"pentanoic acid; 4,4-di£tuoro-5-(4-phenyl-l,3- butadienyl)-4-bora-3a ₅ 4a-diaza-s-indacene-3-pentanoic acid; 4,4-difluoro-5-(4-pher¾yl-l,3~ butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid: 4,4-difluoro-5-(2-thienyl)-4-bora- 3 a,4a-diaza-s-indacen.e-3 -dodecanoic acid; 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-dodecanoyl)-l-hexadecanoyl-sn-glycero~3-phosphoch oline; 2-(4.4-difluoro-5,7- dimethyM-bora-3a,4a-diaza-s-indacene-3-penta^

phosphocholine; 2-(4,4-difluoro-5-methyl-4-bora--3a,4a-diaza-s--indacene-3-d odecanoyl)-l- hexadecanoyl-sn-glycero-3- phosphocholine; 2~(4,4-difluoro-5-octyl-4-bora-3a,4a diaza-s~ indacene-3-pentanoyl)-l-hexadecanoyl-sn-glycero-3-phosphocb. oline; 2-(4,4-difluoro-5-(4- phenyl- 1 ,3 -butadienyl)-4-bora-3 a,4a-diaza-s-indacene-3 -pentanoyl)- 1 -hexadecanoyl-sn-glycero- 3- phospliocIioline: 2-(4,4-diiluoro~5,7-dimethyl-4-b^

hexadecanoyl-sn-glycero-3-phosphate; N-(4,4-difiuoro-5,7~dim^

indacene-3-propionyl)-l,2-dihexadecanoyl-sn-glycero-3-pho sphoethanolamine; N-(4,4-difluoro- 5,7~dimethyl~4~bora~3a,4a-diaza-s~m^

phosphoethanolamine; cholesteryl 4 ₅ 4-dffluoro-5,7-dimethyl-4-bom-3a,4a-diaza-s ndacene-3- dodecanoate; cholesteryl 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3 a,4a-diaza-s-indacene-3 - undecanoate; cholesteryl 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3- undecanoate; 4,4-difluoro- 1 ,3 ,5,7,8-pentame b.yl-4-bora-3 a,4a-diaza-s-indacerie; 4 ₅ 4-difluoro- l,3 _; 5,7-tetrattiethyl-4-bora-3a,4a-diaza-s-indacene; N-(4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a- Diaza-s-li lacene-3-yl)Methyl)Iodoacetamide; N-(4,4-Difluoro~ 1,3,5 ,7-Tetramethyl-4-Bora- 3a,4a-Diaza-s-Indacene-2-yl)Iodoacetamide; 8-Bromomethyl-4,4-Dif{uoro-13 ₅ 5,7-Tetramethy!-

4- Bora-3a,4a-Diaza-s-Indacene; or salts and/or ester derivatives thereof. In some embodiments, a pharmaceutically acceptable salt includes sodium, diammonium, and triethylammonium. in some embodiments, a pharmaceutically acceptable ester derivative includes siiccinimidyl ester and sulfosuccinimidyl ester derivatives.

Also contemplated herein are the BODIP Y compounds described in Ulrich, G. et al.,

Angew. Chem. Int. Ed, 2008, 47; 1184-1201; Ziessel, R. et aL, New J. Chem. 2007, 31 : 496-501 ; and Louclet, A. and Burgess, K., Chem. Rev. 2007, 107: 4891-4932, Jiang, X-D et al, Org.

Letters 2012 14(1): 248-251 ; Zhu, S. et al, Org. Letters, 2011 13(3): 438-441; Bura, T. and Ziessel, ., Org. Letters 2011 13(12): 3072-3075; Niu, S.L. et al, Org. Letters 2009 11(10):

2049-2052; Li, L. et al, JOC 2008 73 : 1963-1970, and Tahtaoui, C. et al, JOC 2007 72: 269-

In some cases, BODIPY fiuorophores can include a group having the structure:

or a salt thereof,

wherein:

R ¹ and R ² are selected from the group consisting of: H, (Ci-O alkyl, (Ci~C6)haloalkyl, (C ₂ - C ₆ )alkenyl, (C ₂ -C ₆ )alkynyl _; (Ci-C ₆ )alkoxy, -COR ¹⁰ , -CO ₂ R ⁱ⁰ , -SOR ¹² , -SO2R ¹² , -NR ⁱ⁰ R ⁿ , - NO2, (C3-Cio)carbocyclyl, (C6-Cio)aryl, 4-10 membered heterocyciyl, and 5-10 membered heteroaiyl, each of which is independently substituted or nsubstltuted, and wherein if both R ¹ and R ^z are present, no more than one of R ¹ and R ² is H;

R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are independently selected from H, (Ci-C6)aikyL (Ci-C ₆ )haloalkyl, (C ₂ - C ₆ )alkenyl, (C2-C6)alkynyl, (Ci-Cejalkoxy, -SO3H, (C3-Cio)carbocyclyl, (C6-Cio)aryl, 4-10 membered heterocyciyl, 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted, and a reactive moiety;

R ^s and R are independently selected from halogen (e.g., fluorine), (Ci-Cgjalkyl, (C ₂ "Cs)alkyrryl, -CO2R ¹⁰ , (Ci-C6)alkoxy, 0(4 membered heterocyciyl), -0-(Ci-C6)alkyl-0(nPEG), each of which is independently substituted or unsubstituted:

each R ⁱ⁰ and R ⁿ are independently selected from H and (Ci-C6)aikyl;

each R ¹² is independently a (C6-Cio)aryl.

In some embodiments, if one of R ¹ or R ² is H, the other can be a (d-C6)alkyl, (C3-

Cio)carbocyclyl, (C6-Cio)aryl, or 5-10 membered heteroaiyl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl, tert-butyl, or isopropyl. In some embodiments, R ^l and R ² are

independently selected from, methyl, ethyl, isopropyl, and tert-butyl.

In some embodiments, R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are independently selected from H, (Ci~

C6)alkyl, (C2-C6)alkenyl, (C6-Cio)aryl, and 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted. For example, R ³ , R ⁴ , R ⁵ , R ^tt , and R ⁷ can be

R ⁸ and R ⁹ are independently selected from halogen (e.g., fluorine), (C2-C ₆ )alkynyL - CO2R ¹⁰ , (Ci-C<s)alkoxy, 0(4 membered heterocyclyl), and -0-(Ci-C6)alkyl-0(iiPEG), each of which is independently substituted or unsubstituted. For example, R ⁸ and R ⁹ can be

independently selected from F. -OCO2H, -OCH3 PEG

, wherein X is O, N, or S, and nPEG is a polyethylene glycol polymer.

Coumarin fluorophores include, for example, 2H-chromen-2-one, iiiiibelliferone (7- hydroxycoumarin), aesculetm (6,7-dihydroxy coumarin), herniatm (7-meraoxycoumarin), psoralen, and imperatorin. In some embodiments, a coumarin fluorophore includes a group selected from:

or a salt thereof,

wherein:

R ¹ and R ² are selected from, the group consisting of: H, (Ci-CejalkyL (Ci-C6)haloalkyl, (C ₂ -

C ₆ )alkenyl, (C ₂ -C ₆ )a.lkynyL (Ci-C6)alkoxy, -C ⁽⁾ R ^W , -CO2 ΌF 10 -SOR ¹² , -SO2R ¹² , -NR^R. ¹ NO?, (C3-C1 o)carbocyciyl, (C6-Cio)aryl, 4-10 membered heterocyclyl, and 5-10 membered heteroaryl, each of which is independently substi tuted or misubsiituted;

R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ are independently selected from H, (Ci-C ₆ )aikyL (Ci-Cejhaloalkyl (C.2-C6)alkenyl. (C ₂ -C6)alkynyl ₅ (C ₁ -C6)alkoxy, -SO3H, (C3-C ₁ o)carbocyclyl, (Ce-Ciojary!,, 4- 10 membered heterocyclyl, 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted, and a reactive moiety; each R ^Rl and R ⁿ are independently selected from H and (Ci-Cejalkyi; and

each R ^l2 is independently a (C6-C ₁₀ )aryl.

In some cases, wherein if both R ¹ and R ² are present, no more than one of R ¹ and R ^z is H (i.e., at least one of R ¹ and R ² is not H). In some of these embodiments, if one of R ¹ or R ² is H, the other can be a (Ci-C6)alkyl, (Cs-Ciojearboeyclyl, (C6-Cio)aiyl, or 5-10 membered heteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl, tert-butyl, or isopropyl. in some embodiments, R ¹ and R are independently selected from H, methyl, ethyl, isopropyl, and tert- butyl. In some embodiments, R ^J , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ , when present, are H.

in some embodiments, a coumarin fluorophore is selected from the group consisting of:

or a salt thereof.

or example, a coumarin lluorophore can include:

_ _{or a sa†} th _ereo £

A fluorophore can be a rhodamine lluorophore. Non-limiting examples include rhodamine, rhodamine B, rhodamine 6G, rhodamine 123, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR) and its isothiocyanate derivative (TRITC), sulforhodamine 101 (and its sulfonyi chloride form Texas Red), Rhodamine Red, Alexa 546, Alexa 555, Alexa 633, DyLight 550, and DyLight 633.

or a salt thereof,

wherein:

X is a counter anion;

R ¹ and R ² axe selected from the group consisting of: H, (Ci~C6)aIkyi, (Ci-C6)haloalkyl, (C ₂ -

Ce alkenyl, (C ₂ -C6)alkynyl _s (Ci-C ₆ )alkoxy, -COR ¹⁴ , -CO2R ¹⁴ , -SOR ¹⁶ , -SC R ¹⁶ , -NR ¹⁴ R ¹⁵ , - NO2, (C3-Cio)car ocyclyl, (C6-C ₁ o)atyl, 4-10 membered heterocyclyl, and 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted;. R\ I ⁴ , R ⁵ , R ⁶ , ^' R ⁷ , R\ It ⁹ , P. ¹⁰ , R ¹¹ , R ¹² , and R ¹³ are independently selected from the group consisting of: H, (Ci-C6)alkyl, (Ci-C6)haloaikyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (Ci~ C ₆ )alkoxy, -SO3, -SO3H, (C3-Cio)carbocyclyl, (Ce-CiojaryL 4-10 membered heterocyclyl, 5- 10 membered heteroaryl, each of which is independently substituted or unsubstituted, and a reactive moiety; wherein, optionally, one or more of the pairs R ³ and R' ^' , R ⁴ and R ⁸ , ⁵ and

R ^Y , and R ⁶ and R ^! come together to form a saturated or unsaturated ring structure with the . carbons or nitrogens to which they are attached, any of which can be substituted or unsubstituted;

each. R ¹⁴ and R ¹⁵ are independently selected from H and (C ₁ -C6)alkyl; and

each R ^{! 6} is independently a (C6-C ₁₀ )aryi.

In some cases, wherein if both R ¹ and R ² are present, no more than one of R ¹ and R ² is H (i.e., at least one of R ¹ and ² is not H). In some of these embodiments, if one of R ¹ or R ² is H, the other can be a (d-Cejalkyl, (C3-Cio)carbocyclyL (Ce-Cio aryL or 5-10 membered heteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyc!opentyl, tert-butyl, or isopropyl. In some embodiments, R ¹ and R ² are independently selected from H, methyl, ethyl, isopropyl, and tert- butyl.

Fluorescein fluorophores can include fluorescein, fluorescein isothiocyanate (FITC), 6- FAM phosphoramidite, esters of fluorescein including succinimidyi esters (NHS-fluorescein), pentaflaorophenyl esters (PFP) and tetrafluorophenyl esters (TFP), eosin, carboxyfluorescem, fluorescein amidite (FAM), merbromin, erythrosine, Rose Bengal, and DyLight Fluor agents. In some embodiments, a. fluorescein fluorophore is a group selected from:

or a salt thereof,

wherein:

R ³ and R ² are selected from the group consistmg of: H, (Ci-C6)alkyl, (Ci-C6)haloalkyl, (C2- C6)alkenyL (C ₂ -C ₆ )alkynyl, (C ₃ -C ₆ )alkoxy, -COR ¹⁰ , -CChR ¹⁰ , -SOR ¹² , -S0 ₂ R ¹² , -NR¾ ⁿ , - NO2, (C3-Cio)carbocyclyl, (Cg-Ciojaryl, 4-10 membered heterocyclyl, and 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted;

R ^J , R ⁴ , R ³ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ are independently selected, from the group consistmg of: H, (O- C6)atkyl, (Ci-C6)haloalkyl, (C2-C6)alkenyl, (C2-C ₆ )aj]cynyi, (Ci-Cejalkoxy, (C3- Ciojcarbocyciyl, (C6-Cio)aryl, 4-10 membered heterocyclyl, 5-10 membered heteroaryl, each of which is independently substituted, or unsubstituted, and a reactive moiety;

each R ¹⁰ and R ⁿ are independently selected from H and (Ci-Cgjalkyi; and

each R ¹² is independently a (C6-Cio)aryl.

In some cases, wherein if both R ¹ and R are present, no more than one of R ¹ and R ² is H (i.e., at least one of R ^{ and R ² is not H). In some of these embodiments, if one of R. ¹ or R ² is H, the other can be a (Ci-C _& jalkyl, (C3-C io)carbocyclyl, (C6~Cio)aryl. or 5-10 membered. heteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl, tert-butyl, or isopropyi. In some embodiments, R ¹ and R ² are independently selected from H, methyl, ethyl, isopropyi, and tert- butyl.

A fiuorophore, as described herein, can include a. combination tluorophore based on a combination of rhodamine and. fluorescein. For example, a fiuorophore can be selected from:

or a salt thereof,

wherein:

X is a counter anion;

R ¹ and R ² are selected from the group consisting of: H, (Ci-CejalkyL (Ci-Cbjhaloalkyl, (C ₂ - Cfijalkenyh (C ₂ -C ₆ )alkynyl _s (Ci-C ₆ )alkoxy, -COR ¹² , -CO2R ¹² , -SOR ¹⁴ , ~80 ₂ R ¹⁴ , -NR ⁱ² R ¹³ , - N0 ₂ , (C3-Cio)carbocyc].yl, (C<5-Cio)aryl, 4-10 membered heterocyclyl and 5-10 membered heteroarvl, each of which is independentl substituted or ^substituted;

R ³ , R ⁴ . R ⁵ , R ⁶ , R ⁷ , R ^s , R ⁹ , R ¹⁰ , and R ¹¹ are independently selected from the group consisting of:

H, (Ci-C ₆ )alkyl, (Ci-Cejhaloaikyl, (C ₂ -C ₆ )alkenyL (C ₂ -C6)alkynyl, {Ci~C ₆ )alkoxy, -SO3, - SO3H, (C3-Cio)carbocyciyl, (C6~Cio)aryl, 4-10 membered heterocyclyl, 5-10 membered heteroarvl, each of which is independently substituted or unsubstituted, and a reactive moiety; wherein, optionally, one or more of the pairs R ⁵ and R ⁶ , and R ⁷ and R ⁸ come together to form a saturated or unsaturated ring structure with the carbons or nitrogens to which they are attached, any of which can be substituted or unsubstituted;

each R ¹² and R° are independently selected from H and (Ci-C6)alkyl; and

each R ¹⁴ is independently a (C6-Cio)aryl.

In some cases, wherein if both R ¹ and R are present, no more than one of R ¹ and R ² is H

(i.e., at least one of R ¹ and R ² is not H). In some of these embodiments, if one of R ¹ or R ² is H. the other can be a (Ci-C ₆ )alkyl, (Cs-Ciojcarbocyclyl, (Ce-Ciojaryl, or 5-10 membered heteroaryl.

For example, phenyl, cyciohexyl, pyridyi, cyclopentyl, text-butyl, or isopropvl. in some embodiments, R ¹ and R are independently selected from H, methyl, ethyl, isopropvl, and tert- butyl.

In some embodiments, a fluorophore can be a group selected, from:

or a salt thereof,

wherein:

R ¹ and R ² are selected from the group consisting of: H, (Ci-C6)alkyL (Ci-CgjhaloalkyL (C ₂ - C ₆ )alkenyl (C2-C ₆ )alkynyl, (Ci-C ₆ >aikoxy, -COR ¹² , -CO2R ¹² , -SOR ¹⁴ , -S0 ₂ R ¹⁴ , -NR ¹² R ¹³ , - NO2, (C3-Cio)carbocyclyi, (C6-Cio)aryL 4-10 membered iieterocyciyl, and 5-10 raembered heteroaryl, each of which is independently substituted or unsubstituted;

R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , and R ⁿ are independently selected from the group consisting of:

H, (Ci-C ₆ )alkyL (Ci-C ₆ )haloalkyL (CVCf a!kem l. (C2-C ₆ )alkynyL (Ci-Cejalkoxy, (C ₃ - Cio)carbocyclyl, (CVCiojaryl, 4-10 membered heterocyclyl, 5-10 membered heteroary!, each of which is independently substituted or unsubstituted, and a reactive moiety; wherein, optionally, one or more of the pairs R ⁴ and R'', and R ⁵ and R ⁶ come together to form a saturated or unsaturated ring structure with the carbons or nitrogens to which they are attached, any of which can be substi tuted or unsubstituted ;

each R ¹² and R are independently selected from H and (Cj-Cejalkyl; and

each R ¹⁴ is independently a ( ¾-Cio)aryi.

In some cases, wherein if both R ^{ and R ² are present, no more than one of R ¹ and R ² is H (i.e., at least one of R. ¹ and R ² is not H). In some of these embodiments, if one of R ^! or R ² is H, the other can be a (Ci-C6)alkyl, (C3-Cio)carbocyclyl, (C6-Cio)aryL or 5-10 membered heteroaryl. For example, phenyl, cyclohexyl, pyridyl, eyclopentyl, tert-butyl, or isopropyl. In some embodiments, R ¹ and R ² are independently selected from H, methyl, ethyl, isopropyl, and tert- butyl.

A fluorophore can include a pyrene fluorophore. Non-limiting examples of a pyrene fluorophore include a group selected from:

or a salt thereof,

wherein:

R. ¹ and R ² are selected from the group consisting of: H, (Ci-C6)alkyL, (Ci~ V)haioalkyl, (C2- C ₆ )alkenyl, (C2-C ₆ )alkyiiyl, (Ci-C ₆ )alkoxy, -COR ¹³ , -CQ ₂ R°, -SOR ¹⁵ , -SO2R ¹⁵ , -NR ⁱ³ R ¹⁴ , - NO2, (C3-Cio)carbocycly.l, (C6-C ₁₀ )aryi, 4-10 membered heterocyclyl, and 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted:

R ³ , R ⁴ , R ⁵ , R ⁶ , R'', R ⁸ , R ⁹ , R ¹ , R ^u , and R ¹² are independently selected from the group consisting of: H, (Ci-C ₆ )alkyh (Ci-C ₆ )haloalkyl, (C ₂ -C6)alkenyl, (C ₂ -C ₆ )alkynyl,.(Ci-C6)alkoxy, -SO.-.H (C3-Cio)carbocyclyl, (C6-Cio)aryl, 4-10 membered heterocyclyl, 5-10 membered heteroaryl, each of which is independently substituted or unsubstituted, and a reactive moiety;

each R ^u and R ¹⁴ are independently selected from H and (C ₁ -C6)alkyl; and

each R ¹⁵ is independently a (Ce-C^aryl.

In some cases, wherein if both R ¹ and R ² are present, no more than one of R ^! and R ² is H (i.e., at least one of R ¹ and. R ² is not H), In some of these embodiments, if one of-R ¹ or R ² is H, the other can be a (Ci-C6)alkyl, (C3-C ₁₀ )carbocyclyl, (Ce-Ciojaryl, or 5-10 membered heteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyi, tert-butyl, or isopropyl. In some embodiments, R ¹ and R ² are independently selected from H, methyl, ethyl, isopropyl, and tert- butyl.

Additional fluorophores may also be used in the compounds described herein, for example, cyanine fluorophores. For example, a fluorophore having the structure: or a salt thereof. In some embodiments, the fiuorophore can be an oxazine tluorophore. For example, the fiuorophore can be selected from:

or a salt thereof , wherein X is a counter anion as described above, A. fiuorophore can also be an acridine fiuorophore. For example, the fiuorophore can be a compound having the strucuture: or a salt thereof. In some embodiments, a fiuorophore can be an auramine O fiuorophore.

example, a fiuorophore can be a compound:

or a salt thereof , As for the fluorophores described previously, the location of attachment of the -L-Tz moiety may vary and the fluorophores may be further substituted to modify the steric and/or fluorescent characteristics of the compound.

A linker moiety can be a conjugated linker. The linker contains alternating single and multiple bonds and can be cyclic, acyclic, linear, or any combination thereof. For example, the linker can include an alkenyl. alkynyl, aryl, and/or lieteroaryl moiety. In some embodiments, the linker can. include an alkenyl and an aryl, an alkynyl and an aryl, an alkenyl and a heteroaryl, or an alkynyl and a heteroaryl. The structure of the linker must be such that the linker bridges and continues the conjugation of the tluorophore with that of the tetrazine so that a conjugated pi- system extends between these three moieties.

In some embodiments, a linker moiety can be an aromatic linker. For example, the aromatic linker can be a (CVCio)aryl or 5-10 membered heteroaryl. In some embodiments, the aromatic linker is selected from the group consisting of:

wherein:

R , R" ^a , R ^Ja , and R are selected from the group consisting of: H, (Ci-Cejalkyl, (Ci~

C ₆ )haloa]jkyl, (C2-C ₆ )alkenyl, (C ₂ -C ₆ )alkynyL (Ci-C ₆ )alkoxy, -COR ¹³ , -C0 ₂ R ¹³ , -SOR ¹⁵ , - S0 ₂ R ¹⁵ , -NR ¹³ R ¹ ₅ -NO2, (C ₃ -Cio)carbocyclyl, (C ₆ -Cio)aryL 4-10 membered heterocyclyl, and 5-10 menibered heteroaryl, each of which is independently substituted or unsubstituted.

Additional examples of aromatic linkers include:

In some embodiments, a lin ker is , wherein the linker is substituted by F and Tz as exemplified above. As above, the linkers described herein can be further substituted to modify the sterics of the linker and the orientation of the L-Tz moiety with respect to the F moiety.

Linkers, as provided herein, can also be selected from a substituted or unsubstituted heteroaryl or heterocyclyls. Non-limiting examples include:

each of the above compounds is independently substituted by F and Tz (e.g., through a carbon- carbon bond). In addition, each of the compounds described abo ve may be unsubstituted or substituted (e.g., by one or more R _x as described below). In some embodiments, the heteroaryl or heterocyclic linkers pro vided herein can include an aikenyl or alkynyl linkage between the fluorophore and/or the tetrazine moiety.

In some embodiments a linker can be selected from the group consisting of:

wherein n is an integer from 1 to 10 (e.g.. 1 to 4); and each R ^x is independently selected from the group consisting of H, (Ci-CsjalkyL (Ci -Cejalkoxyl, and a tetrazine moiety as provided herein. For example, each R ^x can be independently selected from H, methyl, ethyl, propyl, isopropyl, methoxy, and a tetrazine moiety as provided herein.

The tetrazine moiety in the compound of Formula (I) can be a substituted or unsubstituted tetrazine. In some embodiments, the tetrazine moiety is tetrazine. In other embodiments, the tetrazine is substituted with a substituent selected from Cy ¹ , Cw alkyl, C2-6 aikenyl, C2-0 alkynyl, halo, Ci-6 haloalkyl, CN, NO2, OR ^al , SR ^al , C(0)R ^bi , C(0)NR ^c] R ^dl , C(0)OR ^al , OC(0)R ^bl , OC(Q)NR ^cl R ^dl , C( ^:::: NR ^ei )NR ^ci R ^dl , NR ^cl C(-NR ^el )NR ^cl R ^di , NR ^cl R ^di ₅ NR° ³ C(Q)R ^bl ,

NR ^cl C(0)OR ^al , NR ^0l C(O)NR ^cl R ^dl , NR ^cl S(0)R ^bl , NR ^cl S(0) ₂ R ^bl , R ^c S(0) ₂ NR ^ci R ^dl , S(0)R ^bl , S(0)NR ^cl R ^dl , S(0) ₂ R ^bl and S(0) ₂ NR ^cl R ^dl , wherein

each Cy ¹ is independently Ce-io an , C3.10 carbocyclyL 5-10 membered heteroaryl or 4-10 membered heterocyclyl, each of which is unsubstituted or substituted by 1, 2, 3, 4 or 5 substituents independently selected from Ci -s alkyl, C2-6 aikenyl, C2-6 alkynyl, halo, CN, 0 ₂ , OR ^ai , SR ^al , C(0)R ^bl , C(0)NR ^cl R ^dl , C(0)OR ^ai ₅ OC(0)R ^bi , OC(0)NR ^c3 R ^di , C(=NR ^el )NR ^cl R ^dl , R ^cl C(===NR ^cl )NR ^cl R ^dl , R ^cl R ^dl , R ^cl C(0)R ^bl , NR ^cl C(0)OR ^al , NR ^cl C(0)NR ^cl R ^dl ,

NR ^c] S(0)R ^h NR ^cl S(0)2R ^bl _; NR ^ci S(0)2NR ^ci R ^iU ₅ S(0)R ^bl , S(Q)NR ^cl R ^d3 , S(0) ₂ R ^bl _;

S(0)2NR ^ci R ^dl and oxo;

each R ^aI , R ⁱ , R ^cl , R ^di is independently selected from H, C3..5 alkyl, Ct^ haloalkyl, C2-6 alkenyl, C2-6 alkynyl, Ce-io aryl, C3-10 carbocyclyl, 5-10 membered heteroaryl, 4-10 membered heterocyclyl, C ₆ -io aryl-O.4 alkyl,€3-10 carbocyclyl-Ci.4 alkyl (5-10 membered heteroaryl)-Ci- ₄ alkyl or (4-10 membered. heterocyclyl)-Ci-4 alkyl.

For example, a tetrazine can be a moiety having the structure:

R lb

N N

N wherein:

R ^lb is selected from the group consisting of: H, (Ci-C6)alkyl, (Ci-Cejiia!oalkyl, (C6-Cio)aryl, and

5-10 membered heteroaryl, each of which is independently substituted or unsubstituted. For example, the tetrazine can be selected from:

Non-limiting examples of a compound of Formula (I) include:

or a salt thereof, wherein each R is independently selected from H, (Ci-Cejalkyl, (Ci- C6)haloalkyl, (C6-Cio)aryl, and 5-10 membered heteroaiyl. in some embodiments, R is H or (Ci- Cf aikyl, such as C¾.

For example, a compound of Formula (I) can include:

, or a salt, thereof.

Also provided herein is a compound of Formula (II):

F Tz

or a salt thereof, wherein F is a fluorophore and Tz is a substituted or unsubstituted. tetrazine as described previous!}', and wherein the Tz and F moieties form a single conjugated pi-system. In some embodiments, the Tz and F moieties form a non-coplanar pi-system. For example, the Tz moiety can be oriented with, respect, to the F moiety such that the tetrazine transition dipole is either collinear with or parallel to the transition dipole of the fluorophore. The moieties F and ^• Tz can be as defined for any of the embodiments described above.

Without being bound by any theory, the most TBET quenched probe may be one where the tetrazine is directly attached to the fluorophore. In non-planar compounds, the disruption in conjugation between the two systems can be provided by the fluorophore (e.g., by the steric effects of substituents on the fluorophore). In effect, the TBET connection between the two systems is the shortest possible (i.e., the single bond between the two moieties),

Non-limiting examples of a compound of Formula (II) include:

or a salt thereof.

This disclosure further provides a compound of Formula. (Ill):

F L Z or a salt thereof,

wherein:

F is a fluorophore;

L is a conjugated linker; and

Z is a moiety comprising the reaction, product of a diene and a dienophile, wherein die diene is a tetrazine or a derivative thereof:

wherein the linker bridges the Z and F moieties in a single conjugated, pi-system.

The single, conjugated pi-system may, in some embodiments, be non-copianar due to steric factors within the compound that enforce a twist in the interchromophore linkage between the F and L-Z moieties. Such steric factors can originate from substituents on either the linker or on the fluorophore. Without being bound by theory, fluorescence can be increased when the L-Z moiety is oriented with respect to the F moiety such that the transition dipole of Z is either colSinear with or parallel to the transition, dipole of the fluorophore.

The fluorophore. linker, and tetrazine are as described above.

Z is a moiety formed through the reactio of a diene (e.g., tetrazine or a derivative thereof) and a dienophile using bioorthogonal chemistry. Bioconjugation methods using inverse electron, demand Diels- Alder cycloadditions between tetrazines and highly strained dienophiies such as norbomene, strained cyclic alkenes, and frans-cyclooctene are known in the literature, however the tetrazine used has limited stability to aqueous media. (Blackmail et al, 2008, J Am Chem Soc, 130, 13518-9; Devaraj et al., 2009, Ang w Chem Int Ed Engl, 48, 7013-6; Devaraj et al., 2008, Bioconjug Chem, 1.9, 2297-9; Pipkorn et al, 2009, JPept Sci, 15, 235-41).

Dienophiies useful in the compounds described herein include but are not limited to carbon containing dienophiies such as alkenes or alkynes, or compounds containing nitroso, carbonyl or imine groups. In some embodiments, the dienophile is a strained dienophile. As used herein, a "strained" dienophile has a dihedral angle that deviates from the idealized 180 degree dihedral angle. Alternatively, non-strained dienophiies (e.g., styrenes) and/or electron rich electrophiles (e.g., eneamines or vinyl ethers), can also be used with nitroso compounds. Alkenes as used herein refers to an alkyl group having one or more double carbon-carbon bonds such as an ethylene, propylene, and the like. Alkenes can also include cyclic, rmg-strained alkenes such as tttms-cyclooctene or norbomene carrying a double bond which induces significant ring strain and is thus highly reactive. Alkenes can also include more complex structures such as indoles and azaindoles, electron rich enamines. Heterodienophiles containing carbonyl, nitroso or imine groups can also be used. In some embodiments, the dienophile is a trans-cyclooctenol, e.g., (E)-cyclooct-4-enol. Additional examples of dienophiles include those described in Thalhammer, F. et al. Tetrahedron Letters 1990 31(47): 6851-6854. in some embodiments, the dietiophile is a substituted or derivatized trans-cyclooctenol. For example, the nydroxyl moiety on the trans-cyclooctenol can be derivatized with a linker (e.g., a PEG linker) and/or a reactive moiety. In some embodiments, the trans-cyclooctenol is modified with a reactive moiety such as:

wherein n is an integer from 1 to 10; or a carbamate-Hnked PEG (e.g., a carbamate-linked PEGm, wherein m is an integer from 1 to 10) such as a carbamate-linked PEG? (TCOc):

Non-limiting examples of a compound of Formula (III) include:

40

or a salt thereof.

Also provided herein is a compound of Formula (IV):

F Z

or a salt thereof, wherein F is a fluorophore and Z is a moiety comprising the reaction product of a diene and a dienophile, wherein the diene is a tetrazine or a derivative thereof as described previously, and wherein the Z and F moieties form, a single conjugated pi-system.

^' The compounds provided herein can be referred to as Hyper Emissive Ligation-Initiated Orthogonal Sensing (HEL IOS ) probes. Many of the compounds provi ded herein are desi gnated HELIOS XXX, where XXX refers to the absorption maximum of the "turned on" probe. This designation is used to refer to certain compounds within the present disclosure. Synthesis

The compounds provided herein, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.

The reactions for preparing the compounds provided herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the

intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.

Preparation of the compounds provided herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Protecting Group Chemistry, 1 ^st Ed., Oxford University Press, 2000; March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 ^th Ed., Wiley-Interscience Publication, 2001 ; and Peturssion, S, et ah, ' ^' 'Protecting Groups in Carbohydrate Chemistry, ^" ' J. Chem. Ed c, 74(1 1), 1297 (1997).

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such, as nuclear magnetic resonance spectroscopy (e.g.. ^J H or ⁱ³ C), infrared spectroscopy, spectrophotometry (e.g., UV- visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS), or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) ("'Preparative LC-MS Purification: Improved Compound Specific Method Optimization " K.F. Blom, et ah, J. Combi. Chem. 6(6), 874 (2004) and normal phase silica chromatography.

in some embodiments, the compounds provided herein can be prepared as described in the Examples provided herein and as illustrated in Scheme 1. Scheme 1.

23 R— H

1 2b R = CH ₃

5a R = H

5b R = CH

Reaction conditions: i = Zn(OTf , NH2 H2. acetonitrile, 60°C, 18 hrs: ii = NaNC% iii = HC1 (1

As another example, a compound as provided herein can also be prepared as illustrated in

Scheme

As another example, a compound as provided herein ca also be prepared as illustrated Scheme III.

HELIOS 455 e R = CH,

HELIOS 455H R = H

HELiOS 463Me

HELIOS 463H

Reaction conditions: i) N-Broi osuccinimide in acetonitrile, ii) 3-cyanophenylboronic acid, Pd(OAc)2(PPh ₃ )2, K2CO3 in 75 % dioxane(aq) at 100 °C, iii) hydrazine, Zn(OTi) ₂ ,

acetonitrile/dioxane (Me-Tz) or formamidine hydrochloride DMF (H-Tz) at 60 °C for 15 hours followed by NaN0 ₂ and HC1. Pharmaceutical Compositions

Pharmaceutical compositions can include any of the compounds described herein, and can be formulated as a pharmaceutical composition in accordance with routine procedures. As used herein, pharmaceutical compositions can include pharmaceutically acceptable salts or derivatives thereof. "Pharmaceutically acceptable" means that the agent can be administered to an animal without unacceptable adverse effects. A "pharmaceutically acceptable salt or derivative" means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of composition that, upon administration to a recipient, is capable of pro viding (directly or indirectly) a composition of the present disclosure. Other derivatives are those that increase the bioavailability when administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) thereby increasing the exposure relative to the parent species. Pharmaceutically acceptable salts of the therapeutic or diagnostic compositions or compositions of this disclosure include counter ions derived from pharmaceutically acceptable inorganic and organic acids and bases known in the art, e.g., sodium, calcium, N-methylglutamine, lithium, magnesium, potassium, etc.

Pharmaceutical compositions can be administered by any route, including oral, intranasal, inhalation, or parenteral administration. Parenteral administration includes, but is not limited to, subcutaneous, intravenous, intraarterial, interstitial, intrathecal, and intracavity administration. When administration is intravenous, pharmaceutical compositions may be gi en as a bolus, as two or more doses separated in time, or as a constant or non-linear flow infusion. Thus, compositions can be formulated for any route of administration.

Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizmg agent, a stabilizing agent, and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients will be supplied either separately, e.g. in a kit, or mixed together in a unit dosage form, for example, as a dry lyopliilized powder or water free concentrate. The composition may be stored in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent in activity units. Where the composition is administered by mfusion, it can be dispensed with an infosion bottle containing sterile pharmaceutical grade "water for injection," saline, or other suitable intravenous fluids. Where the composition is to be administered by injection, an ampule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration. Pharmaceutical compositions comprise the therapeutic or diagnostic compositions of the present disclosure and

pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable ingredient, excipient, carrier, adjuvant or vehicle.

A pharmaceutical composition is preferably administered to the subject in the form of an injectable composition. The method of administering a therapeutic or diagnostic composition is preferably parenterally, meaning intravenously, intra-arterially, intrathecally, mterstitially or intracavitarilly. Pharmaceutical compositions can be administered to mammals including humans in a manner similar to other diagnostic or therapeutic agents. The dosage to be administered, and the mode of administration will depend on a variety of factors including age, weight, sex, condition of the subject and genetic factors, and will ultimately be decided by medical personnel subsequent to experimental determinations of varying dosage followed by imaging as described herein. In. general, dosage required for diagnostic sensitivity or therapeutic efficacy will range from about 0.001 to 50,000 μ§/1¾, preferably between 0.01 to 25.0 μg' g of host body mass. The optimal dose will be determined empirically follo wing the disclosure herein.

Methods of Use

The compounds described herein can be imaged using methods known in the art. For example, a compound provided herein can be detected by traditional fluorescence imaging techniques allowing for the facile tracking of the compounds by fluorescence microscopy or flow cytometry using methods known in the art, e.g., as described in US 2005/0249668.

The compositions and methods described herein can. be imaged using a variety of modalities that are Imown to one of skill in the art. Detection methods can. include both imaging ex vivo and in vivo imaging methods, e.g., fluorescence reflectance imaging, fluorescence microscopy, super-resolution microscopy (see, e.g., M. Bates et al., Science 2ΘΘ7, 317: 1749; and G. Patterson et ah, Ann . Rev. Phys. Chem, 2010, 61 : 345), and fluorescence molecular tomographic imaging, in. some embodiments, one or more imaging techniques can be used in the methods provided herein. After a compound has been designed, synthesized, and optionally formulated, it can be tested in vitro by one skilled in the art to assess its biological and performance characteristics. For instance, different types of ceils grown in culture can be used to assess the biological and performance characteristics of the compound. Cellular uptake, binding or cellular localization of the agent can be assessed using techniques known in the art, including, for example, fluorescent microscopy and fluorescence-activated cell sorting (FACS) analysis.

By way of example, the compound can be contacted with a sample for a period of time. The sample can then be viewed using an appropriate detection device without the need for washing or clearance steps, A. detection, device can include a fluorescent microscope equipped with appropriate filters matched to the optical properties of a fluorescent agent. Fluorescence microscopy of ceils in culture is also a convenient means for determining whether uptake and binding has occurred. Tissues, tissue sections and other types of samples can also be used in a similar manner to assess the biological and performance characteristics of the compounds.

Formation of a compound of Formula (III) can occur through the reaction of a compound of Formula (I) with the corresponding dienophile. Similarly, the formation of a compound of Formula (IV) can occur throu gh the reaction of a compound of Formula (Π) with the

corresponding dienophile.

In some embodiments, a compound of Formula (I) or (II) exhibits low or no fluorescence prior to activation upon reaction with a dienophile. For example, the turn-on ratio of a compound of Formula (III) or (IV) (i.e., the reaction product of the corresponding compound of Formula (I) or (II) with the corresponding dienophile) can be greater than 50 (e.g., greater than 60, greater than 75, greater than 100, greater than 125, greater than 150, greater than 200, greater than 300, greater than 400, greater than 500, greater than 600, greater than 700, greater than 800, greater than 900, greater than 1000, greater than 1500, greater than 2000, greater than 2500, greater than 3000, greater than 3500, greater than 4000, .greater than 4500, greater than 5000, greater than 5500, greater than 6000, greater than 6500, greater than 7000, greater than 7500, greater than 8000, greater than 8500, greater than 9000, greater than 9500, greater than 10,000, greater than 11,000, greater than 12,000, greater than 14,000, greater than 15,000, greater than 16,000, greater than 18,000, greater than 20,000, greater than 22,000, greater than 24,000, greater than 26,000, greater than 28,000, greater than 30,000). In some embodiments, the turn- on ratio of a compound of Formula (III) or (IV) can range from about 100 to about 2000 (e.g., from about 100 to about 1800: trom about 100 to about 1600; from about 100 to about 1250; from about 100 to about 1000; from about 1.00 to about 800; from about 100 to about 750; from about 100 to about 500; from about 100 to about 400; from about 100 to about 300: from about 200 to about 2000; from about 400 to about 2000; from about 600 to about 2000; from about 800 to about 2000; from abotit 1000 to about 2000; from about 1200 to about 2000: from about 1500 to about 2000; from about 200 to about 1800; from about 400 to about 1600; from about 500 to about 1500; and from about 750 to about 1250). In some embodiments, the turn-on ratio of a compound of Formula (III) or (IV) can range from about 1000 to about 30,000 (e.g., from, about from about 1000 to about 25,000; from about 1000, to about 22,000; from about 1000 to about 18,000; from about 1000 to about 16,000; from about 1000 to about 12,500; from about 1000 to about 10,000; from about 1000 to about 8,000; from about 1000 to about 7,500: from about 1000 to about 5000; from about 1000 to about 4000; from about 1000 to about 3000; from about 2000 to about 30,000: from about 4000 to about 30,000: from about 6000 to about 30,000; from about 8000 to about 30,000; from about 10,000 to about 30,000; from about 12,000 to about _. 30,000; from about 15,000 to about 30,000; from about 20,000 to about 30,000; from about 2000 to about. 12,000; from about 4,000 to about 16,000; from about 2500 to about 17,500; from about from about 10,000 to about 20,000; from about 5000 to about 15,000; and from about 7,500 to about 12,500). Such low or no fluorescence prior to activation can lead to a background signal which is zero or very near the detection limit of the fluorescence instrument.

The bioorthogonal inverse electron demand Diels- Alder reaction can be tailored to provide a straightforward method for the rapid, specific covaient labeling and imaging with ligands such as small molecules and other biomolecules inside living cells. Despite numerous developments in the application of various selective chemistries to extracellular live cell labeling, to date, no method has been universally adapted to intracellular labeling. For example, described herein are a series of "turn-on ^' ' tetrazine-Iirrked fluorescent probes that react rapidly via an inverse electron demand Diels- Alder reaction to strained dienophiles such as trans- cyclooctene. Upon cycloaddition, the fluorescence intensity increases dramatically, in some cases by ~20 fold. This fluorescence "turn-on" significantly lowers background signal. These novel probes for live cell imaging of a ligand such as an antibody, small molecule, or other biornolecule modified with a strained alkene can provide a general method for labeling and imaging a ligand bound to a specific target. For example, this bioorthogonal inverse electron demand Diels-Alder reaction can be applied to an asymmetric tetrazine and a strained alkene, which is physically coupled to a small molecule, i.e. a trans-cyclooctene modified taxol analog and can be used to label and image this small molecule bound to intracellular tubules. The rapid reaction rate coupled with fluorescence "turn-on" makes this a nearly ideal method for revealing small molecules inside living cells.

In some embodiments, the ligand, e.g., an antibody, small molecule therapeutic agent (e.g., a drug), nanoparticles, polymers, or other biornolecule, is physically attached (conjugated) to the dienophile. In some embodiments, the ligand carries a functional group such as an amine, alcohol carboxyiic acid or ester, or other group of atoms on the ligand that can undergo a chemical reaction allowing attachment to the dienophile. Alternatively or in addition, the dienophile possesses a reactive functional group for attachment to the ligand. Thus, the reactive functional group on the ligand and/or dienophile undergoes a chemical reaction to form a link between the two. in some embodiments, e.g., where the ligand is a biopolymer such as a nucleic acid, peptide, or polypeptide, the functional group on the ligand can be a non-natural nucleoside or amino acid, e.g., as described in Xie and Schultz, Nat. Rev. Mol. Cell Biol. 7:775-782 (2006): for example, the diene or dienophile can be incorporated into a non-natural amino acid as the side chain. One of skill in the art could readily synthesize such compounds. For example, a compound as provided herein can be modified to include a reactive moiety (A):

A

Tetrazine ! Tetrazine Tetrazine

Linker Linker i— A Linker

A

Non-limiting examples of the reactive moiety (A) include a reactive ester (e.g., NHS ester ), a primary amine, carboxyiic acid, azide, alkyne, a second tetrazine. maleimide, and thiol. The reactive moiety can be used to couple the compound to additional, groups, including small molecules (e.g., drags and therapeutic agents) and nanoparticles. In the case of a small molecule drug, the fluorescent compo unds provided herein could be used to monitor delivery of the drug following administration.

In some embodiments, a compound as provided herein is imaged in vivo using fluorescent imaging. For example, the use of such methods permits the facile, real-time imaging and localization of cells or tissues labeled with a compound pro vided herein, hi some embodiments, a compound provided herein is imaged in vivo using laparoscopy and/or endomiscroscopy. For example, the use of laparoscopy permits the facile, real-time imaging and localization of cells or tissues labeled with a compound provided herein. In some embodiments, a compound can be imaged using fiber optic endomicroscopy.

A. number of preclinical and clinical applications for a compound provided herein can be envisioned. For example, a compound described here can be used: 1) for the early detection cancers; 2) as an aid to surgeons during surgery (e.g., by allowing for real-time detection of cancer cells); and 3) as a method for monitoring the progress of a cancer treatment (e.g., by quantifying the cancer cells present before, during, and after treatment).

For example, the compounds provided, herein can be administered to a subject in combination with surgical methods, for example, resection of tumors. The compounds can be administered to the individual prior to, during, or after surgery. The compounds can be administered parenterally, intravenous or injected into the tumor or surrounding area after tumor removal, e.g., to image or detect residual cancer cells. For example, the compound may be used to detect the presence of a tumor and to guide surgical resection. In some embodiments, the compound can be used to detect the presence of residual cancer cells and to guide continued surgical treatment until at least a portion (e.g., all) such cells are removed from the subject. Accordingly, there is provided a method of guided surgery to remove at least a portion, of a tumor from a subject comprising providing a compound provided herein; causing the compound to be present in at least some cancer cells; observing the image following activation of the compound (e.g., fluorescence); and performing surgery on the subject to remove at least a portion of the tumor that comprises detected, cancer cells.

With respect to in vitro imaging methods, the compounds and compositions described herein can be used in a variety of in vitro assays. An exemplar}' ^' in vitro imaging method comprises: contacting a sample, for example, a biological sample (e.g., a cell), with one or more compounds provided herein; allowing the compound to interact with a biological target in the sample; illuminating the sample with light of a wavelength absorbable by a.fluorophore of the agents; and detecting a signal emitted from fluorophore thereby to determine whether the agent has been activated by or bound to the biological target. In some embodiments, the compounds provided herein may be used without requiring clearance of unbound fluorophore prior to imaging,

EXAMPLES

The invention is further described, in the following examples, which do not limit the scope of the invention described in the claims.

General Methods

Materials and Methods

All reagents were purchased from Sigma- Aldrich and used without further purification unless otherwise noted. Proton and carbon nuclear magnetic resonance ( ^l E & ^L' C NMR) spectra were recorded on either a Varian AS-400 (400MHz) spectrometer or a Varian 500MHz spectrometer. Silica Gel 60 (40-63 .urn) was used for purification. High performance liquid chromatography-mass spectrometry analysis (HPLC-MS) was performed with on a Waters instrument equipped with a Waters 2424 ELS Detector, Waters 2998 UV-Vis Diode array

Detector, Waters 2475 Multi-wavelength Fluorescence Detector, and a Waters 3100 Mass

Detector. Separations employed Waters XTerra RP Cig 5 urn or Waters Atlantis RP 5μηι columns, with a watenacetomtrile solvent gradient (0.1% formic acid added). Fluorescence measurements were conducted with a Perkin Elmer LS50B Luminescence Spectrometer, and UV-VIS absorption spectra on an Agilent Technologies Gary 100 UV-Vis Spectrophotometer.

Fluorescence assays

The purity of all compounds was verified by LCMS prior to quantitative activation experiments and a fresh aliquot of the fluorophore collected from the analytical HPLC elution. Stock solutions in acetonitrile were diluted into 2 niL or 3 mL of the appropriate solvent in a 1 cm x 1 cm quartz cuvette. Measurements of solvent and pre-activation emission spectra for baseline values were made .in at least triplicate, prior to addition of transcyclooctenol (TCO) to initiate the fluorogenic reaction. Activation ratios were calculated from the peak emission intensi ty of the reacted dihydropyridazine product and the corresponding baseline intensity, integration of the area under the emission intensity curves was used to validate the activation ratios. For the time-course experiments (FIG. 2), the normalized baseline was calculated from, the mean emission intensity during 30-60 seconds of observation of the solvent blank followed by the pre-TCO fluorophore solution. After addition of TCO, the fluorescence emission intensity was monitored until a plateau was reached. The rate of activation of 5b was attenuated relative to the other methyltetrazines (2b,4b); a 2.5-fold additional excess of TCO was added for facile comparison of the magnitude of fluorescence turnon.

For quantum yield determinations, fluorescem in O.lM NaOH was used as a reference, with an excitation wavelength of 470 or 480 nm (ex slit 2.5 ^' nm); a value of 0.925 was assigned to the quantum yield of fluorescein (Magde, D., Wong, R., & Seybold. P. G. Photochemistry and Photohiology, 2002, 75(4), 327-334), and calculations made according to the methods described by Crosby and. Demas (Chemical Reviews, 1971, 75(8), 991-1024).

Example 1 - Preparation of Compounds 2a and 2b

To -cyanophenyl-BODIPY 1 (100 mg, 0,28 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf>2 (52.3 mg, 0.14 mmol), MeCN (0.16 mL, 3.0 mmol), DMF (0.34 mL) and NH2NH2 (0,55 mL, 16,0 mmol). The vessel was sealed and allowed, to stir at 60°C for 24 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added NaN h (300. trig, 4.34 mmol) in 10 mL of water follo wed by 1 M HC1 until the pH 3. The aqueous phase was extracted three times with CH2CI2 (100 mL). The combined organic extracts were dried with MgS04 and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography (toluene :hexane gradient 3: 1, 4: 1, 100% toluene) to give 2a and 2b. Compounds 2a and 2b were further purified using flash column chromatography (hexanes:ethyl acetate gradient 6: 1 , 4: 1) to give H-tetrazine 2a (6.2 mg, ,015 mmol, 5.5%) and Me-tetrazine 2b (9.5 mg, .022 mmol, 8.1%) as dark red solids. In sharp contrast to the parent compound i, both 2a and 2b are modestly soluble in aqueous solution and almost completely non-fluorescent.

Compound 2a: ¾ NMR (400 MHz, CD2C12) δ 10.29 (s, H i). 8.80 (d, J - 8.4 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 6.07 (s, 2H), 2.55 (s, 6H), 1.50 (s, 6H): ¹³ C NMR (100 MHz, CD2CI2) δ 166.1, 158.1, 155.9, 143.1, 140.3, 139.8, 132.6, 130.8, 129.4, 128.9, 121.4, 14.4 (4C). ESIMS [Μ+ΗΊ+ calcd for C21H20BF2N6 405.18, found 405.23.

Compound 2b: Ή NMR (400 MHz, CD2C12) δ 8.76 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 6.10 (s, 2H), 3.11 (s, 3H), 2.54 (s, 6H), 1.50 (s, 6H); ¹³ C NMR (100 MHz, CD2C12) δ 167.7, 163.7, 155.9, 143.2, 140.5, 139.1, 132.8, 130.9, 129.2, 128.5, 121.4, 21.0, 14.3 (4C). ESIMS | NT-I t j - calcd for C22H22BF2N6 419.20, found 419.29.

Example 2 - Preparation of Com ound 3

To 3-formylbenzonitrile (1.0 g, 7.6 mmol) and 2,4-dimethylpyrrol.e (1.7 mL, 16.5 mmol) in 200 mL of CH ₂ CI2 under argon atmosphere was added four drops of TFA and allowed to stir at room temperature. After 30 minutes thin layer chromatography showed the

disappearance of 3~formylbenzonitrile after which 2,3-dicWoro-5,6-dicyano-l,4-benzoquinone (DDQ) (1.73 g, 7.6 mmol) In CH2CI2 (200 mL) was added followed by N,N- diisopropylethylamine (15.5 mL, 88.9 mmol) and BF3 · OEt2 (15.5 mL, -45% BF3 content). The reaction was allowed to stir overnight after which water (100 mL) was added and the aqueous phase was extracted three times with CH2CI2 (300 mL). T he combined organic extracts were dried with MgS0 ₄ and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography (toluene:hexane gradient 3 : 1, 100% toluene) to give 3 (822.7 n g, 2.3 mmol, 30.2%) as a red solid. ¾ NMR (500 MHz, (CD ₃ ) ₂ SO) δ 8.04 (m, 1H), 7.98 (m, l l n. 7.75 (m, 2H), 6.19 (s, 2) 1), 2.45 (s. 6H), 1.32 (s, 6H) ); ¹³ C NMR (125 MHz, (CD3)2SO) δ 155.9, 144.5, 143.0, 139.5, 135.6, 133.7, 133.5, 132.2, 131.0, 122.2, 121.1, 112.9, 14.7 (4C). ESIMS [M+H]+ calcd for C20H19BF2N3 350.16, found 350.22.

Exam le 3 - Preparation of Com ounds 4a and 4b

To 3 (100 mg, 0,28 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf) ₂ (523 mg, 0.14 mmol), MeCN (0.16 mL, 3.0 mmol), DMF (0.34 mL) and NH2NH2 (0,55 mL, 16.0 mmol). The vessel was sealed and allowed to stir at 60°C for 24 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added NaNC (300 mg, 4.34 mmol) in 10 mL of water followed by I M HCI until the pH = 3. The aqueous phase was extracted three times with CH2.CI2 (100 mL). The combined organic extracts were dried with MgS04 and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography (toluene racetonitrile gradient 100% toluene,

100:0.5) to give 4a and 4b as dark orange solids. Compounds 4a and 4b were further purified using flash column chromatography (hexanes: ethyl acetate 4:1) to give H-tetrazine 4a (9.6 mg, .023 mmol, 8.4%) and Metetrazine 4b (10.1 mg, .024 mmol, 8.6%) as dark red. solids.

Compound 4a; Ή ΝΜΚ (500 MHz, (CD ₃ ) ₂ SG) δ 10.63 (s, IH), 8.67 id. J - 8.0

Hz, 1 I I I 8.40 (s, 1 H), 7.89 (t, J = 8.0 Hz, IH), 7.78 (d, J = 8.0 Hz, i l l). 6.21 (s, 2H),

2.47 (s, 6H), 1.41 (s, 6H); ¹³ C NMR (125 MHz, (CD ₃ ) ₂ SO) δ 165,6, 158.7, 155.8, 143.1, 140,9, 135.5, 133.6, 132.7, 131.1, 131.0, 129.0, 127.6, 122.1, 14.8 (4C). ESIMS calcd for C21H20BF2N6 405.18, found 405.27.

Compound 4b : ) NMR. (500 MHz, (CD^SO) δ 8.64 (dt, J = 8.0, 1.5 Hz, IH), 8.37 (t, J - 2.0 Hz, IH), 7.87, (J = 7.5 Hz, IH), 7.75 (dt J = 7.5, 1.5 Hz, IH), 6.21 (s,

2H), 3.00 (s, 3H), 2.47 (s, 6H), 1.40 (s, 6H); ^r C NMR. (125 MHz, (CD ₃ )2SO) δ 167.8,

163.4, 155.8, 143.0, 140.9, 135.5, 133.6, 132.3, 132.2, 132.0, 129.1, 127.2, 122.1, 21.3, 14.8 (4C). ESIMS [M+H]+ caicd for C22H22BF2N6 419.20, found 419.27. Example 4 - Preparation of Compound 6

S

To 2-iodo-pentamethyl BODIPY (800 mg, 2.06 mmol) dissolved in 60 mL of

toluene:methanol (5:1) under argon atmosphere was added Na ₂ C03 (874 mg, 824 mmol), Pd(OAc) ₂ (46.2 mg, 0.20 mmol), 2-methyl-4-cyanopheri lboronicacid (994.8 mg, 6.2 mmol) and 2 lieyclohexylphosp.W (121.6 mg, 0.31 mmol). The mixture was heated, to 100°C and stirred. After 2.5 hours, thin layer chromatography showed, the disappearance of 2-iodo-pentamethyi BODIPY and the reaction was diluted with 100 mL of water. The aqueous phase was extracted three times with toluene (300 mL). The combined organic extracts were dried with MgS0 ₄ and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography (toluene) to give 6 (606.3 mg, 1.6 mmol, 77.7%) as a red solid.

Ή NMR (400 MHz, Cl M. b) δ 7.34 (s, HI), 7.57 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 7.6 Hz, lH),

6.17 (s, IH), 2.67 (s, 3H), 2.52 (s, 3H), 2.48 (s, 3H), 2.28 (s, 3H), 2.19 (s, 3H), 2.18 (s, 3H); C NMR (100 MHz, CD2CI2) δ 154.9, 150.2, 142.5, 142.4, 139.6, 138.9, 137.1 , 133.5, 1.32.6, 1.31.9, 131.6, 130.8, 129.4, 121.8, 118.9, 111.5, 19.4, 17.2, 16.7, 15.0, 14.2, 12.7. ESIMS [M+H]+ calcd for C22H23BF2N3 378.20, found 378.27.

Example 5 - Preparation of Compounds 5a and 5b

5b

To 6 (100 mg, 0.26 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf) ₂ (49.5 mg, 0.13 mmol), MeCN (0.15 mL, 2.8 mmol), DMF (0.32 mL) and NH2NH2 (0.52 mL, 15.1 mmol). The vessel was sealed and allowed to stir at 60°C for 24 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added NaNCb (283.8 mg, 4.1 mmol ) in 10 ml . of water followed by 1 M HC1 until the pH = 3. The aqueous phase was extracted three times with CH2CI2 (100 mL), The combined organic extracts were dried with MgS0 ₄ and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography

(tolueneracetonitrile gradient 100% toluene, 100:0.2) to give 5a and 5b. Compounds 5a and 5b were further purified using flash column chromatography (hexanes: ethyl acetate 4:1) to give H-tetrazine 5a (8.9 mg, 0.023 mmol, 8.0%) and Me-tetrazine 5b (11.6 mg, 0.026 mmol, 10.0 %) as dark red solids.

Compound 5a: Tl NMR (500 MHz, i ^' XH ^' ) δ 10.20 (s, I I I }. 8.61 (s, 1 1 i ), 8.51

(dd, J = 8.0, 2.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 6.17 (s, 1H), 2.71 (s, 3H), 2.54 (s,

31 !). 2.49 (s, 3H), 2.36 (s, 3H), 2.30 (s, 3H), 2.27 (s, 3H); ¹³ C NMR (125 MHz, CD K b) δ

166.5, 157.9, 154.4, 150.9, 142.3, 142.1, 139.5, 138.9, 137.5, 132.5, 132.2, 131.8, 131.7, 131.2,

129.6, 125.5, 121.6, 19.8, 17.2, 16.7, 15.2, 14.2, 12.8. ESMS [M+HJ+ ca!cd for C23H24BF2N6 433.2, found 433.26.

Compound 5b: ^! ! ( NMR (400 VI 1 (/. CDCI3) δ 8.54 (s, 1H), 8.46 (dd, J = 7.6, 1.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 6.11 (s, 1H), 3.12 (s, 3H), 2.67 (s, 3H), 2.56 (s, 3H),

2.46 (s, 3H), 2.38 (s, 3H), 2.26 (s, 3H), 2.23 (s, 3H); ⁱ³ C NMR (100 MHz, CDCI3) δ 167.2, 164.1, 154.5, 151.2, 141.7, 141.6, 139.2, 138.4, 137.2, 132.6, 132.1, 132.1, 131.8, 131.2, 129.4, 125.3, 121.7, 21 .2, 20.1 , 1 7.5, 16.7, 1 5.4, 14.5, 13.1. ESIMS [M-Hj- calcd for C24H24BF2N6 445.21, found 445.21. Example 6 - Characterization of TCO-reacted 2b, 4b and 5b

Characterization of TCO-reacted 2 b

An aliquot of TCO-reacted 2b in MeCN was purified via flash column chromatography to remove excess TCO (dichloromethane:methanol gradient 20:0.1, 20:0.2, 20:0.3). Ή NMR (400 MHz, CDCI3) 5 7.50 (d, J = 6.4 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.01 (s, IPI), 6.00 (s, 2H), 3.93 (m, 1H), 3.02 (dd, J = 10, 6.4 Hz, 1 H), 2.56 (s, 6H), 2.07 (s, 3H), 2.01 -1.80 (m, 3H), 1.71- 1.59 (m, 4H), 1.40 (s. 6H), 1.35-1.25 (m, 4H).

ESIMS i VI · 1 i ; · calcd for C30H36BF2N4O 517.30, found 517.34.

haracterization ofTCO-reacted 4b

An aliquot of TCO reacted 4b in MeCN was purified via flash column chromatography to remove excess TCO (dichloromethaneimethanol gradient 20:0.1, 20:0.2, 20:0.3). ¾ NMR (400 MHz, CDC13) δ 7.90 (d, J = 8.4 Hz, 1H), 7.63 (t, J = 1.6 Hz, i l l}. 7.23 (m, 1H), 7.22 (m, 1H), 7.05 (s, H i). 5.98 (s, 2H), 3.85 (m, 1H), 3.56 (dd, J = 11.6, 3.6 Hz, i l l). 2.56 (s, 6H), 2.19 (m, 2H), 1.98-1.89 (m, 2H), 1.85 (s, 3H), 1.60 (m. 3H), 1.41 (s, 61 ! }. 1.31 (rn, 4H). ESIMS [M+H]+ calcd for C30H36BF2N4O 517.30, found 517.32.

Characterization ofTCO-reacted 5b

An aliquot of TCO reacted 5b in MeCN was purified via flash column chromatography to remove excess TCO (dichioromethane:rnethanol gradient 20:0.1 , 20:0.2, 20:0.3). ^l E NMR (400 MHz. CDC13) δ 7.69 (s, 1H), 7.57 (d, J = 8 Hz, 1H), 7.09 (s, 1H), 7.07 (s, 1H), 6.08 (s, 1H), 3.90 (rn, 1 ) I). 3.54 (dd, J = 9.6, 6.8 Hz, 1H), 2.64, (s, 3H), 2.54 (s, 3H), 2.44 (s, 3H), 2.33 (s, 3H), 2.18 (s, 3H), 2.13 (s, 3H), 1.94 (s, 3H), 1.86 (m, 2H), 1.82-1.75 (m, 4H), 1.64-1.58 (m, 4H), 1.32 (rn, III). ESIMS [M+BJ+ calcd for C32H39BF2N4O 545.33, found 545.38. The quantum yield and fluorogenic activation of the compounds were measured and compared to the pre-TCO compounds. As shown in Table 1, the compound exhibited a significant increase in fluorescence as compared to the precursor compounds.

Table 1.

a Quantum yield for dihydropyridazine product; fluorescein in 0.1 M NaOH (pH 13, =0.925) was used as the standard.

b Increase in peak fluorescence intensity at reaction completion; for experiments in water, 400 nM BODIPY-Tz, and 1 uM TCO were used.

0 Compound 5b is insufficiently soluble in water for this determination.

The exceptional fluorogenic turn-on of 2 and 4— as much as 100-fold greater than flexibly linked fiuorophore-Tz conjugates— suggested that FRET may not be the sole quenching mechanism for these compounds. Example 7 - Absorption and Fluorescence of 4b and TCO-reacted 4b

Figure 5 provides the absorption and fluorescence emission spectra of 4b in toluene (dielectric constant 2.4) and acetomtrile (dielectric constant 37.5). Samples were prepared by matched dilutions from a concentrated stock solution of 4b. For the fluorescence spectra, the excitation wavelength was offset from peak absorbance in that solvent by 10 nm; in toluene, the excitation wavelength was 496 nm; in acetomtrile,

489 nm. Emission spectra are the mean of three scans and dashed lines correspond to +/- SEM.

The activation, of 4b upon reaction, with TCO is demonstrated in Figure 6, which shows the emission spectra (excitation 490 nm) of compound 4b in water, before and after addition of TCO. For baseline and pre-TCO spectra, data plotted are means of 3 scans and dashed lines indicate +/- SEM. Given that the fluorescence emission intensity of 4b was found to be independent of solvent polarity, redox-based quenching, such as via photomduced-electron transfer (PET) from the excited BODIPY to the relatively electron-poor tetrazine ring, was judged unlikely. Example 8 - Biological application of activatable BODIPY

Having advanced our understanding of the quenching mechanism, die utility of fluorogenic BODIPY-tetrazines for biological imaging was explored further (see FIG. 7). Figure 7A shows fluorogenic imaging of EGFR expression on both fixed and live A431 ceils. Cells were incubated with TCO-conjugated monoclonal antibodies (see S. S. Agasti et al . Small 2013, 9, 222-227), washed, and then imaged immediately after the addition of lOGnM BODIPY- Tz in PBS. As shown in Figure 7B, Fluorogenic live-cell imaging of intracellular nanoparticles internalized by RAW 264.7 cells. The nanoparticles are labeled with both TCO and with the near-infrared dye VT680 (see S. S. Agasti et al . Small 2013, 9, 222-227), and were imaged in two channels after addition of 100 liM BODIPYTz, demonstrating co-localization. Both extracellular and intracellular TCO-labeled targets were readily visualized, with excellent signal intensity, very low background, and wi th no washing steps required after addition of the dye solution.

Example 9 - Preparation of compound 7

To hrornocoumarin 7a (245.0 mg, 0.733 mmol) in 8.0 mL of dioxane:water (3: 1) was added 3-cyanophenylboronic acid (215.4 mg, 1.47 mmol), Pd(OAc)2(PPl¾)2 (27.4 mg, 0.037 mmol), and K.2CO3 (202.6 mg, 1.46 mmol). The reaction mixture was refluxed for 7 hours after which it was concentrated using a rotary evaporator and purified using flash column chromatography (hexanes; ethyl acetate gradient, 6:1 to 4:1) to give 7b (105.0 mg, 0.29 mmol, 40.1 %) as a yellow solid.

¾ NMR (400 MHz, CDC1 ₃ ) δ 7.61-7.47 (m, 4H), 7.04 (s, 1H), 3.25 (m, 4H), 2.89 (t, J = 6.4 Hz, 2H), 2.76 (t, J = 6.0 Hz, 2H), 2.16 (s, 3H), 1.97 (m, 4H); ¹³ C NMR (100 MHz, CDCI3) δ 161.9, 150.5, 149.5, 146.2, 137,4, 135.6, 134.5, 131.2, 129.2, 122.6, 1 18.9, 118.5, 118.1, i 1 2.6. 109.0, 106.7, 50.1 , 49.7, 27.9, 21.7, 20.8, 20.6, 16.6. ESIMS | · 1 1 j ^' calcd for C23H21N2O2 357.42, found 357.16.

Bromocoumarin 7a was prepared from literature protocol (Gong, et aL, PCX int. Appl. (2006), WO 2006026368). Example 10 - Preparation of HyperEmissive Ligation- Initiated Orthogonal Sensing (HELIOS) probe 400 Me

To nitrile 7b (100.0 nig, 0.28 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf)2 (51.3 mg, 0.14 mmol), MeCN (0.15 mL, 2.80 mmol), dioxane (0.22 mL) and NH2NH2 (0.44 mL, 14.0 mmol). The vessel was sealed and allowed to stir at 60 °C for 15 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added NaN0 ₂ (386.4 mg, 5.60 mmoi) in 10 mL of water followed by 1 M HC1 until the pH = 3. The aqueous phase was extracted three times with methylene chloride (100 mL). The combined organic extracts were dried with MgS0 ₄ and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography (hexanes: ethyl acetate gradient 5: 1 to 1 : 1) to give HELIOS 400Me (55.3 mg, 0.13 mmol, 46.4%) as an orange solid.

¾ NMR (400 MHz, CDCI3) δ 8.55 (d, J = 8.0 Hz, 1H), 8.50 (s, H i). 7.63 (t J - 8.0 Hz, 1H), 7.55 (d, J = 7.6 Hz, IH), 7.06 (s, 1H), 3.26 (m, 4H), 3.07 (s, 3H), 2.93 (t, J = 6.4 Hz, 2H), 2.79 (t, J = 6,4 Hz, 21 i ). 2,23 (s, 3 PI), 1.99 (m, 4H); ¹³ C NMR (100 MHz, CDCI3) δ 167.4, 164.3, 162.1, 150.4, 149.1 , 145.6, 137.2, 135.2, 132.0, 130.3, 129.4, 127.2, 122.6, 1 19.9, 1 18.6, 109.7, 107,2, 50,2, 49.8, 27.9, 21 ,8, 21.3, 20,9, 20.7, 16.7. ESIMS [M+H] ⁺ calcd for C25H24N5O2 426.19, found 426.24.

Example 11 - Preparation of HELIOS 40011

To nitrile 7b (110.4 nig, 0.31 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf)2 (56.6 nig, 0.15 mmol), formamidine acetate (322.7 mg, 3.1.0 mmol), DMF (0.24 mL) and NH ₂ NH ₂ (0.49 mL, 15.5 mmol). The vessel was sealed and allowed to stir at 60 °C for 15 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added NaNCb (427.8 mg, 6.20 mmol) in 10 mL of water followed, by 1 M HCI until the pH = 3. The aqueous phase was extracted three times with methylene chloride ( 100 mL). The combined organic extracts were dried with MgS0 ₄ and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography (hexanes: ethyl acetate gradient, 4: 1 to 2: 1 ) to give HELIOS 400H (12.1 mg, 0.029 mmol, 9.5%) as an orange solid.

³ H NMR (400 MHz, CDCI3) δ 10.19 (s, 1 H), 8.60 (d, J = 7.6 Hz, I S ! }. 8.54 (s, 1 H), 7.66 (t, J = 7.6 Hz, 1 H), 7.60 (d, J - 8.0 Hz, 1H), 7.08 (s, 1 H), 3.28 (m, 4H), 2.94 (t, J - 6.4 Hz, 2H), 2.81 (t, J = 6.4 Hz, 2H), 2.24 (s, 3H), 2.01 (m, 4H); ¹³ C NMR (100 MHz, CDCI3) δ 166.5, 161.9,

157.8, 150.3, 148.9, 145.6, 137.2, 135.6, 131.6, 130.6, 129.4, 127.4, 122.4, 1 19.6, 1 18.4, 109.4,

106.9, 50.0, 49.6, 27.8, 21.6, 20.7, 20.5, 16.5. ESIMS M+H] ⁺ calcd for C24H22N5O2 412.17, found 412.1 8.

Example 12 - Preparation of 7c

To bromocoumarin 7a (105.5 mg, 0.31 mmol) in 4.0 niL of dioxane: water (3:1) was added 3-cyanophenylboronic acid (91.1 mg, 0.62 mmol), Pd(OAc)2(PPh.3)2 (H -6 mg, 0.015 mmol), and K2CO3 (85.6 mg, 0.62 mmol). The reaction mixture was refluxed for 7 hours after which it was concentrated using a rotary evaporator and purified using flash column

chromatography (hexanes: ethyl acetate gradient, 6:1 to 4: 1) to give lc (37.7 mg, 0.10 mmol, 34, 1%) as a yellow solid.

⁵ H NMR (400 MHz, (CD ₃ )2SO) δ 7.86 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.20 (s, 1H), 3.25 (m, 4H), 2.75 (m, 4H), 2.14 (s, 3H), 1.89 (m, 4H); ¹³ C NMR (100 MHz, (C! ⁾ , ) >SO) δ 160.2, 149.7, 149.3, 145.6, 140.8, 131.8 (2C), 122.8, 118.8, 118.0, 1 17.3, 109.9, 108.0, 105.2, 49.2, 48.6, 27.0, 20.9, 20.1, 19.9, 16.1. ESMS [M+H] ⁺ calcd for C23H21N2O2 357.16, found 357.16. Exampl 13 ^■■■ · Preparation of HELIOS 400pMe

To nitrile 7c (37.7 mg, 0.10 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf)2 (19.3 mg, 0.52 mmol), MeCN (0.055 mL, 1.05 mmol), dioxane (0.083 ml.) and NH2NH2 (0.16 mL, 5.28 mmol). The vessel was sealed and allowed to stir at 60 °C for 15 hours after which it was allowed, to cool and the septum removed . To the reaction mixture was added NaN0 ₂ (145.9 mg, 2.11 mmo!) in 10 mL of water followed by 1 M HC1 until the pH = 3. The aqueous phase was extracted three times with methylene chloride (100 mL). The combined organic extracts were dried with MgSCn and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography (hexanesiethyl acetate gradient, 4: 1 to 2: 1) to give HELIOS 400p!Vie (31.1 mg, 0.073 mmol, 73.1%) as an orange solid.

Ή NMR (400 MHz, CDCb) δ 8.63 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.06 (s, 1H), 3.26, (m, 4H), 3.08 (s, 3H), 2.93 (t, J = 6.0 Hz, 2! I). 2.79 (t, J = 6.4 Hz, 21 1 ). 2.34 (s, 3H), 1.99 (m, 4H); ¹³ C NMR (100 MHz, CDCI3) δ 167.4, 164.2, 161.9, 150.5, 149.0, 145.8, 140.7, 131 .9 (2C), 130.9, 127.9 (2C), 122.6 , 119.7, 1 18.5, 109.4, 106.9, 50.2, 49.8, 28.0, 21.8, 21.4, 20.9, 20.7, 16.7. ESIMS [M+Hf calcd for C25H24N5O2 426.19, found 426.19.

Example 14 - Preparation of 8a

To coumarin 339 (8) (410.6 mg, 1.90 mmol) dissolved in 25 mL of acetonitrile was added NBS (373.7 mg, 2.10 mmol) and the reaction mixture allowed to stir for 2 hours. The crude mixture was concentrated using a rotary evaporator and purified using flash column chromatography (methylene chloride to methylene chloride:methanol 250: 1) to give 8a (276.4 mg, 0.94 mmol, 49.4%) as a yellow solid.

¾ NMR (400 MHz, (CD ₃ ) ₂ 80) δ 7.27 (s, 1H), 6.93 (bs, 1H), 6.29 (s, 1H), 3.24 (t, J = 4.8 Hz, 2H), 2.72 (t, J = 5.6 Hz, 2H), 2,44 (s, 3H), 1 .79 (m, 2H); ¹³ C NMR (100 MHz, (CD ₃ )2SO) δ 156.9, 152.2, 152.1, 149.4, 125.6, 118.1 , 107.9, 103.2, 97.0, 40.3, 26.3, 20.6, 18.9. ESIMS

[M+lTf calcd for Ci ₃ Hi ₃ BrN02 294.01, found 294.00. Coumarin 339 (8) was prepared from, literature protocol (R. L. Atkins, D. E. Bliss, J. Org. Chem. 1978, 43, 1975). Example 15 - Preparation of 8b

To bromocoumarin 8a (150 mg, 0.51 mmol) in 5.2 mL of dioxane: water (3 : 1) was added 3-cyanophenylboroiiie acid (151.6 mg. 1.01 mmol), Pd(OAc j;;( PPh ;); (19, 1 mg, 0.025 mmol), and K2CO3 (140.9 mg, 1.01 mmol). The reaction mixture was refluxed for 7 hours after which it was concentrated using a. rotar evaporator and purified using flash column chromatography (hexanes: ethyl acetate gradient, 4: 1 to 2: 1) to give 8b (74.5 mg, 0,23 mmol, 46.2%) as a yellow solid.

f H NMR (400 MHz, CD3OD) δ 7.72 (m, H i). 7.67 (s, I H), 7,60 (m, 2H), 7.33 (s, H I ). 6.36 (s, 1H), 3.36 (t, J = 5,6 Hz, 2H), 2.83 (t, J = 6.4 Hz, 21 ! }. 2.23 (s, Π ). 1.93 (quin, J - 5,6 Hz, 21 S ): ¹³ C NMR (100 MHz, (CD ₃ ) ₂ SO) δ 160.4, 152.9, 149.5, 149.1, 136.9, 135.7, 134.2, 131 .0, 129.2, 125.7, 1 1 8,7, 117.7, 116.9, 11 1.1, 108.2, 97.1, 40.4, 26.4, 20.7, 16.1. ESIMS \ \V \ i y calcd for C20H17N2O2 317,12, found 317.18.

Lxaisjple

To nitrile 8b (64.8 mg, 0,20 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf) ₂ (37.4 mg, 0.10 mmol), MeCN (0.11 mL, 2,10 mmol), dioxane (0.16 mL) - and BbNEb (0.32 mL, 10.20 mmol). The vessel was sealed and allowed to stir at 60°C for 15 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added NaN0 ₂ (276.0 mg, 4.0 mmol) in 10 mL of water followed by 1 M HC1 until the pH = 3, The aqueous phase was extracted three times with methylene chloride (100 mL). The combined organic extracts were dried with MgSQ ₄ and concentrated using a rotar evaporator. The crude mixture was purified using flash column chromatography (hexanes: ethyl acetate gradient, 3:1 to 1 : 1) to give HELIOS 388Me (33.3 mg, 0.086 mmol, 43.2%) as an orange solid.

H NMR (400 MHz, CDC1 ₃ ) δ 8,56 (d, J = 8.0 Hz, 1H), 8.50 (s, 1H), 7.64 (t, J = 7.6 Hz,

1 H), 7.56 (d, J = 7.6 Hz, 1H1 7.18 (s, lH), 6.37 (s, IH), 4.55 (s, 1H), 3.37 (m, 2H), 3.07 (s, 3H), 2.80 (t, J = 6.0 Hz, 2! !).. 2.25 (s, 3H), 1.95 (quin, J = 5.6 Hz, 2H); ¹³ C NMR (100 MHz, CDCb) 6 167.5, 164.3, 162.1 , 153.6, 149.1, 148.3, 136.9, 135.1, 132.1, 130.3, 129.5, 127.3, 125.7, 120.5, 118.5, 110.6, 99.2, 41.8, 27.2, 21.7, 21.4, 16.7. ES1MS [M+Hf calcd for C22H20N5O2 386.16, found 386.13.

Example 17 - Preparation of HELIOS 388H

To nitriie 8b (76.7 mg, 0.24 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf)2 (44.3 mg, 0.12 mmol), fonnamidine acetate (251,9 mg, 2.42 mmol), DMF (0.19 mL) and NH2NH2 (0.38 mL, 12.1 mmol). The vessel was sealed and allowed to stir at 60°C for 15 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added aN0 ₂ (333.9 mg, 4.84 mmol) in 10 mL of water followed by 1 M HC1 until the pH = 3. The aqueous phase was extracted three times wit methylene chloride (100 mL). The combined organic extracts were dried with MgSC and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography (hexanes: eth l acetate gradient, 3 : 1 to 1 :1) to give HELIOS 388H (7.48 mg, 0.020 mmol, 8.4%) as an orange solid.

' f ! NMR (400 MHz, (C! ), bSO) δ 10.62 (s, 1H), 8.50 (d. J = 8.0 Hz, i l l). 8.38 (s, 1 H), 7.74 (t, J = 7.6 Hz, U l i 7.62 (d, J = 7.6 Hz, i l l ). 7.33 (s, 1H), 6.86 (s, 1H), 6.36 (s, 1H), 3.28 (m, 2H), 2.76 (t, J = 6.0 Hz, 2H), 2.23 (s, 3H), 1.82 (m, 2H); ¹³ C NMR (100 MHz, (CD ₃ ) ₂ SO) δ 1.65.4, 160.6, 158.1, 152.9, 149.2, 149.0, 136.8, 135.1 , 131.7, 129.9, 129.2, 126.7, 125.7, 118.1, 117.7, 108.4, 97.2, 40.4, 26.5, 20.8, 16.2. ESIMS [M+H] ⁺ calcd for C21H18N5O2 372.14, found 372.12.

le 18 - Preparation

To bromocoumarin a (5.25 g, 20.7 mmol) in 133.0 mL of dioxane: water (3 : 1) was added 3-cyanophenylboronic acid (4.56 g, 31.0 mmol), Pd(OAc)2(PPb.3)2 (775.3 mg, 1.03 mmol), and 2CO3 (5.71 g, 41.4 mmol). The reaction mixture was refmxed for 2 hours after which it was concentrated using a rotary evaporator. The crude was partitioned between water and methylene chloride and extracted 3 times (250 mL), concentrated, using a rotar evaporator and purified using flash column chromatography (methylene chloride methanol, 10:0.05) to give 3b (2.38 g, 8.6 mmol, 41.5%) as a white solid.

Ή NMR (400 MHz, (CD ₃ )2.80) δ 7.82 (m, iH), 7.78 (s, 1H), 7.64 (m, 2H), 7.50 (d, J = 8.4 Hz, IH) 6.63 (d, J = 8.8 Hz, IH), 6.46 (s, IH), 6.19 (s, 2H), 2.15 (s, 3H); ¹³ C NMR (100 MHz, (CD ₃ )2SO) δ 160.4, 154.6, 153.1, 149.6, 136.7, .135.7, 134.2, 131.1, 129.3, 126.9, 118.7, 117.6, 111.6, 111.2, 108.9, 99.3, 16.2. ESIMS [M+H] ⁺ calcd for O7H13N2O2 277.09, found 277.06.

Bromocoumarin 9a was prepared from literature protocol (M. S. Schiedel, C. A. Briehn, P. Bauerle, Angew. Chern. Int. Ed 2001, 40, 4677-4680).

>Ie 19 - Preparation of HELIOS 327M

To nitrile 9b (39.0 mg, 0.14 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf)2 (25.8 mg, 0.070 mmol), MeCN (0.073 ml,, 1 .40 mmol), dioxane (0.11 mL) and NH2 H2 (0.22 mL, 7.00 mmol). The vessel was sealed and allowed to stir at 60°C for 15 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added NaNQ ₂ ( 193.2 mg, 2.80 mmol) in 10 mL of water followed by 1 M HO until the pH ^:::: 3. The aqueous phase was extracted three times with methylene chloride (100 mL). The combined organic extracts were dried with MgS0 ₄ and concentrated using a rotary evaporator, The crude mixture was purified using flash column chromatography (hexanesrethyl acetate gradient, 2: 1 to 1 :1) to give HELIOS 347Me (5.94 mg, 0.017 mmol, 12.3%) as a red solid.

^! H NMR (400 MHz, (CDs^SO) δ 8.46 (d, J = 7.6 Hz, 1H), 8.35 (s, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.60 (d, J - 7.6 Hz, 1H), 7.52 (d, J = 8.4 Hz, lH), 6.62 (d, J = 8.4 Hz, 1H), 6.48 (s, 1H), 6.16 (s, 2H), 3.00 (s, 3H), 2.22 (s, 3H); ^B C NMR (100 MHz, (CD ₃ ) ₂ SO) δ 167.1, 163.2, 160.6, 1.54.6, 152.9, 149.2, 136.5, 134.6, 131.8, 129.5, 129.2, 126.9, 126.4, 118,7, 111.5, 109.0, 98.3, 20.8, 16.2. ESIMS [M+H] ⁺ calcd for C19H16N5O2 346.13, found 346.11.

Example 20 - Preparation of HELIOS 3471 !

To nitrite 9b (200.0 mg, 0,72 mmol) in a microwave reaction tube under a stream of argon was added Zn(OTf)2 (132.2 mg, 0.36 mmol). formamidine acetate (749.6 mg, 7.2 mmol), DMF (0.56 mL) and NH2 H2 (1.13 mL, 36.0 mmol). The vessel was sealed and allowed to stir at 60°C for 15 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added NaNOa (993.6 mg, 14.4 mmol) in 15 mL of water followed by 1 M HC1 until the pH = 3. The aqueous phase was extracted three times with methylene chloride (150 mL). I ^' he combined organic extracts were dried with MgS0 ₄ and concentrated using a rotary evaporator. The crude mixture was purified using flash column chromatography (hexanesrethyl acetate gradient, 2:1 to 1:1) to give HELIOS 347H (17.6 mg, 0.053 mmol, 7.4%) as a red solid.

/ I ^l H NMR (400 MHz, (CD ₃ )2SO) δ 10.61 (s, l ! h. 8.50 (d, J = 8.0 Hz, 1 Π }. 8.39 (s, I I ). 7.75 (t, J = 8.0 Hz, 1H), 7.63 (d, J - 7.6 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 6.62 (dd, J ^« 8.8, 2.4 Hz, i l l ). 6.48 d (J = 2.0 Hz, 1 H , 6.16 (s, 21 1 2.23 (s, 3H); ¹³ C NMR. (1 00 MHz, (CD ₃ ) ₂ SO) δ 165.4, 160.6, 158.1 , 154.6, 152.9, 149.2, 136.6, 135.0, 131.8, 129.8, 129.2, 126.9, 126.7, 1 18.6, 111.5, 109.0, 98.3, 16.2. ESIMS [M+H] calcd for ^U _^ iS-X 332.1 1 , found 332.09.

Example 21 - Preparation of 10a

To difluorinated hvdroxycoumarin (Marina Blue®) (10) ( 1.06 g, 4.99 mmol) dissolved in 50 mL of acetonitrile was added NBS (0.93 g, 5.24 mmol) and the reaction mixture allowed to stir for 1 hour. The crude mixture was concentrated using a rotary evaporator and purified using flash column chromatography (hexanes: ethyl acetate, 2: 1) to give 10a (1.32 g, 4.52 mmol, 90.6%) as a white solid.

Ή NMR (400 Mi l/, MeOD) δ 7.30 (d, J - 1 1.2 Hz, 1H), 2.51 (s, 3H); ³³ C NMR (100 MHz, MeOD) δ 147.9, 143.3 (t, J = 2.8 Hz), 141.0 (dd, J = 234.7, 5.0 Hz), 131.3 (dd, J = 216.0, 6.4 Hz), 129.9 (m, 2C), 102.7 (d, J = 9.1 Hz), 101.7, 97.7 (dd, J = 19.0, 3 ,3), 10.4. ESMS [M- H]- calcd for Ci ₀ H ₄ BrF ₂ O3 288.93, found 288.88.

Example 21 - Preparation of 10b

To bromocoumarin 10a (475.9 mg, 1.63 mmol) in 15.0 mL of dioxane:water (3 : 1) was added 3-cyanophenylboronic acid (479.0 mg, 3.26 mmol), Pd(OAc) ₂ (Pl¾)2 (61.2 mg, 0.082 mmol), and K.2CO3 (225,3 mg, 3.26 mmol). Th.e reaction mixture was refluxed for 7 hours after which it was concentrated using a rotary evaporator and purified using flash colum chromatography (hexanes:ethyl acetate gradient, 2: 1 to 1 : 1) to give 4b (142.9 mg, 0.45 mmol, 28.0%) as a white solid.

³ H NMR (400 MHz, (CD ₃ )2SO) δ 7.89 (ΐ, J = 3.6 hz, 1H), 7.83 (s, 1H), 7.69 (m, 2H), 7.60 (d, J = 1 1 .6 Hz, 1 H), 2.21 (s, 3H); ¹³ C NMR (100 MHz, (CD ₃ ) ₂ SO) δ 158.7, 148.8 (t, J = 2.6 Hz), 148.6 (dd, J - 232.6, 5.2 Hz), 139.1 (dd, J = 235.8, 6.8 Hz), 138.5 (dd, J = 7.6, 1.8 Hz), 137.6 (dd, J - 12.7, 5.3 Hz), 135.7, 135.3, 133.8, 131.8, 129.5, 122.6, 1 18.5, 1 11.4, 11 1.1 (d, J = 8.9 Hz), 107.0 (dd, J = 18.6, 2.9 Hz), 16.6. ESIMS [M +H] ⁺ calcd for 314.06, found 314.01.

Example 22 - Preparation of 4c

To nitrile 10b (91.1 mg, 0.29 mmol) in 3.0 mL of methylene chloride was added N,N- Diisopropylethyiamine (0.15 mL, 0.87 mmol) and. DMAP (1.77 mg, 0.014 mmol). The mixture was then cooied to 0°C and bromomethyl methyl ether (0.059 mL, 0.72 mmol) was added dropwise. The ice hath was removed and the reaction was allowed to stir at room temperature for one hour after which it was concentrated using a rotary evaporator and purified using flash colum chromatography (hexanes:ethyl acetate 4: 1) to give 10c (99.9 mg, 0.28 mmol, 96.4%) as a white solid.

¾ NMR (400 MHz, CDCI3) δ 7.71 (d, J === 7.6 Hz, 1H), 7.58 (s, 1H), 7.54 (m, 2H), 7.20 (dd, J = 8.8, 2.0 Hz, 1H), 5.28 (s, 11 1). 3.59 (s, 3H), 2.25 (s, 3H); ⁱ³ C NMR (100 MHz, CDCI3) 5 159.0, 152.5 (dd, J = 241.0, 4.0 Hz), 147.8 (v. J = 2,6 Hz), 144.1 (dd, J = 246 7. 5.8 Hz), 139. 1 (dd, J = 8.1, 2.4 Hz), 136.4 (dd, J = 11.2, 4.7 Hz), 135.4, 134.8, 133.9, 132.3, 129.7, 125.7, 118.5, 116.1 (d, J = 8.8 Hz), 113.2,. 106.5 (dd, J - 18.6, 3.8 Hz), 99.3 (t, J = 3.8 Hz), 57.6, 17.1. ESIMS [MHTf calcd for C19H14F2NO4 358.08, found 358.04. >le 23 - Preparation of HELIOS 370Me

To mtrile 10c (144.0 mg, 0.40 mmol) in a -microwave reaction tube under a stream of argon was added Zn(OTf)2 (73.6 mg, 0.201 mmol), MeCN (0.21 mL, 4.03 mmol), dioxane (0.32 mL) and NH ₂ NH ₂ (0.63 mL, 20.1 mmol). The vessel was sealed and allowed to stir at 60°C for - 15 hours after which it was allowed to cool and the septum removed. To the reaction mixture was added NaNO? (556.1 mg, 8.06 mmol) in 10 mL of water followed by 1 M HO until the pH = 3. The aqueous phase was extracted three times with methylene chloride (100 ml.). The combined organic extracts were dried with MgSCH and concentrated using a rotary evaporator. The crude mixture was filtered through l Og of silica (methylene chloride:methanol, 100:0.5,

1 OOmL) and concentrated using a rotary evaporator. This mixture was then dissolved, in 8 mL of methylene chloride and TFA (1 mL) was added and the reaction was allowed to stir at room temperature for 30 minutes, after which it was concentrated under a stream of nitrogen. The crude mixture was purified using flash column chromatograp hy (methylene chloride methanol, 10:0.1) to give HELIOS 370Me (77.4 mg, 0.20 mmol, 50.6%) as a red solid.

Ή NMR (400 MHz, (0)3)280 δ 8.50 (d, J = 7.60 Hz, 1H), 8.40 (s, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.64 (d, J = 8.0 Hz, IH), 7.55 (m, 1H), 3.01 (s, 3H), 2.26 (s, 3H); ¹³ C NMR (100 MHz, (CD ₃ )2SO) δ 167.2, 163.1, 158.9, 148.6 (dd, J = 233.0, 5.3 Hz), 148.3 (t, J = 2.6 Hz), 139.1 (dd, J - 233.1 , 9.3 Hz), 138.5 (dd, J = 7.5, 2.0 Hz), 137.4 (m), 135.5, 134.2, 131.9, 129.4, 129.1, 126.9, 123.6, 1 11.3 (d, J = 9.3 Hz), 107.0 (dd, J = 19.0, 2.7 Hz), 20.8, 16.7. ESIMS j -! ! j- calcd for C19H11F2N4O3 381.08, found 381.06.

Example 24 - Preparation of HELIOS 370H

To nitrile 10c (144,0 mg, 0.40 rnmol) in a microwave reaction tube under a stream of argon was added Zn(OTf)2 (73.6 mg, 0.201 rnmol), formamidine acetate (419.6 mg rnL, 4.03 rnmol), DMF (0.32 mL) and NH2NH2 (0.63 mL, 20.1 rnmol). The vessel was sealed and allowed to stir at 60°C for 15 hours after which it was allowed to cool and the septum removed.. To the reaction mixture was added NaN0 ₂ (556.1 mg, 8.06 rnmol) in 10 mL of water followed by 1 M HQ until the pH - 3, The aqueous phase was extracted three times with methylene chloride (100 mL). The combined organic extracts were dried with MgSQ ₄ and concentrated using a rotary evaporator. The crude mixture was filtered through 10 g of silica (methylene chloride methanol, 100:0.5, lOOmL) and. concentrated using a rotary evaporator. This mixture was then dissolved in 8 mL of methylene chloride and TFA (1 mL) was added and the reaction was allowed to stir at room temperature for 30 minutes, after which it was concentrated under a stream of nitrogen. The crude mixture was purified using flash column chromatography (methylene

chloride methanol, 10:0.1) to give HELIOS 370H (26.9 mg, 0.073 rnmol, 18.3%) as a red solid.

1 1 NMR (400 MHz, (CD ₃ ) ₂ SO) δ 10.61 (s, Ϊ 1 1 }. 8.53 i d. J = 8.0 Hz, Hi), 8.44 (s. 1 1 0, 7.78 (t, J = 7.6 Hz, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.60 (d, J - 11.6 Hz, 1H), 2.27 (s, 3H); °C NMR (100 MHz, (CD ₃ ) ₂ 8()) δ 165.3, 158.9, 158.2, 148.5 (dd, J = 232.0, 5.1 Hz), 148.5 (t, J = 2.4 Hz), 139.1 (dd, J = 235.6, 7.2 Hz), 138.5 (dd, J = 9.4), 137.4 (m), 135.5, 134.6, 131.9, 129.5 (2C), 127.3, 123.6, 111.3 (d, J = 8.9 Hz), 107.0 (dd, J = 21.8, 2.8 Hz), 16.7. ESIMS [M-Hp calcd for C18H9F2N4O3 367.06, found 366.96.

Example 25 ~ Preparation of TCOc

To TCO-NHS (45.4 mg, 0.17 mmol) dissolved in 2 mL of methylene chloride was added triethylamine (0.05 mL, 0.35 mmol), and 2-(2-Aminoethoxy)ethatiol (0.07 mL, 0.71 mmol). The mixture was allowed to stir at room temperature for thiity mmutes after which it was

concentrated under a stream of nitrogen. The crude mixture was purified using flash column chromatography (hexanes: ethyl acetate gradient, 1 :1 to 100% ethyl acetate) to give TCOc (43.3 mg, 0.073 mmol, 98.8%) as a clear oil.

Ή NMR (400 MHz, CDC1 ₃ ) δ 5.50 (in, 2H), 5.03 (111, lH), 4.31 (dd, J = 9.6, 6.0 Hz, 1H), 3.71 (m, 2H), 3.53 (m, 4H), 3.33 (m, 2H), 2.31 (m, 4H), 2.01-1.85 (m, 4H), 1.77-166 (m, 2H) ₅ 1.55-1.51 (m, IH): ^l3 C NMR (100 MHz, CDCI3) δ 156.6, 135.1, 133.2, 80.9, 72.4, 70.4, 61.9, 41.3, 40.9, 38.8, 34.5, 32.7, 31.1. ESIMS [M+H] ⁺ calcd for C13H24NO4 258.17, found 258.37.

Example 26 - Preparation of PhaIIoidiH~TCO

To a 1.0 raM solution of TCO-PEG4- HS (70 uL, 0.7 umoles, Click Chemistry Tools, Scottsdale, AZ) in DMF in a microvial was added amino-phalloidin (60 μg, 0.07 umoles, American Peptide Company, Sunnyvale, CA) and diisopropylethyl amine (DIPEA, 0.2 pL, 1.1 μιηοΐε). After 30 minutes at room temperature with occasional vortex agitation, the reaction mixture was purified by reverse phase chromatography on a Waters Xterra C I 8, 2.5 |im, 10 mm x 50 mm, column (waterraeetonitrile, both with 0.1% formic acid; gradient elution from 5% to 75% acetonitrile) to give phalloidin TCO (58 p.g, 0.05 umoles, 70%). The amounts of aminophailoidin and phalloidin-TCO product were determined spectrophotometrically, based on. the known extinction coefficient of phalloidin at 291nm (13,500). Reverse phase LCM8 characterization of the purified material: ESI-MS [M+HJ ^;' calculated for C55H82 10O178 1187.56, found 1187.43.

Example 27— Preparation of Marina Blue - Tz

To a solution of Marina Blue - succcinirnidyl ester (LifeTechnologies, Ml 0165, Grand Island, NY) at 10 mM in DMF (50 LIL, 0.5,umoles) was added luL of diisopropylethylamine, followed by a small aliquot of dry benzylaminotetrazine-HCl (MW 223.06). After 30 minutes at room temperature with occasional vortex agitation, the reaction mixture was purified by reverse phase chromatography on a Waters Xterra C18, 2.5 urn, 10 mm x 50 mm, column

(watenaeetonilrile, both with 0.1% formic acid; gradient elution from 5% to 75% acetonitrile) to give Marina Blue - Tz (yield not determined). Reverse phase LCMS characterization of the purified material: ESI-MS [M-H] ^" calculated for C21H15F2N5O4 438.1 1 , found 438.01.

Example 28 - Fhtorogenic characterization- of HELIOS probes

As described above, the fluorescence purity of all HELIOS compounds was verified by

LCMS prior to quantitative activation experiments, for which a fresh aliquot of the fJuorophore collected from the analytical HPLC elution was used. Exceptionally pure material is required to obtain the peak measured turn-on ratios, as the presence of trace bright contaminants limits the maximum observable ratio.

Stock solutions of the freshly-purifi ed tetrazine dyes were prepared in MeCN and stored in the dark at 4°C during experiments. For fluorescence measurements, the probes diluted Into 2 niL or 3 mL of phosphate buffered saline (PBS), pH 7.4 (Corning, cellgro) in a standard 10 mm quartz cuvette. Working at peak excitation and emission wavelengths for each probe, data were collected as a continuous time series to enable accurate measurement of the baseline intensity values and optimize signal to noise. Fluorescence experiments were conducted at a range of dye concentrations spanning 100 riM-750 nM, with 500nM being a typical working concentration. The time to peak turn on ratio (but not the final magnitude) is a function of the added TCOc (see Example 25) concentration; for the time courses presented herein, 10 μΜ TCOc was used.

Measurements of solvent and pre-activation emission intensity for baseline values were collected serially over at least 30 seconds, prior to addition of TCOc to Initiate the fluorogenic reaction. After addition, of TCOc (typically a 20-fold excess, as above), the fluorescence emission Intensity was monitored until a plateau was reached. Activation ratios were calculated from the peak emission intensity of the dihydropyridazine product and the corresponding baseline intensity over background. Data were normalized to set the initial background fluorescence of the HELIOS probe to one unit over background, as plotted in FIG. 9 in the main text. Quantum yield determinations: quinine sulfate dihydrate (Fluorescence Reference Standard grade, AnaSpec, Inc) in 0.5 M H2SO4 was used as a reference, with an excitation wavelength of 370 nm; a value of 0.546 was used for the reference quantum yield (Eaton, D. F., Pure and Applied Chemistry, 1988 60(7), 1107-1114). Calculations were made according to the methods described by Crosby and Demas (Chemical Reviews, 1971, 75(8), 991-1024).

HELIOS 400Me and HELIOS 400pMe were both soluble in phosphate buffered saline (PBS) at micromolar concentrations and negligibly fluorescent in their native state. After rigorous purification to remove trace fluorescent impurities, the fluorogenic properties of these new coumarin-Tz conjugates was evaluated on reaction with TCOc, a novel trans-cyclooctene derivative (TCOc) that incorporates a carbamate-linked. PEG ₂ side chain for improved water solubility. Addition of TCOc to HELIOS 400Me in PBS yielded a 4,000-fold peak turn-on ratio (FIG. 9). Reaction of HELIOS 400/ e with TCOc yielded a turn on ratio of 1,000-fold, fourfold lower than its mem-linked counterpart. Mechanistically, the observation of 1000-fold turn- on in a perpendicular-dipole configuration argues against FRET playing a significant role in the energy transfer mechanism. As shown below ⁷ , HELIOS 388Me in PBS yielded a remarkable 11 , 000-fold turn-on upon reaction with TCOc, the highest turn-on ratio reported to date. When ligated to TCOc in PBS, HELIOS 347Me and HELIOS 370Me displayed 2,500-fold and 2,900- fold turn-on ratios, respectively, in spite of minimal spectral overlap with the tetrazine absorption band at 520nm. By comparison, FRET-based coumarin-tetrazine interactions are dramatically less efficient: a flexibly linked analogue of HELIOS 370H displayed only a 60-fold turn-on (Marina Blue-Tz), corroborating the dipole-orientation analysis described previously. All four HELIOS probes exhibit very good postclick quantum yields in PBS, in agreement with structurally similar- coumarins (Table 2).

HELSOS 400Me

HELIOS 388Me

HELIOS 347Me

HELIOS 370Me

Table 2. HELIOS 347 347 / 455 18,500 0.29 2,500-fold

- - _ND -

Marina Blue - Tz 362 / 459 ND 60-fold ^a At peak excitation wavelength in PBS, p i-I 7.4.

⁰ Quantum yield for the dihydropyridazine product after complete reaction of the indicated compound with TCOc in PBS at pH 7.4; quinine sulfate in H2S04 (0.5 M, Φ = 0.546) was used as the standard.

c Fluorogenk tum-on ratio of the Me-tetrazines upon reaction with TCOc.

Example 29 - Solvent polarity effects

Fluorescence emission spectra of HELIOS 400Me in PBS (pH 7.4), acetonitrile (dielectric constant 37,5), and toluene (dielectric constant 2.4) was measured (see FIG. 10). Emission spectra are the mean of 2 or 3 scans and the dashed lines represent +/- SE L

Instrument settings were adj usted to optimize sensitivity given the minimal fluorescence of the native HELIOS probe, and samples were prepared by matched dilution of a concentrated stock solution of HELIOS 400Me into the respective solvents. Redox-based quenching, such as through photoinduced electron transfer (PET) from the excited eouniarm to the relatively electron-poor tetrazine ring, was j udged unlikely to contribute significantly, because the fluorescence emission intensity was largely independent of solvent polarity, with less than a twofold change between PBS and the organic solvents, and no intensity difference between toluene (ε = 2.4) and acetonitrile (ε = 37.5). PET is characteristically enhanced by the relative stabilization of chargeseparated states in polar solvents [E. E. Neuteboom, S. C. J. Meskers, E. H. A. Beckers, S. Chopin, R. A. J. Janssen, J. Phys. Chem. A 2006, 110, 12363].

Example 30 - In vitro imaging

General Methods

Microscope: Multichannel images were collected on an Olympus F!noview FVIOOO confocai laser microscope. Coumarin probes were excited with a 405 nm laser, with alternate excitation, sources used as relevant for reference channels, paired with appropriate emission filter sets.

Cell culture: A-431 cells (ATCC CRL 555) and COS-1 cells (ATCC CRL-1650) were cultivated in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10 % fetal bovine serum and STO in standard culture conditions in 10 cm dishes. OVCA-429 cells were cultivated in RPMI-1640 supplemented with 10 % fetal bovine serum under standard culture conditions. For imaging experiments cells were plated on Millicell EZ slides (EMD Millipore, Ine, Billerica, MA).

Antibody reagents: Monoclonal antibody-TCO conjugates were prepared by incubation of commercially available monoclonal antibodies TCO-PEG4- HS (Click Chemistry Tools, Scottsdale, AZ). Anti EGFR (Cetuximab, Imclone), Anti cytochrome c oxidase (COXIV, Cell signaling Technology, #4844, Danvers, MA).

An aliquot, of antibody in the manufacturer-supplied storage solution was buffer exchanged into PBS with lOmM sodium bicarbonate, pH 8.0, on a 40K ZebaSpin desalting column. (0.5m.L, Thermo Fisher Scientific, Rockford, IL). To this solution was added 20 equivalents of TCO- PEG4-NHS; the mixture was allowed to react at 25 °C for 30 minutes, with continuous shaking. The reaction mix was loaded onto a 40K ZebaSpin column to remove organic solvent and small molecule fractions: this eluate was loaded onto a second 40K ZebaSpin column to ensure comprehensive removal of any excess TCO.

EGFR Imaging: Fixed A431 cells were prepared by treatment with 4 % paraformaldehyde solution (10 niin, room, temperature), followed by .3 washes with PBS. Fixed cells were stored at 4 °C until the time of imaging, when they were incubated for 20 minutes with 20 ^ig/niL cetuximab-TCO, then rinsed three times with PBS.

For optimal image quality, HELIOS 370H probe must be purified on the day of imaging by reversed phase HPLC-MS. The concentration of stock solutions in PBS were calculated by absorbance spectrometry, based on the measured extinction coefficient of 19000 M^cm ^"1 . Prior to imaging, the purified stock solutions were subjected to tum-on testing, verifying a fluorogenic turn-on ratio of >1000-fold for HELIOS 37GPL For imaging experiments, the acetonitrile stocks were diluted into PBS to yield a 100 iiM solution. Image acquisition: Immediately prior to imaging, buffer was replaced, with a 100 nM solution of HELIOS 370H probe in PBS. Specific staining was evident within 10 seconds and reached maximum signal/background intensity over a time course of 3-5 minutes.

Mitochondria Imaging: At -70% confluence, OVCA-429 cells were incubated with 3% v/v of CellLight Fluorescent mitochondria-targeted red fluorescent protein BacMam reagent, reconstituted according to the manufacturer's guidelines (CI 0601, Invitrogen, Carsbad, CA), in complete medium for 24 h. Following incubation, cells were washed in PBS and. incubated in growth media a further 24 hrs before fixation. The cells were fixed with 4% parafoiiiialdehyde and permeabilized with 0.5% Triton-X-100 in PBS. Cells were incubated with anti-COX IV-TCO (lO.ug/raL) for 40 minutes, then washed twice with PBS prior to imaging. HELIOS 388H was freshly purified as described above and added to cells at lOOnM concentration. Mitochondrial labeling was evident within 2 minutes and stable target to background ratios were observed on serial images collected up to an hour from dye addition.

Actin Cytoskeleton Imaging: carried out per procedures developed by Mitchison and coworkers (e.g. Cramer, L., and Mitchison, T.J., J Cell Biol 1993 Aug;122(4):833-43,

http://mitchison.med.harvard.edu/protocols.html). In brief, phalloidin-TCO was dissolved in methanol to prepare a stock solution at 250-1000 ^xg/mL (stored at -80°C); this stock was diluted into the labeling buffer to give a final staining solution at l .ug/raL. Labeling buffer: 10 mM Tris buffered saline, pH 7.4 (TBS), with 0.1% triton X-100 and 2% bovine serum albumin.

COS-1 cells were grown in standard culture conditions as described above and then fixed and permeabilized per the procedures of Mitchison and coworkers (vide supra). In brief, cells were fixed in 4% formaldehyde in cytoskeleto buffer for 20 minutes, and the permeabilized with 0.1% Triton-X-lOO in TBS. Cytoskeleton buffer: lOmM MES, pH6.1, 138mM C1, 3mM MgCL 2mM EGTA, 0.32M sucrose. Nuclear staining was performed by incubating the fixed and permeabilized cells with DRAQ5 (Biostatus, DR50G50) diluted to a final concentration of 1 uM for 3-5 minutes at room temperature. After 20-40 minutes incubation with phalloidin-TCO (l,ug/mL), cells were washed, once with PBS and then imaged after addition of lOOnM HELIOS 388H or HELIOS 370H.

Results.

HELIOS 370H was used as a model to determine the applicability of the HELIOS probes as a native bioorthogonal fluorogenic imaging agent. This model system was used to assess HELIOS probe kinetics in the extracellular context: imaging of the epidermal growth factor receptor (EGFR) on. the surface of cancer cells. EGFR o verexpression plays a cri tical role in the most common molecularly-defined subtype of lung cancer, where it is a key treatment target, and drives proliferation in other epithelial malignancies, including colon cancer and pancreatic cancer. As noted above, A431 cells were incubated with a TCO labeled anti-EGFR antibody (20_ug/mL, Cetuximab, ImClone) for 20 minutes and then washed briefly with PBS. Addition, of 100 nM HELIOS 370H in PBS revealed bright, membrane specific staining coinciding with the known distri bution of the receptor (FIG. 11.A). Images were generated within seconds of dye addition and exhibited no nonspecific binding even after extended incubation, nor any membrane staining in the presence of a control antibody-TCO conjugate (FIG. 11B).

To further explore the imaging potential of HELIOS probes, mitochondria were selected as a target; their structures have features at the diffraction limit of conventional light microscopy. OVCA-429 cells expressing mitochondria-specific red fluorescent protein (RFP) were incubated with an anti-mitochondria antibody-TCO conjugate and visualized with HELIOS 388H, yielding high spatial resolution images with good, colocalizatioii (FIG. 12A). Intracellular imaging of small molecule targets is another area of intense interest, given the potential, applications in drug- development, chemical biology, and optical pharmacology . To demonstrate the utility of HELIOS probes in this context with a structurally validated model system, the ability of the probes to image the actin cytoskeleton was tested with a phalloidin-TCO conjugate (Example 26). Sequential addition of phalloidin-TCO and several HELIOS probes produced vivid fluorogenic images of the cytoskeleton; control experiments revealed negligible background. (FIG. 12.B). Example 31 - In vivo imaging with HELIOS probes

Nude mice (Cox7, Massachusetts General Hospital) were surgically implanted with a dorsal skin window chamber. A-431 cells (ATCC CRL-1555, Manassas, VA), a human epidermoid carcinoma cell line with overexpression of the epidermal growth factor receptor (EGFR), were then implanted into the window chamber as a suspension of 2-3 million cells in a 1:1 mixture of phosphate buffered saline (PBS) and matrigel. Tumors were allowed to develop for ~ 2 weeks, by which, time they had become vascularized and attained a diameter of 2-3 mm.

In parallel, a TCO conjugated anti-EGFR antibody was prepared, as previously reported (Haun et al., Nat. Nanoteck , 2010, 5, 660-665). 24 hours prior to imaging mice were, injected via tail vein IV catheter with 100 xL of a 1 mg/iiiL solution of the antibody in PBS. On the day of imaging, a deri vative of compound 2a:

, was purified by HPLC. The pure dye was dried by evaporation formulated by dissolving the dry powder in 20 μΕ of a 1 :1 mixture of dimethylaeetamide:solutoI HS-15, followed by slow dilution in PBS to a final volume of 300 uL. The concentration of the clear orange solution was determined spectrophotometrically (between 100-200 μΜ) and stored at 4 °C until imaging.

Mice were anesthetized with 2% isofluxane in 2 L/min oxygen on a heated microscope stage and a. tail vein catheter was placed, in some instances, Angiosense-680 (Perkm Elmer, Walthani, MA, USA) was injected to delineate the vasculature. Static and time series images were collected using a customized. Olympus FV1000 confocal microscope (Olympus America). A XLUMPLFLN 20x water immersion objective (NA 1.0) water immersion objective was used, for data collection (Olympus America). Pre-treatment images of the turner cells were collected, and then time-lapse imaging was initiated synchronously with injection of 150 μΕ of the fluorogenic BQDIPY-tetrazine dye by tail vein IV, followed by a second 150 \iL injection of dye ten minutes later. Images in Figure 13 illustrate bright membrane-specific staining of tumor cells within the window chamber, consistent with the known distribution of EGFR, as well as punctate foci within the cells at site of endocytic receptor internalization, all with, excellent target to background ratios.

OTHER EMBODIMENTS

It is to be understood that while the invention has bee described in conjunction with, the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended cl aims. Other aspects, advantages, and modifications are within the scope of the following claims.