Fluorescent BIS-trifluoromethyl carborhodamine compounds

Abstract

The disclosure provides for the design, synthesis, and characterization of fluorophores useful for imaging.

Description

Attorney docket No.00012-089WO1 FLUORESCENT BIS-TRIFLUOROMETHYL CARBORHODAMINE COMPOUNDS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 from Provisional Application Serial No. 63 / 688,220, filed August 28, 2024, the disclosures of which are incorporated herein by reference. GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under Grant Number GM153237 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The disclosure provides for the design, synthesis, and characterization of synthetic fluorophores. BACKGROUND

[0004] Fluorophores are considered as optical “paints” to allow direct visualization of biological structures and processes with high spatial-temporal resolution. Ever since Herschel discovered blue-emitting quinine in 1845, small molecule fluorophores have been extensively studied and the color palette of these paints has expanded to the whole visible region of electromagnetic spectrum. These fluorophore scaffolds, including the well-established fluorescein, boron-dipyrromethene (BODIPY), rhodamine, cyanine, porphyrin, etc., have found great utilities in various applications including molecular imaging, sensor design, and photosensitizing. SUMMARY

[0005] Synthetic fluorophores built on a classic rhodamine scaffold are essential for modern microscopy. An attractive feature of synthetic fluorophores is their potential to access long wavelength excitation and emission profiles (>650 nm) that are difficult to achieve through genetically encoded methods like fluorescent proteins. The disclosure provides a new strategy to achieve excitation and emission above 650 nm: bis(trifluoromethyl)carborhodamine dyes, or BF dyes. In BF dyes, the geminal methyl groups of carborhodamines are replaced with trifluoromethyl (CF) groups. This accomplishes two things. First, CF groups substantially red shift in the optical profile by overAttorney docket No.00012-089WO1 90 nm compared to classic, oxygen-bridged rhodamine dyes, resulting in a dye framework with excitation and emission profiles >650 nm and high brightness (extinction coefficient >140,000 M cm and fluorescent quantum yield of 33%). Second, CF groups render BF dyes fluorogenic, by shifting the position of the open-closed equilibrium of the colorless lactone and colored zwitterion form, resulting in a 10- to 20-fold improvement in fluorogenicity compared to silicon-bridged rhodamines. The disclosure provides the design and computational analysis of BF dyes; synthetic studies to access over a dozen new BF dyes through a unique, late-stage functionalization strategy; spectra characterization; and applications in advanced fluorescence microscopy including no-wash intracellular labeling, functional imaging with chemigenetic indicators, and single molecule tracking in living cells. Together, this shows that bis(trifluoromethyl)carborhodamine dyes provide a complementary approach to achieving long-wavelength, fluorogenic dyes for live cell microscopy that does not rely on dimethyl silicon rhodamines.

[0006] The synthesis and characterization of an NIR xanthene- based fluorophore: bis-(trifluoromethyl)carborhodamines are provided. DFT was used to determine that bis- (trifluoromethyl)carborhodamines are red-shifted compared to the parent compound, CarboTMR. Bis-(trifluoromethyl)carborhodamines could be accessed in 10 steps proceeding through a carbocation cyclization. The is the first instance of the fluorophore synthesis utilizing a customizable, penultimate trityl alcohol intermediate (19) for late-stage diversification. Bis-(trifluoromethyl) carborhodamines were found to possess similar NIR spectral characteristics to silicon rhodamines, while also offering chemical modularity for spectral fine-tuning. Synthesis of the probes was accomplished with HaloTag ligands (BF and BF ), for fluorogenic targeting. In vitro,a >10-fold turn-on after binding to HaloTag protein than SiTMR, due to their much lower background fluorescence. One could also use the bis- (trifluoromethyl)carborhodamines HaloTag ligand probes for cellular voltage measurements (Voltron) and super resolution microscopy.Attorney docket No.00012-089WO1

[0007] The disclosure provides a xanthene-based fluorophore comprising a structure of Formula I or Formula II:or a pharmaceutically acceptable salt or solvate thereof, wherein, X-X are independently selected from N or C, wherein when an X group is an N, then the R group is absent; and R-R and R-R are independently selected from H, D, optionally substituted functional group (FG), optionally substituted (C-C )alkyl, optionally substituted (C-C )heteroalkyl, optionally substituted (C-C )alkenyl, optionally substituted (C-C )heteroalkenyl, optionally substituted (C-C )alkynyl, optionally substituted (C- C )heteroalkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted mixed ring system, wherein one or more adjacent R groups can be linked together to form one or more substituted rings selected from the group comprising cycloalkyl, cycloalkenyl, heterocycle, aryl, and mixed ring system;Attorney docket No.00012-089WO1 R16 18F3C CF3Ror a X is selected from O, S, NH or CH; X is selected from O, S, NH, or CH; X is a halo; R and R are selected from an optionally substituted (C-C)alkyl, or can be joined together to form a heterocycle comprising 3, 4, or 5 carbon atoms; R and R are selected from an optionally substituted (C-C)alkyl, or can be joined together to form a heterocycle comprising 3, 4, or 5 carbonatom; R is ; R -R are each individuallyselected (C-C) alkyl, optionallysubstituted (C-C) alkenyl, optionally substituted (C-C) alkynyl, alkoxy, hydroxyl, ketone, ester, aldehyde, amino, azide, cyano, halo, thiol; v is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; y is an integer selected from 0, 1, 2, 3, 4, 5, and 6. Z is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In one embodiment, the xanthene-based fluorophore has a structure of Formula I(a):Attorney docket No.00012-089WO1or a pharmaceutically acceptable salt or solvate thereof, wherein, R-R are independently selected from H, D, optionally substituted FG, optionally substituted (C-C)alkyl, optionally substituted (C-C)heteroalkyl, optionally substituted (C- C)alkenyl, optionally substituted (C-C)heteroalkenyl, optionally substituted (C-C)alkynyl, optionally substituted (C- C)heteroalkynyl, optionally substituted (C-C)cycloalkyl, optionally substituted (C-C)cycloalkenyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted mixed ring system, wherein one or more adjacent R groups can be linked together to form one or more substituted rings selected from the group comprising cycloalkyl, cycloalkenyl, heterocycle, aryl, and mixed ring system. In another embodiment, the xanthene-based fluorophore has a structure selected from Formula IIa, IIb, IIc and IId: ,Attorney docket No.00012-089WO1a biomolecule tethered to a xanthene-based fluorophore of any one of Formula I, Ia, II, IIa, IIb, IIc or IId. In one embodiment, the biomolecule is selected from an antigen, a receptor, a steroid, a protein, an antibody, a scFV, and a peptide. In a further embodiment, the biomolecule is an antibody or a scFV.

[0009] The disclosure also provides an imaging or diagnostic assay comprising the xanthene-based fluorophore of any one of Formula I, Ia, II, IIa, IIb, IIc or IId. In a further embodiment, the imaging or diagnostic assay is an in vitro assay.

[0010] The disclosure also provides a method to image cells, comprising contacting a cell with a xanthene-based fluorophore of any one of Formula I, Ia, II, IIa, IIb, IIc or IId; illuminating the cells with light having a first wavelength; imaging the cells by detecting light having a second wavelength, wherein the first wavelength and second wavelength of light have different wavelengths, and wherein the light having the second wavelength is in the far red to near infrared region. In one embodiment, the cell is contacted to measure a biological process. In a further embodiment, the biological process is an enzymatic reaction. In still another embodiment, the method measures the localization of a molecule within the cell, wherein the molecule is tagged with a xanthene-based fluorophore of any one of Formula I, Ia, II, IIa, IIb, IIc or IId.

[0011] The disclosure also provides a kit comprising a plurality of aliquots which comprise a compound of any one ofAttorney docket No.00012-089WO1 Formula I, Ia, II, IIa, IIb, IIc or IId in a buffered solution, or a concentrated solution. DESCRIPTION OF DRAWINGS

[0012] Figure 1 Frontier molecular orbital energies of CarboTMR (2), SiTMR (3), and bis-(trifluoromethyl) carborhodamine (4a) (Left to Right). Performed at the B3LYP / def2TZVP level.

[0013] Figure 2 shows absorbance and emission spectra of 4a in 1X dPBS, pH = 7.2.

[0014] Figure 3A-C presents HaloTag binding in vitro.

[0015] Figure 4A-D provides cellular imaging and localization of CF3 HaloTag in HEK cells.

[0016] Figure 5 shows frontier molecular orbitals of synthesized 2-Me BF dyes 4a-m. Computed at the B3LYP / def2TZVP (IEF- PCM Ethanol) level.

[0017] Figure 6A-M shows absorption and emission spectra of bis-CF rhodamines. Normalized absorbance (solid) and emission (dashed) spectra of bis-CF carborhodamines in 1X dPBS pH 7.2. a) SiTMR (600 nm excitation), b) 15 (600 nm excitation), c) 16 (605 nm excitation), d) 17 (575 nm excitation), e) 18 (600 nm excitation), f) 19 (585 nm excitation), g) 20 (565 nm excitation), h) 21 (605 nm excitation), i) 22 (610 nm excitation), j) 600 (600 nm excitation), k) 27 (555 nm excitation), l) 30 (600 nm excitation), m) 31 (600 nm excitation). Spectra are normalized to respective max values for each dye.

[0018] Figure 7A-F shows normalized absorbance (solid) and emission (dashed) spectra of bis-CF carborhodamines in 2,2,2- trifluoroethanol with 0.1% trifluoroacetic acid. A) 4a (592 nm excitation), B) 4g (575 nm excitation), C) 4h (595 nm excitation), D) 4j (575 nm excitation), E) 17a (598 nm excitation), F) 17b (600 nm excitation). Spectra are normalized to respective max values for each dye. 4a shown here as a reference for other dyes.

[0019] Figure 8A-C shows the effect of pH on bis-CF Me dyes. Effect of pH on bisCF electrophilic Me dyes. a) Scheme showing how pH dependent absorbance could be affected by nucleophilic attack at the C-9 carbon of BF dyes with electron withdrawing auxochromes (4g, 4h, and 4j). b) Structure and absorbance spectra of 2-Me piperazine BF probe (4g) at varying pH. 4g shows pH dependentAttorney docket No.00012-089WO1 absorbance intensity, meaning that the increased electron withdrawing character of the auxochrome makes the c-9 carbon susceptible to nucleophilic attack. c) Structure and absorbance spectra of 2-Me tetraethyl BF probe (4b) at varying pH. 4b does not display pH dependent absorbance, and therefore is not susceptible to nucleophilic attack at the C9 position. All dyes were measured in 1X dPBS at the designated pH at 2.5 μM. Data shown are average absorbance (n = 3).

[0020] Figure 9A-B shows determination of D values for BF (A) and BF (B). Plots of normalized absorbance of the different diacid dyes as a function of the dielectric constant of various dioxane / milliQ water mixtures. A) A plot of the normalized absorbance of the SiTMR-dicarb and BF (22a). B) A plot of the normalized absorbance of SiTMR-dicarb and BF (22b). The absorbance values in both (A) and (B) were normalized to each dyes respective fully open 2-Me pendant ring (3, 4a, and 4c). Data shown is the average normalized absorbance (n = 3).

[0021] Figure 10A-H shows the characterization of HaloTag ligands SiTMR , BF and BF in vitro. Structure and intensity of SiTMR (A, D),BF (B, E), and BF (C, F)unbound. Raw (G) absorbance and (H) emission intensity data for SiTMR , BF , and BF both with (+) and without (−) HaloTag protein.

[0022] Figure 11A-C shows voltage imaging with Voltron and bis- CF dyes. Voltage imaging with BF and Voltron. A) Epifluorescence image of expressing Voltron andstained with 250 nM BF . Scale bar is 20 um. B) Plot of relative change influorescence vs time for voltage-clamped HEK293T cell atpotentials. Holding potential was –60 mV and 100 ms steps were provided at +100 to –100 mV in 20 mV increments. C) Plot of relative change in BF fluorescence vs final membrane potential in mV. Data are+ / - standard error of the mean (n = 2 cells). Lines of best fit are for the steady-state change (solid line) or peak change (dashed line).Attorney docket No.00012-089WO1

[0023] Figure 12A-N shows fluorescent lifetime measurements of Bis-CF3 rhodamines. All spectra were recorded in pH 7.2 PBS buffer except for the following compounds: 4g, 4h, and 4j were recorded in trifluoroethanol with 0.1% trifluoroacetic acid, and 4m was recorded in pH 7.4 PGB buffer with 0.1% SDS. DETAILED DESCRIPTION

[0024] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a xanthene-based fluorophore" includes a plurality of such fluorophores and reference to "the voltage sensitive dye" includes reference to one or more voltage sensitive dyes or equivalents thereof known to those skilled in the art, and so forth.

[0025] Also, the use of “or” means “and / or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

[0026] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

[0027] All publications mentioned herein are incorporated herein by reference in their entirety for the purposes of describing and disclosing methodologies that might be used in connection with the description herein. Moreover, with respect to any term that is presented in the publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although many methods and reagents similar to or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are now described.Attorney docket No.00012-089WO1

[0029] The term "alkyl", refers to an organic group that is comprised of carbon and hydrogen atoms that contain single covalent bonds between carbons. Typically, an "alkyl" as used in this disclosure, refers to an organic group that contains 1 to 30 carbon atoms, unless stated otherwise. Where if there is more than 1 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 2 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkyl may be substituted or unsubstituted, unless stated otherwise.

[0030] The term "alkenyl", refers to an organic group that is comprised of carbon and hydrogen atoms that contains at least one double covalent bond between two carbons. Typically, an "alkenyl" as used in this disclosure, refers to organic group that contains 1 to 30 carbon atoms, unless stated otherwise. While a C alkenyl can form a double bond to a carbon of a parent chain, an alkenyl group of three or more carbons can contain more than one double bond. It certain instances the alkenyl group will be conjugated, in other cases an alkenyl group will not be conjugated, and yet other cases the alkenyl group may have stretches of conjugation and stretches of nonconjugation. Additionally, if there is more than 1 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 3 carbons then the carbons may also be linked in a branched fashion so that the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkenyl may be substituted or unsubstituted, unless stated otherwise.

[0031] The term "alkynyl", refers to an organic group that is comprised of carbon and hydrogen atoms that contains a triple covalent bond between two carbons. Typically, an "alkynyl" as used in this disclosure, refers to organic group that contains 1 to 30 carbon atoms, unless stated otherwise. While a C alkynyl can form a triple bond to a carbon of a parent chain, an alkynyl group of three or more carbons can contain more than one triple bond. Where if there is more than 1 carbon, the carbons may be connected in a linear manner, or alternatively if there are more than 4 carbons then the carbons may also be linked in a branched fashion so thatAttorney docket No.00012-089WO1 the parent chain contains one or more secondary, tertiary, or quaternary carbons. An alkynyl may be substituted or unsubstituted, unless stated otherwise.

[0032] The term "aryl", as used in this disclosure, refers to a conjugated planar ring system with delocalized pi electron clouds that contain only carbon as ring atoms. An "aryl" for the purposes of this disclosure encompass from 1 to 12 aryl rings wherein when the aryl is greater than 1 ring the aryl rings are joined so that they are linked, fused, or a combination thereof. An aryl may be substituted or unsubstituted, or in the case of more than one aryl ring, one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof.

[0033] The term "cylcloalkyl", as used in this disclosure, refers to an alkyl that contains at least 3 carbon atoms but no more than 12 carbon atoms connected so that it forms a ring. A "cycloalkyl" for the purposes of this disclosure encompass from 1 to 12 cycloalkyl rings, wherein when the cycloalkyl is greater than 1 ring, then the cycloalkyl rings are joined so that they are linked, fused, or a combination thereof. A cycloalkyl may be substituted or unsubstituted, or in the case of more than one cycloalkyl ring, one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof.

[0034] The term "cycloalkenyl", as used in this disclosure, refers to an alkene that contains at least 3 carbon atoms but no more than 12 carbon atoms connected so that it forms a ring. A "cycloalkenyl" for the purposes of this disclosure encompass from 1 to 12 cycloalkenyl rings, wherein when the cycloalkenyl is greater than 1 ring, then the cycloalkenyl rings are joined so that they are linked, fused, or a combination thereof. A cycloalkenyl may be substituted or unsubstituted, or in the case of more than one cycloalkenyl ring, one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof.

[0035] The term "heterocycle", as used in this disclosure, refers to ring structures that contain at least 1 non-carbon ring atom. A "heterocycle" for the purposes of this disclosure encompass from 1 to 12 heterocycle rings wherein when the heterocycle is greater than 1 ring the heterocycle rings are joined so that theyAttorney docket No.00012-089WO1 are linked, fused, or a combination thereof. A heterocycle may be a hetero-aryl or nonaromatic, or in the case of more than one heterocycle ring, one or more rings may be nonaromatic, one or more rings may be hetero-aryls, or a combination thereof. A heterocycle may be substituted or unsubstituted, or in the case of more than one heterocycle ring one or more rings may be unsubstituted, one or more rings may be substituted, or a combination thereof. Typically, the non-carbon ring atom is N, O, S, Si, Al, B, or P. In case where there is more than one non-carbon ring atom, these non- carbon ring atoms can either be the same element, or combination of different elements, such as N and O. Examples of heterocycles include, but are not limited to: a monocyclic heterocycle such as, aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazolidine, pyrazolidine, pyrazoline, dioxolane, sulfolane 2,3-dihydrofuran, 2,5-dihydrofuran tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydro- pyridine, piperazine, morpholine, thiomorpholine, pyran, thiopyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dihydropyridine, 1,4- dioxane, 1,3-dioxane, dioxane, homopiperidine, 2,3,4,7-tetrahydro- 1H-azepine homopiperazine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepin, and hexamethylene oxide; and polycyclic heterocycles such as, indole, indoline, isoindoline, quinoline, tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline, 1,4-benzodioxan, coumarin, dihydrocoumarin, benzofuran, 2,3-dihydrobenzofuran, isobenzofuran, chromene, chroman, isochroman, xanthene, phenoxathiin, thianthrene, indolizine, isoindole, indazole, purine, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, phenanthridine, perimidine, phenanthroline, phenazine, phenothiazine, phenoxazine, 1,2-benzisoxazole, benzothiophene, benzoxazole, benzthiazole, benzimidazole, benztriazole, thioxanthine, carbazole, carboline, acridine, pyrolizidine, and quinolizidine. In addition to the polycyclic heterocycles described above, heterocycle includes polycyclic heterocycles wherein the ring fusion between two or more rings includes more than one bond common to both rings and more than two atoms common to both rings. Examples of such bridged heterocycles include quinuclidine, diazabicyclo[2.2.1]heptane and 7-oxabicyclo[2.2.1]heptane.Attorney docket No.00012-089WO1

[0036] The terms "heterocyclic group", "heterocyclic moiety", "heterocyclic", or "heterocyclo" used alone or as a suffix or prefix, refers to a heterocycle that has had one or more hydrogens removed therefrom.

[0037] The term "hetero-" when used as a prefix, such as, hetero-alkyl, hetero-alkenyl, hetero-alkynyl, or hetero- hydrocarbon, for the purpose of this disclosure refers to the specified hydrocarbon having one or more carbon atoms replaced by non-carbon atoms as part of the parent chain. Examples of such non- carbon atoms include, but are not limited to, N, O, S, Si, Al, B, and P. If there is more than one non-carbon atom in the hetero- based parent chain then this atom may be the same element or may be a combination of different elements, such as N and O.

[0038] The term "substituted" with respect to hydrocarbons, heterocycles, and the like, refers to structures wherein the parent chain contains one or more substituents.

[0039] The term "substituent" refers to an atom or group of atoms substituted in place of a hydrogen atom. For purposes of this disclosure, a substituent would include deuterium atoms.

[0040] The term "unsubstituted" with respect to hydrocarbons, heterocycles, and the like, refers to structures wherein the parent chain contains no substituents.

[0041] Rhodamine dyes have long been a hallmark of the biomedical research community due to their high brightness and excellent biocompatibility. First synthesized in the 19 century, the prototypical member of the rhodamine family is tetramethyl rhodamine (1, Figure 1A). TMR features two dimethyl nitrogen auxochromes and an oxygen atom at the 10' position which locks the two rings of the xanthene core in place. This rigidity contributes to the high fluorescence quantum yield (Φ ) of xanthene dyes. TMR absorbs and emits light in the yellow-orange region (~560 to 590 nm), which scatters in thick tissue and overlaps strongly with endogenous chromophores.

[0042] In the last quarter century, modern rhodamine dyes have reached the far-red and near infrared (NIR) window region (650–900 nm) by replacing the 10' bridging oxygen atom with other elements like C, Si, P, S, or B. As predicted by the Dewar-Knott color rule,Attorney docket No.00012-089WO1 substitutions at the 10' position primarily influence the lowest unoccupied molecular orbital (LUMO) energy for the fluorophore and largely explain the bathochromic shifts associated with 10' substitution.

[0043] Generally, replacing the 10' oxygen of rhodamine with other atoms stabilizes the LUMO, since the 2p orbitals of the oxygen lone pair overlap with the carbon-centered π* orbitals. The overlap of the 2p oxygen orbital with the ^* orbitals destabilizes the LUMO, increases the energy of the gap between the highest occupied molecular orbital (HOMO) and LUMO, and shifts the wavelength toward blue. For carborhodamines, in which oxygen is replaced with dimethyl carbon, the decreased electron-donating character of the C-C σ bond relative to the 2p lone pairs of oxygen results in a ~50 nm bathochromic shift (2, Figure 1A). Silicon rhodamine (SiR) dyes are approximately 90 nm farther red compared to oxygen-containing rhodamine dyes, owing to less σ donation into the π* orbitals: a result of the poor overlap between the 3sp orbitals of silicon and 2sp orbitals of carbon (3, Figure 1A). This large shift pushes SiR the far-red / NIR window, with excitation just below 650 nm and emission at 660 nm.

[0044] SiR dyes can be chemically modified and appended to antibodies, small molecules, lipids, self-labeling enzyme ligands, and ion receptors for a variety of applications in light microscopy, super-resolution microscopy, and functional imaging. Especially powerful is the ability to pair with self-labeling enzymes for chemical-genetic labeling of fusion proteins at far red wavelengths that are complementary to existing green- and red- fluorescent proteins. Even more attractive for live-cell imaging is the potential for no-wash labeling. The standard carboxy SiR dye used for HaloTag labeling is mildly fluorogenic; binding to HaloTag shifts the close-open equilibrium to favor the open form.

[0045] SiRs can be rendered even more fluorogenic by tuning the close-open equilibrium (K ) through chemical modification. Introduction of fluorinated azetidine auxochrome renders SiR dyes fluorogenic. Alternatively, TMR-SiRs can be made fluorogenic by incorporation of arylsulfonamides at the 2 position to control the close-open equilibrium. Both strategies render SiR dyesAttorney docket No.00012-089WO1 fluorogenic, but either restricts the choice of auxochromes, blue- shifts the dye, or requires bulky arylsulfonamides.

[0046] The strategy to achieve both far-red excitation and emission along with high fluorogenicity is different. Rather than rely of formation of a Si-C bond, the C-C bond framework of carborhodamines was preserved and the introduction of strong electron withdrawing groups in the form of bis(trifluoromethyl) groups was used (4a. Fogire 1A). It was hypothesized that the strong electron withdrawing character of the CF groups stabilizes the LUMO inductively, resulting in a bathochromic shift. Further, it was postulated that the now-electron deficient xanthene core will favor the closed, spirocyclic form and enable fluorogenic labeling (8a, Figure 1D).

[0047] The disclosure provides a class of fluorophores, bis(trifluoromethyl)-carborhodamine dyes, which feature a bis-CF group at the 10' position. The disclosure describes the design, synthesis, and characterization of these new BF (bis-CF or BerkeleyFluors (BF)) dyes. The synthetic route is scalable and produces gram scale quantities of a precursor that enables rapid diversification of the BF dye class across a range of auxochromes. The parent BF dye, with tetramethyl auxochrome substitution, features excitation and emission in the far- to near-infrared window. When combined with HaloTag ligands, BF dyes display high brightness and fluorogenicity that is 10- to 20-fold greater than the corresponding SiR dyes. BF dyes can be paired with HaloTag fusion proteins to enable no-wash labeling and single molecule tracking in living cells.

[0048] In a particular embodiment, the disclosure provides for a xanthene-based fluorophore comprising a structure of Formula I or Formula II:Attorney docket No.00012-089WO1or a or thereof, wherein, X-X are independently selected from N or C, wherein when an X group is an N, then the R group is absent; and R-R and R-R are independently selected from H, D, optionally substituted functional group (FG), optionally substituted (C-C )alkyl, optionally substituted (C-C )heteroalkyl, optionally substituted (C-C )alkenyl, optionally substituted (C-C )heteroalkenyl, optionally substituted (C-C )alkynyl, optionally substituted (C- C )heteroalkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted mixed ring system, wherein one or more adjacent R groups can be linked together to form one or more substituted rings selected from the group comprising cycloalkyl, cycloalkenyl, heterocycle, aryl, and mixed ring system; R16R18Attorney docket No.00012-089WO1 or a pharmaceutically acceptable salt or solvate thereof, X is selected from O, S, NH or CH; X is selected from O, S, NH, or CH; X is a halo; R and R are selected from an optionally substituted (C-C)alkyl, or can be joined together to form a heterocycle comprising 3, 4, or 5 carbon atoms; R and R are selected from an optionally substituted (C-C)alkyl, or can be joined together to form a heterocycle comprising 3, 4, or 5 carbon ;R -R are each individually(C-C) alkyl, optionallysubstituted (C-C) alkynyl, alkoxy, hydroxyl, ketone, ester, aldehyde, amino, azide, cyano, halo, thiol; v is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; y is an integer selected from 0, 1, 2, 3, 4, 5, and 6. Z is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The xanthene-based fluorophore can have a structure of Formula I(a):or a pharmaceutically acceptable salt or solvate thereof, wherein, R-R are independently selected from H, D, optionally substituted FG, optionally substituted (C-C)alkyl, optionally substituted (C-C)heteroalkyl, optionally substituted (C- C)alkenyl, optionally substituted (C-C)heteroalkenyl, optionally substituted (C-C)alkynyl, optionally substituted (C- C)heteroalkynyl, optionally substituted (C-C)cycloalkyl,Attorney docket No.00012-089WO1 optionally substituted (C-C)cycloalkenyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted mixed ring system, wherein one or more adjacent R groups can be linked together to form one or more substituted rings selected from the group comprising cycloalkyl, cycloalkenyl, heterocycle, aryl, and mixed ring system. In another embodiment, the xanthene-based fluorophore has a structure selected from Formula IIa, IIb, IIc and IId: ,

[0049] It is envisaged herein that minor structural changes can be made to the exemplary fluorophore described in the Examples to provide additional functionality.

[0050] In various embodiments, the disclosure provides for fluorophores and probes incorporating said fluorophores for use in in vitro and in vivo applications. The fluorophores of the disclosure may be used to fluorescently tag biological molecules or structures of interest, or used as optical reporters (i.e., activatable molecular probes, fluorescent dyes) for specific biomarkers / analytes. A fluorophore of the disclosure is useful for cellular assays and in vivo imaging.Attorney docket No.00012-089WO1

[0051] The xanthene-based fluorophore disclosed herein can be used as a fluorescent tag to identify structures or analytes of interest (e.g., biomolecules). As a tag, the fluorescence remains “on”. Various reagents can be used to append a variety of substituents at various positions of a xanthene-based fluorophore of the disclosure using a standard Friedel-Crafts acylation mechanism in order to introduce handles that are commonly used for bioconjugation and / or bioorthogonal ligation reactions (e.g., Staudinger ligation, inverse Diels-Alder reactions, oxime and hydrazone formation, aminothiol-CBT condensations, alkyne-azide Click chemistry, and etc.).

[0052] In a particular embodiment, the xanthene-based fluorophore disclosed herein can be utilized as part of an activatable molecular probe or sensor. In a representative example, the fluorophore would be in an “off” fluorescence state until a specific event occurs (e.g., presence of a substrate, enzymatic cleavage, interaction with a biomolecule, and etc.), which transforms the fluorophore to an “on” fluorescence state. There are many benefits to activatable probes, especially in studies that do not allow for a washing step (e.g., in whole body animals), which is a common strategy to help facilitate the removal of any residual dye that could otherwise contribute to the observed fluorescence response (i.e., background signal) when the fluorophore is part of a non-activatable probe. There are several signal transduction mechanisms that can be used to turn the fluorescence from an “off” to “on” state which includes intramolecular charge transfer (ICT), photoinduced electron transfer (PeT), and several variations of Förster resonance energy transfer (FRET).

[0053] In a particular embodiment, a xanthene-based fluorophore of the disclosure can be tethered to a biomolecule using various conjugation chemistries known in the art. Examples of biomolecules that can be attached to the xanthene-based fluorophore disclosed herein, include but are not limited to, antigens, receptors, steroids, proteins, antibodies, scFVs, and peptides. Fluorophores that have been tethered to a biomolecule can be used in any number of applications, including imaging (in vitro, ex vivo or in vivo), and diagnostics.Attorney docket No.00012-089WO1

[0054] In a certain embodiment, the disclosure provides methods for using the compounds disclosed herein, comprising exciting the compound with incident light; measuring the emission of far red to near infrared light by the compound. In a further embodiment, light emitted by the compound at 650 to 800 nm is quantitated. In yet a further embodiment, light emitted by the compound at 650 nm to 685 nm is quantitated. In another embodiment, the compound can be excited by exposing the compound to incident light having a wavelength from 380 nm to 640 nm. In yet another embodiment, the incident light has a wavelength around 630 nm (e.g., 590 to 650 nm). In particular embodiments, the compounds disclosed herein can be used in methods for cell imaging, drug screening and voltage sensing. In yet a further embodiment, the compounds disclosed herein are useful for screening drug safety and / or efficacy.

[0055] The disclosure provides methods for screening test samples such as potential therapeutic drugs which affect biological cells. These methods involve measuring the cell comprising a xanthene-based molecule of the disclosure in the presence and absence (control measurement) of a test sample or agent. Control measurements are usually performed with a sample containing all components of the test sample except for the putative drug. Detection of a change in the biological activity or viability of a cell in the presence of the test agent relative to the control indicates that the test agent changes the biological activity of the cell.

[0056] Biological cells which can be screened include, but are not limited to, primary cultures of mammalian cells, cells dissociated from mammalian tissue, either immediately or after primary culture. Cell types include, but are not limited to, white blood cells (e.g. leukocytes), hepatocytes, pancreatic beta-cells, neurons, smooth muscle cells, intestinal epithelial cells, cardiac myocytes, glial cells, and the like. The disclosure also includes the use of recombinant cells that have been genetically engineered. The cells are typically mammalian cell.

[0057] The screening methods described herein can be made on cells growing in or deposited on solid surfaces. A common technique is to use a microtiter plate well wherein the fluorescenceAttorney docket No.00012-089WO1 measurements are made by commercially available fluorescent plate readers. The disclosure includes high throughput screening in both automated and semiautomated systems.

[0058] Kits are also a feature of this disclosure. Embodiments of the kits include at least one compound according to any one of general formulas described herein. In other embodiments, such kits are suitable for drug screening. In some embodiments, the kits also include at least one buffer solution in which the compound, when used in conjunction with cells, will allow for sensing changes in the cell. Alternatively, the buffer may be provided as a concentrated solution, which is subsequently diluted prior to use. In certain embodiments, the compound may be premeasured into one or more containers (e.g., test tubes or cuvettes), and the detection is subsequently performed by adding the buffer and test sample to the container.

[0059] The kits also may include one or more containers, such as a disposable test tube or cuvette, in which the detection can be performed. The kits may further include instructions for performing the detection.

[0060] The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used. EXAMPLES

[0061] Computational determination of energy gaps. To evaluate the hypothesis that bis-(trifluoromethyl) carborhodamines would have red-shifted spectral properties relative to the parent CarboTMR derivatives, the HOMO-LUMO gaps of a series of tetramethylrhodamine (TMR) derivatives with dimethyl carbon, dimethyl silicon, or bis-CF groups were compared (Figure 1B). To do this, density functional theory (DFT) calculations were performed at the B3LYP / def2TZVP level (Figure 1B). The HOMO-LUMO gap of 2.46 eV for bis-(trifluoromethyl) carborhodamines was substantially smaller than the gap for CarboTMR (2.60 eV) and marginally smaller than dimethyl silicon (2.48 eV), suggesting that BF rhodamines would possess absorption maxima in the NIR region.Attorney docket No.00012-089WO1

[0062] Retrosynthesis and initial synthesis attempts. To synthesize BF rhodamines, several strategies were considered (Scheme S1). The retrosynthesis targets dianiline 9 as a precursor to BF rhodamines (Figure 2A). The anilines of 9 derive from the reduction of nitro groups, the carbonyl through a benzylic oxidation of 10. It was hypothesized that one could install the nitro groups into the desired positions by a dinitration of 11 through synergistic direction of the para-directing methylene and meta-directing bis-(trifluoromethyl)carbon. The dihydroanthracene 11 was the target of this synthetic strategy. Its formation was envisioned through a carbocation cyclization of intermediate 12, which would arise from hexafluoroisopropanol (HFIP)-derivative 13. Although formation of carbocation 12 seemed improbable due to the strongly withdrawing trifluoromethyl groups, earlier reports suggested similar trifluoromethyl-substituted carbocations could be generated (Scheme S2(A)).

[0063] Scheme S1: Initial Retrosynthetic Design and Synthesis of IntermediatesAttorney docket No.00012-089WO1Attorney docket No.00012-089WO1

[0064] Scheme S2: α-CF Carbocation Precedent and Proposed Cyclization Mechanism A) Previous Examples of -CF3Carbocations Richard (1989): OMs I pseudo first order 3F3C CF3KH, Et F3C CF32O, 0 COH F33 37 steps and 31% overall yield and can be performed on the multi-gram scale (Figure 2B). First, benzoic acid 14 is activated by conversion to phenyl ester 15 in 92% yield. The addition of trifluoromethyl groups to 15 via TMSCF / CsF, followed by desilylation with TBAFAttorney docket No.00012-089WO1 provides 13 in 82% yield over two steps. Triflation of alcohol 13, followed by TFA-mediated cyclization, likely through a carbocation intermediate (12, Scheme S2(B)), gives dihydroanthracene 11 in 92% yield. Traditional conditions to generate the carbocation (concentrated HCl, sulfuric acid) led to recovered 13. Regioselective nitration of 11 with fuming nitric acid to affords the desired regioisomer 10 in 48% yield. The mass balance of material in the reaction is a statistical combination of nitration isomers; nitrating conditions with KNO and sulfuric acid led to lower selectivity for the desired regioisomer. Oxidation of the weakly acidic 10, followed by iron mediated reduction affords the key intermediate 9 in 92% yield. Permethylation of 9 with methyl iodide affords the tetramethyl ketone 16 in 94% yield. Finally, addition of organometallic reagent to 15 provides bis- (trifluoromethyl)carborhodamine derivatives 4a (R = Me) and 17a (R = -COH) in 90% and 69% yields respectively (Figure 2C).

[0066] The new bis-(trifluoromethyl) carborhodamines display far-red absorption and emission, with a λ of 650 nm and an emission maximum of 661 nm (Table 1, Figure 2E). This is approximately 40 nm bathochromic shift than the parent CarboTMR, and slightly redder than the comparable SiR (3, Table 1). These absorption maxima values strongly correlate with the initial computational data (Figure 1A).

[0067] Table 1. Scope of 2-tolyl bis-(trifluoromethyl) carborhodamine Synthesis Using Different Amine Sources Entry[a]Dye R1RN– Yield λabs,maxλem,maxε (M-1c-1 [b](nm) (nm) m ) Φ [d]Attorney docket No.00012-089WO1 8 4g 44% 621[d]635[d]145,000[d]0.94[d]d]5 d]e]. trifluoroacetic acid. Data was recorded in PBS buffer with 0.1% w / w SDS, pH = 7.4

[0068] An alternative strategy was also developed that increases the reactivity of the carbonyl towards nucleophilic addition, while also enabling rapid access to a number of desirable auxochromes (Scheme A). It was hypothesized that conversion of the anilines of 9 to bromides would decrease the urea-like character of the electrophilic carbonyl and enhance its reactivity towards organometallic species. Dianiline 9 can be converted to dibromide 18 via a Sandmeyer reaction with CuB and tert-butylnitrite in 76% yield. The dibromide 18 readily reacted with aryl rings in the presence of LaCl-2LiCl to generate trityl alcohol 19 in 81% yield. Trityl alcohol 19 provides a useful intermediate for diversification of the nitrogen substitution of rhodamine dyes via a Buchwald-Hartwig coupling (Scheme A).

[0069] Scheme A – Modular synthesis of bis-(CF3) rhodamines via C-N coupling penultimate trityl alcohol intermediate::Attorney docket No.00012-089WO1 19. 68% and 74% yield from 19 (Table 1, Entry 2,3). The yield for 4a via this route is lower than the first route (42% yield from 14, 3 steps; vs 85% yield from 14, 2 steps), but affords substantially more synthetic flexibility. Cyclic secondary amines like azetidine, difluoroazetidine, and 7-Azabicyclo[2.2.1]heptane were also tolerated, generating rhodamines 4c, 4d, and 4e in 50%, 65%, and 81% yields, respectively (Table 1, Entry 4–6). Cyclic amine auxochromes of this type have been used to improve the fluorescence quantum yield of fluorophores are therefore an attractive target.

[0071] Heterocyclic auxochrome dyes are also accessible by this method: morpholine (4f), piperazinium (4g), tosyl protected piperazine (4h), thiomorpholine (4i) and sulfone morpholine (4j) derived dyes could be generated in yields ranging from 21% to 63% (Table 1, Entry 7–11). Electron-withdrawing, heterocyclic auxochromes have been used to blue-shift the spectra of rhodamine dyes. Additionally, amine-functionalized piperazines can be used for subsequent bioconjugation and other synthetic elaboration.

[0072] One could also extend this methodology to generate di- substituted and unsubstituted auxochromes. The primary aniline 4k was isolated in 30% yield when using a benzophenone imine coupling partner (Table 1, Entry 12). Primary amines such as ethylamine (4l) and trifluoroethylamine (4m) could also be introduced (Table 1, Entry 13,14). Fluorine-substituted auxochromes have been used toAttorney docket No.00012-089WO1 decrease oxidative photobluing rates and are therefore of synthetic interest.

[0073] In general, the spectral properties of the bis- (trifluoromethyl) carborhodamines are similar to the reported properties of their analogous silicon rhodamine counterparts (Table 1, Figure 6, 7). Diversification of the auxochromes from common intermediate 19 enables tuning of absorption and emission profiles by over 70 nm. Of note, the tetramethyl variant 4a has similar max absorption and emission wavelengths to the analogous silicon rhodamine 3, while also having a slightly higher extinction coefficient and quantum yield (Table 1, Entry 1,2). BF dyes show a linear relationship between HOMO-LUMO (H-L) gap and experimental absorption and emission values, when using DFT to model the 4a–m series of BF dyes (Figure 5, 12).

[0074] BF dyes with heterocyclic auxochromes (4g, 4h, and 4i, Table 1, Entries 8,9,10) showed near-zero absorption intensity in aqueous buffer. This could be caused by nucleophilic attack of water at the C-9 xanthene position (Figure 8a), due to the largely electron withdrawing auxochromes making C-9 extremely electrophilic. Consistent with this hypothesis, the absorbance of piperazine 4g varies inversely with pH (Figure 8b), while tetraethyl 4b displays no absorption intensity dependence on pH (Figure 8c). To avoid C-9 attack and deactivation of absorbance, the spectral properties of 4g, 4h, and 4i in trifluoroethanol with 0.1% v / v trifluoroacetic acid were recorded (Figure 7). Switching to organic solvent resulted in disproportionately high values for quantum yield and molar extinction coefficient, and so the values of 4a in the same solvent were recorded as a point of comparison. Finally, although there were no challenges with the unsubstituted 4k and diethyl 4l in PBS buffer, some broadening with the trifluoroethyl BF dye 4m was observed, but not for closely related diethyl 4l or unsubstituted 4k. This aggregation be addressed by using PBS buffer with sodium dodecyl sulfate (SDS) as a surfactant additive. Together these data show that BF rhodamines can incorporate a variety of amine substitutions that shift the spectra over a range of ~70nm (592 nm, 4k – 661 nm, 4i).Attorney docket No.00012-089WO1

[0075] Pleased with the wide scope of amines that could be coupled onto 19, experiments were performed to explore dyes with additional substitution on the pendant ring (Scheme 6). Although singly substituted protected carboxylates could be added to 18 (Scheme S5), aryl-rings doubly substituted with protected carboxylic acids were not substrates (Scheme S4). Less bulky dimethyl aryl rings could be added, however. Using (2,5- dimethylphenyl)magnesium bromide as a nucleophile followed by benzylic oxidation mediated by Cr(VI) gives (20), Subsequent t-Bu protection afforded access to doubly functionalized intermediate 21. The bromines of 21 could be converted to amines via C-N Pd- mediated coupling, which provided TMR BF (8a) and azetidine BF (8b) dyes in 66-73% yield after t-Bu deprotection. Monocarboxylate BF dyes could also be accessed in a similar route (Scheme S5).

[0076] Scheme 6: Synthesis of doubly functionalized CF3 rhodamines and HaloTag ligandsdibromide Xanthone 17BF dyes from dibromide xanthone 17Attorney docket No.00012-089WO1

[0079] emission, nearly identical to the corresponding SiR (Table A, below). One difference is that the new BF dyes have lower extinction coefficients, indicating an equilibrium shifted towards the transparent lactone. Dyes with low K values (favoring the closed lactone) or high D (indicating a low propensity to open in high dielectric medium) have potential for fluorogenic binding of self-labeling enzymes like HaloTag. BF dyes have low K values (0.0022 to 0.0001) and D values >79 (Table A, Figure 9). Both values suggest BerkeleyFluors dyes would show high fluorogenicity. Carboxy-substituted BF Dyes (17a and 17b) show low absorbance at physiological pH and are stable to millimolar concentrations of glutathione indicating compatibility for live-cell imaging.

[0080] Experiments were performed to synthesize and examine the fluorogenicity of the HaloTag dyes BF and BF (Figure 3, Scheme S6, Figure 10). Silicon rhodamine SiTMR shows a 2.6x turn on in absorbance and a 4x turn on in fluorescence when upon binding purified HaloTag, in line with reported values of 7- and 5-fold, respectively. This low degree of turn on a result of the considerable background signal that SiTMR exhibits in PBS buffer. The tetramethyl BF dye (BF ) exhibited a 25x absorbance turn on and 24x fluorescence turn-on in the presence of HaloTag (Figure 3). This turn on is due to exceptionally low background signal in the absence of HaloTag. The corresponding azetidine (BF ) exhibits even greater fluorogenicity, with a 52x fold increase in absorbance and a 56x increase in fluorescence. Importantly, the overall fluorescence of HaloTag-bound BF remains high, at about 64% ofAttorney docket No.00012-089WO1 SiTMR bound to HaloTag (Figure 10g,h). Together, these data establish that BR rhodamines 10-20x improvement in fluorogenicity compared to SiTMR while retaining >60% of the fluorescence intensity of SiTMR .

[0081] Scheme S6: Synthesis of HaloTag Ligand (HTL) BF Dyes

[0082] withhigh fluorogenicity and high brightness make BF dyes attractive candidates for live cell imaging. Both BF dyes effectively label cells expressing HaloTag. HEK293T cells expressing nuclear- localized HaloTag and stained with either TMR or azetidine BF show bright fluorescence. The BF dye fluorescence is localized with the nucleus, as evidenced by co-localization with nucleus-specific Hoechst 33342 staining. Cells that do not express HaloTag show negligible fluorescence (Figure 4b). Together, this establishes that BF dyes are cell permeable and effectively label HaloTag in living cells.

[0083] It was hypothesized that BF dyes would be good candidates for fluorogenic HaloTag labeling in living cells, based on their low K values, high D50 values, and large in vitro fluorogenicity. All of the SiR (SiTMR and azetidine-functionalized JF ) and BF rhodamine (TMR-derived BF and azetidine-derived BF ) dyes show fluorogenic labeling in cells, as evidenced by the scant difference in cell brightness under wash or no-wash imaging conditions.

[0084] The fluorescence intensity of BF dyes in HaloTag- expressing cells is 37 to 85% as bright as the cognate SiR dyes,Attorney docket No.00012-089WO1 consistent with in vitro HaloTag binding results. However, the fluorogenicity of BF dyes is exceptionally high. fluorogenicity is defined as the ratio of the brightness of dye in HaloTag expressing cells vs. the brightness of the same dye in non- expressing cells. The fluorogenicity of BF dyes in living cells is >1200× (BF ) and >10,000× (BF ), whereas SiTMR and JF show a cellular fluorogenicity of 60× and 466×, respectively. Together, BF dyes show a >30× to >20× increase in fluorogenicity.

[0085] Localization of the CF3 dye signal can be controlled by expression of HaloTag fusions. Cells expressing different HaloTag fusion proteins enables labeling of sub-cellular structures like nucleus, mitochondria, endoplasmic reticulum, lysosome, and plasma membrane (Figure 4e-h).

[0086] This high fluorogenicity stems from the near-zero background cellular fluorescence of BF dyes in cells that lack HaloTag. The fluorescence from cells that do not express HaloTag is indistinguishable from background, even up to concentrations as high as 2500 nM. The increase in non-specific background fluorescence in SiR dyes is greater than the increase for BF . SiR dyes show non-specific fluorescence associated with internal membranes in cells that do not express HaloTag. The bleach rates of BF dyes are lower than their cognate SiR dyes. Together, these experiments show that BF dyes effectively label HaloTag in living cells, display photostability that exceeds SiR, and possess substantial fluorogenic character in both in vitro and cellular imaging contexts.

[0087] Despite the somewhat lower cellular fluorescence intensity of BF dyes compared to SiR dyes, BF dyes still perform well under demanding, photon-starved microscopy conditions, such as voltage imaging and single molecule tracking in living cells. BF dyes can pair with chemigenetic sensing approaches in which HaloTag is fused to a functional motif, rather than a targeting motif. This allows synthetic fluorophores to be used with genetically encoded indicators for a variety of biologically relevant phenomena. Co- expression of Voltron, a fusion of a voltage-sensitive rhodopsin Ace2N, and HaloTag demonstrate that changes in the absorptionAttorney docket No.00012-089WO1 spectrum of Ace2N change the fluorescence of a HaloTag ligand, enabling optical detection of membrane potential changes.

[0088] HEK293T cells expressing Voltron and labeled with BF show bright plasma membrane-associated fluorescence (Figure 11a). Fluorescence from BF -Voltron (BFV ) is voltage- sensitive. Dual whole-cell patch clamp electrophysiology and fluorescence imaging reveals a voltage sensitivity of approximately -49% ΔF / F per 100 mV (±0.5%, standard error of the mean, peak response) (Figure 11b). As previously reported, some electrochromic FRET indicators like Voltron show voltage-dependent hysteresis. It was observed that the same phenomenon, and the voltage sensitivity of the steady state voltage step is slightly lower, at -3.3% ΔF / F (±0.4%) (Figure 11c). This compares favorably to Voltron , which has a voltage sensitivity of between -3 and -5% for steady-state and peak responses. Together, these data establish that BF dyes perform admirably under phot-starved conditions of voltage imaging.

[0089] In a related fashion, single molecule localization microscopy and tracking requires bright and photostable fluorophores to precisely localize single particles in live cells. BF rhodamines enable single particle tracking of HaloTag fusions in living cells when paired with highly inclined laminated optical sheet (HILO) microscopy.

[0090] BF dyes can be localized to the nuclei or via HaloTag fusions to a variety of targets, including histones (H2B), transcription factors (Sox2), or RNA-binding proteins (HEXIM1). At the single-molecule level, the distribution of fluorescence intensity of individual localizations for H2B labeled with BF or BF are similar, although BF is about 3% higher (mean, 10,200; median, 9,500) than the TMR-derived BF646-HTL (mean, 9,800; median, 9200). This tracks well with in vitro brightness determination, in which the brightness of BF bound to HaloTag is about 10% larger than the brightness of BF . Single molecule localizations of BF dyes in U2OS cells can be used to track the single molecule trajectories of these molecules, allowing dissection of transcriptional biochemistry in living cells.

[0091] Using established single molecule tracking microscopy and analysis methods, BF reveals differences in the mobilitiesAttorney docket No.00012-089WO1 of histone H2B and the RNA-binding protein HEXIM1. The majority of H2B localizations show low diffusion coefficients (D = 0.01 μm2 / s), consistent with chromatin-associated histone. On the other hand, HEXIM1 shows only a small population of slow diffusers (D = 0.01 μm2 / s) and two faster diffusing sub-populations at 2.7 and 7.4 μm2 / s, consistent with a slow-diffusing population of HEXIM1 molecules bound to the P-TEFb:7SK ribonucleoprotein complex and a faster-diffusing population unbound to P-TEFb:7SK.

[0092] The disclosure provides the design, synthesis, characterization, and application of a class of xanthene-based fluorophore: bis-(trifluoromethyl)carborhodamines, or BF dyes. BF dyes can be accessed in 10 steps proceeding through a carbocation cyclization. The synthetic design features a trityl alcohol intermediate (19) for late-stage diversification to BF dyes with a wide array of auxochromes. This unique synthetic approach offers chemical modularity for spectral fine-tuning across a nearly 70 nm wavelength range within the NIR window. BF dyes can be converted into fluorescent HaloTag ligands. BF HaloTag ligands show >10-20× improvement in fluorogenicity upon HaloTag binding in vitro and >10-30× in live cell imaging compared to their SiR counterparts.

[0093] BF rhodamines perform well, even under demanding, photon-starved microscopy conditions and can be integrated into existing state-of-the-art imaging modalities, like voltage imaging or single particle tracking with HiLo microscopy. Both BF646 and BF648 HaloTag ligands enable single particle tracking of HaloTag fusion proteins in living cells and reveal differences in the mobilities of essential nuclear components histone H2B and the RNA- binding protein HEXIM1. Together these data show that BF rhodamines provide a powerful complement to existing approaches to achieve far-red / NIR imaging.

[0094] Chemical Synthesis and Characterization. Chemical reagents and anhydrous solvents in Sureseal bottles were purchased from commercial suppliers and used without further purification. The exceptions were toluene and diethyl ether, which were stored over freshly activated 4Å MS for three days before their use. Thin layer chromatography (TLC) (silica gel, F254, 250 mm) and preparative thin layer chromatography (PTLC) (Silicycle, F254, 1000Attorney docket No.00012-089WO1 mm) were performed on precoated TLC glass plates and were visualized by fluorescence quenching under UV light. Flash column chromatography was performed on Silicycle Silica Flash F60 (230–400 Mesh) for normal phase or 60 RP-18 (200-400 mesh) for reverse phase, using a forced flow of air at 0.5–1.0 bar. NMR spectra were recorded on BrukerAV-300 MHz, BrukerAVB-400 MHz, Bruker AVQ-400 MHz, Bruker NEO-500 MHz, and Bruker AV-600 NMR spectrometers. Chemical shifts (δ) are expressed in parts per million (ppm) and are referenced to CDCl3 (7.26 ppm), DMSO (2.50 ppm), MeCN-d3 (1.94 ppm), or Acetone-d6 (2.05 ppm). Coupling constants are reported as Hertz (Hz). Splitting patterns are indicated as follows: s, singlet; d, doublet; t, triplet; q, quartet; qq, quartet of quartets; dd, doublet of doublet; p, pentet; heptet, heptet; m, multiplet; br, broad singlet. High resolution mass spectra (ESI EI) were measured by the QB3 / Chemistry mass spectrometry service at University of California, Berkeley. High performance liquid chromatography (HPLC) and low-resolution ESI Mass Spectrometry were performed on an Agilent Infinity 1200 analytical instrument coupled to an Advion CMS-L ESI mass spectrometer. The column used was Phenomenex Luna 5 μm C18(2) (4.6 mm I.D. × 150 mm) with a flow rate of 1.0 mL / min. The mobile phase was MilliQ-H2O with 0.05% trifluoroacetic acid (TFA) (eluent A) and HPLC grade MeCN with 0.05% TFA (eluent B). Signals were monitored at 210, 254, 370, 645 and 655 nm over 13 min, with a gradient of 10 to 100% eluent B for 10 min, then held at 100% B for 3 min. Preparative HPLC method was run on an Agilent Technologies 1260 Infinity system, using a Phenomenex Luna 5 μm C18(2) (10 mm I.D. x 150 mm) with a flow rate of 20.0 mL / min. Mobile phase used was MilliQ-H2O with 0.05% TFA (eluent A) and HPLC grade MeCN with 0.05% TFA (eluent B). Signals were monitored at 254, 625, and 650 nm over 30 min. Solvent was held at 30% elutent B from 0 to 1 min, ran at a linear gradient of 30% eluent B to 100% eluent B from 1 to 28 min, was held at 100% eluent B from 28 to 29 min, and then decreased back to 30% eluent B from 29 to 30 min. Loading solutions were ~10 to 20 mg / mL compound in 30% MeCN in MQ-H2O with 0.05% TFA.

[0095] Spectroscopic Studies. UV-Vis absorbance and fluorescence spectra were recorded using a 2501 SpectrophotometerAttorney docket No.00012-089WO1 (Shimadzu) and a Quantamaster 4 L-format scanning spectrofluorometer (Photon Technologies International). The fluorometer is equipped with an LPS-220B 75-W xenon lamp and power supply, A-1010B lamp housing with integrated igniter, switchable 814 photon-counting / analog photomultiplier detection unit, and MD5020 motor driver. Fluorescence lifetime measurements were recorded on a Horiba Fluorolog-QM spectrometer equipped with either a DeltaDiode-510LB Laser Diode 510 nm 25MHz laser or a DeltaDiode- 635L 635 nm pulsed laser as a light source, a photomultiplier tube (920 PMT) detector, and related power suppliers and controllers. Samples were measured in 1-cm path length quartz cuvettes (Starna Cells). The maximum absorption wavelength (λmax), maximum emission wavelength (λem), extinction coefficients (ε), and quantum yields (Φ) were taken in either 1X dPBS pH 7.2, 1X dPBS with 0.1% Sodium Dodecyl Sulfate (SDS), or trifluoroethanol (TFE) with 0.1% trifluoroacetic acid (TFA).

[0096] Fluorescence Lifetime Measurements. Fluorescence lifetime data was collected in triplicate and are reported as the amplitude-weighted averages in Table 1. The raw data was then globally fitted using the FelixFL software (version 1.0.48.0). The data was fitted with one-, two- and three-component exponential decay curves, with the data reported being the balance between fewest components used for the fit and the lowest χ2 value. For the compounds, this resulted in primarily reporting component fitsas the decrease in χ2 was minimal with the three-component fit. A minority of samples saw sizeable decreases in χ2 when analyzed with a three-component fit, leading us to report fit. The averagelifetime is an amplitude-weighted average.

[0097] HaloTag binding. These studies were performed in a sub- micro 50 μL quartz fluorometer cell. In a separate glass vial a solution was prepared using PBS buffer and the dye stock in DMSO (maximum 0.5% DMSO concentration in solution). When measuring the background absorbance, nothing else was added to the solution. When measuring the absorbance when bound to HaloTag, we added the purified HaloTag protein (2 equivalents relative to the dye) to the solution and incubated for a minimum of 15 minutes before measuring the absorbance. The final volume of the total solution was alwaysAttorney docket No.00012-089WO1 100 μL, with or without the HaloTag protein. When transferring the solution to the cuvette, we used 70 μL of the solution; less than 65 μL could result in a meniscus in our cuvette that would alter the baseline absorbance. The absorbance values of the bound and unbound dye were recorded in triplicate and averaged in order to record the absorbance and fluorescence turn-on. The quantum yields (Φ) of the HaloTag complexes were measured via the same method as the other dyes.

[0098] D50 Determination. To measure the D value, absorbance values of the diacid dye SiTMR-dicarboxy (see Table A for structure), BF (22a), and BF (22b) in dioxane / milliQ water mixtures (100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100 dioxane / milliQ water) were recorded. These were measured in a cuvette on a spectrometer. The peak absorbance values for each dye in every dioxane / water mixture were averaged before normalizing to the absorbance of the fully open dyes 3, 4a, and 4c in PBS buffer. Si and BF rhodamines have different extinction coefficients which would preclude normalizing the diacid absorbance to SiTMR-dicarboxy. Instead, the absorbance was normalized to the fully open 2-Me pendant ring dyes 3, 4a, and 4c in PBS buffer. Once the normalized absorption data was obtained, this data was plotted as a function of the dielectric constant of the different dioxane / milliQ water mixtures. From this data one could calculate the D values as the dielectric constant at which the normalized absorbance was 50% of the corresponding 2-Me pendant ring dyes.

[0099] Table A: Properties of mono-carboxy, di-carboxy, and HaloTag Ligand (HTL) BF dyes Dye[a]Abs Em ε (M-1cm-1) φ[b]KL-Z[c]D50[d]A / A0[e]F / F0[f](nm) (nm) 2Attorney docket No.00012-089WO1 BF646-HTL651 664 N.D. 0.35 - - 24.8 ± 0.7 24.1 ± 2.1 BF648-HTL653 665 N.D. 0.55 - - 52.3 ± 1.1 55.8 ± 3.2HaloTag Binding for method) than when unbound in 1X dPBS buffer, pH = 7.2.eX-fold turn-on for fluorescence when bound to HaloTag protein (see HaloTag Binding for method) than when unbound in 1X dPBS buffer, pH = 7.2.gdata from Ref3,13.hData was recorded in trifluoroethanol with 0.1% (v / v) of trifluoroacetic acid.iData was recorded in PBS buffer with 0.1% w / w SDS, pH = 7.4.

[0100] Kextinction coefficient of the dye wasfound that 8a and 8b displayed in trifluoroethanol with 0.1% v / v trifluoroacetic acid. This is in contrast to previous studies of silicon rhodamines, which used ethanol with 0.1% v / v trifluoroacetic acid. The extinction coefficient of the mono-carboxy dyes in a 50:50 mixture of dioxane / milliQ water was then obtained. For the azetidine mono- carboxy 8b, no measurable absorbance in 50:50 dioxane / milliQ water was observed, so the estimate of the K was <0.0001. When absorbance in 50:50 dioxane / water was observable, the following equation was used to calculate K : K = [Z] / (1-[Z])*; *The value for [Z] was determined through the following calculation: [Z] = ε(50:50 dioxane / milliQ water) / ε(TFE w / 0.1% TFA)

[0101] Computations. Models were created in GaussView 6 (Table A). All DFT optimizations were done with Gaussian 16, using B3LYP / def2TZVP, IEF-PCM Ethanol (ε = 24.852). Visualizations of MOs (Figure 5) were generated using VMD.

[0102] Cell Culture. Human embryonic kidney 293T (HEK293T) cells were passaged and plated onto 12 mm #1.5 glass coverslips pre-coated with Poly-D-Lysine (PDL; 0.1 mg / ml; in 10 mM NaBO;Attorney docket No.00012-089WO1 Sigma-Aldrich) to provide a density of ~44,000 cells / cm. HEK293T cells were plated and maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 4.5 g / L D-glucose (high glucose), 10% FBS and 1% Glutamax. Unless otherwise stated, all cell culture for imaging experiments were done in high glucose DMEM. For loading cells, dyes were diluted in DMSO to 1000x the final indicated concentration and then diluted 1:1000 in HBSS. All imaging experiments were performed in HBSS.

[0103] Transient Transfections. Transfections were done using Lipofectamine 3000 (Invitrogen) 24 h after initial plating. To each well containing a 12 mm coverslip in a 24-well plate, 500 ng DNA / lipofectamine solutions per coverslips were added. The cells were then left untouched for another 24 hours (~48 hours after initial plating) to result in a final confluency of ~175,000 cells / cm (~75%) for epifluorescence imaging. Non-transfected cells were plated at the same time as transfected cells, but the addition of DNA / lipofectamine solutions was omitted.

[0104] Transfections for electrophysiology experiments began with plating cells at a density of ~61,000 cells / cm in a 6-well plate. After 24 hours, cells were transfected with the same lipofectamine reagents at a concentration of 1000 ng DNA / lipofectamine solution per well of the 6-well plate. The cells were then allowed to grow for another 24 hours before being plated onto 25 mm #1.5 glass coverslips precoated with PDL at a density of 26,000 cells / cm in DMEM supplemented with D-glucose (low glucose), 10% FBS and 1% Glutamax to achieve single cell confluency. These cells were then allowed to grow for a final 24 hours before imaging and patch clamp electrophysiology experiments.

[0105] Fluorescence microscopy. Epifluorescence imaging was performed on an AxioExaminer Z-1 (Zeiss) equipped with a Spectra-X Light engine LED light (Lumencor), controlled with Slidebook (v6, Intelligent Imaging Innovations). Images were acquired with a W- Plan-Apo 63x / 1.0 water objective (63x; Zeiss). Images were focused onto an OrcaFlash4.0 sCMOS camera (sCMOS; Hamamatsu). Confocal imaging was performed with a Nikon Ti Inverted Yokogawa spinning disk microscope with 405 nm, 488 nm, 561 nm, and 637 nm laser lines. Live-cell imaging was done with incubation and CO. ImagesAttorney docket No.00012-089WO1 were acquired with a Plan Apo VC 60x oil objective and DAPI, FITC, and Cy5 filter sets and controlled via Nikon imaging elements

[0106] HaloTag localization. HEK293T cells were transfected with HaloTag fusions expressed in the mitochondria, nucleus, and endoplasmic reticulum (ER). These HaloTag expressing cells were then co-stained with 200 nM BF and a respective organelle marker for 20 min at 37°C 5% CO. Mitochondrial localization was assessed with HaloTag fusion in mitochondria (Su9) co-stained with HTL and 100 nM MitoTracker Green (Invitrogen, cat. M714). Nuclear localization was assessed with nuclear HaloTag fusion (3xNLS) co- stained with HTL and 1 μg / mL Hoechst 33342 (Invitrogen, cat. H1388). Finally, ER localization was assessed with ER HaloTag fusion (Sec61β) co-stained with HTL and 100 nM ER Tracker Green (Invitrogen, cat. E34251). BF was excited at 637 nm and emission was collected with a Cy5 filter set. MitoTracker Green and ER Tracker Green were excited at 488 nm and emission was collected with a FITC filter set. Hoechst 33342 was excited at 405 nm and emission was collected with a DAPI filter set. Finally, a ND image files were then directly analyzed in ImageJ.

[0107] Wash Protocol. Transfected (T) and non-transfected (NT) HEK29T cells were co-stained with 1 mg / mL Hoechst 33342 and 200 nM HaloTag ligand (BF , BF , SiTMR , or JF ) in HBSS for 20 min at 37°C, 1 volume ofHBSS before being transferred to a 3 cm imaging dish containing 3 mL HBSS. Images were acquired with a W-Plan-Apo 63x / 1.0 water objective (Zeiss) and OrcaFlash4.0 sCMOS camera (Hamamatsu). For HaloTag ligand imaging, the excitation light was delivered from an LED with a power of 0.09 W / cm (ND: 75) for 2 ms exposures at 631 / 28 nm (bandpass) and emission was collect through a quadruple emission filter (430 / 32, 508 / 14, 586 / 30, 708 / 98 nm) after passing through a quadruple dichroic mirror (432 / 38, 509 / 22, 586 / 40, 654 nm LP). For Hoechst 33342 imaging, the excitation light was delivered from an LED with a power of 0.018 W / cm (ND: 75) for 100 ms exposures at 390 / 22 nm (bandpass) and emission was collected with the same quadruple emission filter. Finally, a DIC image was also taken for each field of view (FOV). Images were then exported as tiff files to be analyzed in ImageJ.Attorney docket No.00012-089WO1

[0108] No wash protocol. Transfected (T) and non-transfected (NT) HEK29T cells were co-stained with 1 mg / mL Hoechst 33342 and 200 nM HaloTag ligand (BF , BF , SiTMR , or JF ) directly in a 3 cm imaging dish for 20 min at 37°C, 5% CO. After 20 min, coverslips were imaged directly in the staining solution. HaloTag ligand and Hoechst 33342 images were collected using the same capture settings described in the wash protocol above.

[0109] Saturation Assay. HEK293T cells excessing a nuclear HaloTag fusion (3xNLS) were co-stained with HaloTag ligand (BF , BF , SiTMR , or JF646). Cells were co-stained directly in the imaging dish (no wash) at increasing concentrations from 1 nM to 2500 nM and 1 μg / mL Hoechst 33342 and imaged with the previously mentioned capture settings.

[0110] Photostability Assay. HEK293T cells transfected with nuclear HaloTag (pTG735) were prepared with “no wash” staining protocol with coverslips in a 3 cm imaging dish. For low power photostability experiments, a continuous excitation was delivered with an LED power of 0.09 W / cm (ND: 75) at 631 / 28 nm (bandpass) for 5 minutes. For high power photostability experiments, a continuous excitation was delivered with an LED power of 0.25 W / cm2 (ND: 255) at 631 / 28 nm (bandpass) for 1 minute. Emission was collected for 2 ms exposures every 1000 ms with a quadruple emission filter (430 / 32, 508 / 14, 586 / 30, 708 / 98 nm) after passing through a quadruple dichroic mirror (432 / 38, 509 / 22, 586 / 40, 654 nm LP). Time courses were exported as tiff files for further analysis in ImageJ.

[0111] Image analysis. Exported tiff images were analyzed in ImageJ to extract raw fluorescence values which were then analyzed in Microsoft Excel. ROI selection and measurements were done via ImageJ Macros. For transfected cells, images with HaloTag ligand (HTL channel) were converted to a binary mask using a Gaussian blur followed by Huang thresholding. The same filter and thresholding were applied to Hoechst (nuclear channel) images to create a nuclear mask. The HTL mask and nuclear mask were then combined using the “AND” function in image calculator. ROIs were then created from the resulting mask and raw fluorescence intensity values were measured in HTL channels. A similar route was used forAttorney docket No.00012-089WO1 non-transfected nuclear ROI selection, however only Hoechst (nuclear channel) images were used to create binary masks. Nuclear ROIs were then created from these masks and raw fluorescence intensity values were measured in the corresponding, non- transfected HTL image. In both cases, non-cellular ROIs were manually selected using transmitted light images and measured in HTL images for background correction of nuclear ROI measurements.

[0112] For epifluorescence voltage sensitivity, regions of interest (ROIs) were drawn around patch-clamped cells and mean fluorescence values calculated using ImageJ (FIJI, NIH). Background fluorescence values were calculated from a 10x10 pixel ROI where no cells grew. Briefly, the cell ROI was applied to all acquired images and ROI fluorescence values were calculated by ImageJ at both holding potential and at voltage step epochs. ΔF / F values were calculated by subtracting the mean background intensity for each frame acquired to obtain a background-corrected fluorescence trace. The baseline fluorescence value (F0) was calculated from the mean of all holding potential frames and subtracted from ROI fluorescence measurements at each timepoint to yield a ΔF trace. This was then divided by the F0 trace to give ΔF / F traces. Signal- to-noise-ratio (SNR) was calculated as the average ΔF signal divided by the standard deviation of the F0 noise.

[0113] Electrophysiology with simultaneous imaging. Pipettes were pulled from 1.5mm OD / .86 mm ID borosilicate glass with filament (Sutter Instruments, Novato, CA) to resistances ranging from 2 to 5 MΩ with a P97 pipette puller (Sutter Instruments). Internal solution composition, in mM (pH 7.25, 285 mOsmol / L): 125 potassium gluconate, 10 KCl, 5 NaCl, 1 EGTA, 10 HEPES, 2 ATP sodium salt, 0.3 GTP sodium salt. EGTA (tetraacid form) was prepared as a stock solution in 1 M KOH before addition to the internal solution. Pipettes were positioned with an MP-225 micromanipulator (Sutter Instruments).

[0114] Electrophysiology recordings were made with an Axopatch 200B amplifier and digitized with a Digidata 1440A (Molecular Devices). For epifluorescence voltage sensitivity measurements, cells were held at -60 mV and potentials from +100 to -100 mV were applied in 20 mV steps in descending order using the pClampAttorney docket No.00012-089WO1 software (Molecular Devices). Signals were sampled at 50 kHz and filtered with a 5 kHz low-pass Bessel filter. Pipette capacitance correction was performed in the cell-attached configuration. Recordings were only included if they maintained a 30:1 ratio of membrane resistance (Rm) to access resistance (Ra).

[0115] To perform simultaneous epifluorescence imaging, BF was loaded at 250 nM onto HEK293T cells previously transiently transfected with Voltron, in HBSS (Gibco) for 20 minutes at 37°C in a humidified incubator with 5% CO. Coverslips were washed once with HBSS and transferred to fresh HBSS for imaging. All epifluorescence imaging was conducted under ambient atmosphere. Imaging was performed using an AxioObserver (Zeiss) equipped with a Spectra-X Light engine LED light (Lumencor) and an ORCA-Flash4.0 sCMOS camera (sCMOS; Hamamatsu) controlled with Micro-Manager, using a 40x oil / 1.3 NA Apochromat objective (Zeiss) immersed in Immersol 518F (Zeiss). The field of view was set to 100x100 pixels, with 4x4 binning applied. Excitation light was delivered from an LED (635 / 22 nm or 550 / 15 nm BP, 2ms exposure time) and filtered through a quad-band emission filter (409 / 493 / 573 / 652 nm, Semrock) after passing through a quad-band dichroic mirror (432 / 525 / 595 / 730 nm LP, Semrock). Images were collected at 1100 timepoints, and patch-clamp stimulation was synced to image acquisition using the pClamp software.

[0116] Single Molecule Microscopy. For single-molecule experiments, the same U2OS cell line was labeled for 15 min with 1 nM of each dye in phenol red-free DMEM, washed twice with 1x PBS, and destained for 20 min in medium without dye. Imaging was performed using HILO illumination using the previously described microscope for 3000 frames at a frame rate of 7.48 ms / frame with a single stroboscopic 1 msec red pulse in each frame. The EM gain multiplier was set to 300. Data was acquired using an automated pipeline. Prior to imaging, cells were non-stroboscopically photobleached for 3 sec to reduce the density of labeled emitters. Single molecule localization, intensity estimation, and tracking were performed using quot, and cells were subjected to quality control using cellpicker.Attorney docket No.00012-089WO1

[0117] A homozygous HEXIM1-Halo U2OS knock-in cell line (clone H-D3) was stained overnight with 50 nM CF3-azetidine Halo ligand. Cells were briefly washed twice with 1x phosphate-buffered saline and then destained in medium without dye for 1 hour. Molecules were tracked using quot and analyzed using SASPT. A total of 91885 trajectories from 232 cells were included in the analysis.

[0118] DNA Constructs / Molecular Biology. Target Plasmid Plasmid Sequence World wide web at: b hli - - m [00 Int. to use. A flame-dried flask was charged with 3,6-dibromo-10,10- bis(trifluoromethyl)anthracen-9(10H)-one (18, 1.0 equiv.) and vac cycled three times. Anhydrous THF (0.3 M to 18) was then added to the flask at room temperature and stirred for 5 min to dissolve the xanthone. LaCl·2LiCl (0.6 M in THF, 1.0 equiv.) was added and stirred at room temperature for 1 hr. The flask was then cooled to -40 °C and Grignard reagent (8.0 equiv.) was added dropwise to the reaction. The now blood red reaction was stirred at -40 °C for 5 min and then warmed to 0 °C, where it was stirred for 1 to 1.5 hr. Once TLC showed full consumption of 18, the reaction was quenched with sat. NHCl to yield a yellow crude. The aqueous layer was extracted three times with EtOAc. The combined organics were washed with brine, dried over NaSO, and concentrated in vacuo onto silica. The crude was purified via flash column chromatography (dry load on silica, 0 to 10% EtOAc / Hex, 2.5% increments) to yield theAttorney docket No.00012-089WO1 product as a viscous oil with significant amount of EtOAc. Dissolving the oil in pentane and sonicating before removing the pentane in vacuo, yielded the products as a white, foamy solid. These intermediates were stable at room temperature for extended periods of time (6-12 months).

[0120] General Procedure 2: Buchwald-Hartwig Amination for 2- Methyl BF Dyes. A 1-dram vial with a septa top was flame-dried and cooled under vacuum. The vial was then charged with starting material (19, 1.0 equiv.), palladacycle XPhos G4 (0.20 equiv.), XPhos (0.20 equiv.), CsCO (10 equiv.), and amine(8.0 equiv.; 3,3- Difluoroazetidine (for 4d), 2,2,2-trifluoroethanamine (for 4m), and 7-azabicyclo[2.2.1]heptane HCl (for 4e) were all purchased and utilized as their respective HCl salts) and vac cycled three times (Solid amines were added prior to vac cycle. Amines liquid at room temperature or solutions were added after the vac cycle). Anhydrous toluene (0.03 M to 19)(Toluene was dried over 3 Å molecular sieves for at least 3 days prior to use) was added and the cap was quickly swapped for a non-septa Teflon screw top. The reaction was brought up to 110 °C under pressure and refluxed overnight. The next day the reaction would appear metallic gray, indicating complete consumption of the starting material. The reaction was then quenched with 0.1 M HCl, yielding a bright blue solution, which was then extracted with CHCl three times. The combined organics were dried over NaSO and concentrated in vacuo. All compounds were then purified via preparative HPLC (following the general HPLC method above), pure fractions were concentrated in vacuo. All compounds were then drawn up in MeOH and passed through a short C18 silica plug to remove any grease from the compound, then concentrated in vacuo to yield products as waxy blue compounds. Purified products were stored either as a solid or a stock solution in DMSO at −20 °C.

[0121] General Procedure 3: Buchwald-Hartwig Amination for 2- Carboxy BF Dyes. A 1-dram vial with a septa top was flame-dried and cooled under vacuum. The vial was then charged with starting material (SXII, 1.0 equiv.), Palladacycle XPhos G4 (0.20 equiv.), XPhos (0.20 equiv.), CsCO (10 equiv.), and vac cycled three times. Anhydrous toluene (0.03 M to SXII) and amine (8.0 equiv.) wereAttorney docket No.00012-089WO1 added, and the cap was quickly swapped for a non-septa Teflon screw top. The reaction was brought up to 110 °C under pressure and refluxed overnight. The next day, the reaction would appear metallic gray, indicating complete consumption of the starting material. The crude was then diluted with CHCl and filtered through a celite plug to remove palladium. All compounds were then purified via preparative TLC (5% EtOAc / Toluene), where they were then scrapped from the plate, dissolved in EtOAc and concentrated in vacuo to yield products as white solids. Purified products were stored either as a solid or a stock solution in DMSO at -20 °C.

[0122] Detailed Synthetic Procedures.

[0123] Note: The bis-(trifluoromethyl)methylene group require special consideration when performing NMR experiments. The advantage of this functional group is the ability to quickly verify the purity when examining the NMR spectra. The intermediates prior to 19 and SI-3 all contain a single peak in the F NMR. For compounds 19, SI-3 and molecules further downstream, there exists two signals in the F NMR due to the diastereotopic trifluoromethyl carbons. There may also be a third signal due to a trifluoroacetate counteranion, depending on what conditions were used to isolate the dye. Since the F NMR is extremely quick to acquire and very sensitive, it is very useful for accessing purity of the sample. The downsides of this functional group are that the coupling of F and C nuclei results in carbon signals containing multiplicity, significantly decreasing their intensity in the NMR spectra. Even with a highly sensitive NMR instrument containing a cryoprobe, it was challenging to identify the peaks corresponding to the trifluoromethyl carbons and the methylene carbon bearing the trifluoromethyl groups. In some instances it was necessary to perform F decoupled C NMR experiments in order to identify these signals.

[0124] Phenyl 2-benzylbenzoate (15)Attorney docket No.00012-089WO1

[0125] A flame dried 1L α-Phenyl-o- toluic acid (14, 4.98 g,, DCC (5.05 g, 24.5 mmol, 1.05 equiv.), and DMAP (0.861 g, 7.05 mmol, 0.3 equiv.). The flask was evacuated and backfilled with nitrogen. Anhydrous CHCl (200 mL) was added to the flask and the reaction was allowed to stir at room temperature for 1 hr. While that stirred, phenol (2.41 g, 25.6 mmol, 1.1 equiv.) was added to a separate flame dried flask and dissolved in anhydrous CHCl (15 mL). After 1 hr had elapsed, the phenol solution was added to the reaction mixture slowly at room temperature. The flask containing the phenol solution was washed with additional anhydrous CHCl (15 mL, 230mL total, 0.1 M of toluic acid) and added to the reaction. The cloudy, white reaction mixture was stirred at room temperature overnight. The next day, the crude mixture was filtered through a glass frit to remove excess DCU. The filtrate was concentrated in vacuo, brought up in ACN (~100 mL), stored at -20 °C for 30 min, then filtered again to remove DCU, then concentrated in vacuo to produce a crude yellow oil. The crude was purified via flash column chromatography (dry load on celite, 0 to 10% EtOAc / Hex, 2% increments) to yield 15 as a clear oil (5.68 g, 19.7 mmol, 84% yield).

[0126] H NMR (600 MHz, Acetone) δ 8.13 (dd, J = 7.8, 1.5 Hz, 1H), 7.59 (td, J = 7.6, 1.5 Hz, 1H), 7.47 – 7.39 (m, 4H), 7.30 – 7.24 (m, 3H), 7.22 – 7.16 (m, 3H), 7.15 – 7.12 (m, 2H), 4.46 (s, 2H)

[0127] C NMR (151 MHz, Acetone) δ 166.5, 151.9, 143.6, 141.9, 133.5, 132.7, 131.8, 130.3, 130.2, 129.7, 129.1, 127.4, 126.8, 126.6, 122.7, 39.9.

[0128] HR-EI-MS m / z for C H O [M] : 288.1151. Calculated: 288.1150.

[0129] (SI-2 ((2-(2-benzylphenyl)-1,1,1,3,3,3-hexafluoropropan- 2-yl)oxy)trimethylsilaneAttorney docket No.00012-089WO1

[0130] A flame dried 250 charged with 15 (5.68 g, 19.7 mmol, 1.0 equiv.) and0.197 mmol, 0.01 equiv.). Anhydrous dioxane (66 mL, 0.3 M to 15) the flask (the dioxane should be either freshly distilled or from a new bottle to achieve high yield). TMSCF (9.07 mL, 59.1 mmol, 3.0 equiv.) was added dropwise and the reaction was allowed to stir at room temperature. After 4 hours, TLC showed 1 still left in the reaction, so additional CsF (~0.029 g, 0.197 mmol, 0.01 equiv., 0.02 equiv. total) and TMSCF (9.07 mL, 59.1 mmol, 3.0 equiv., 6.0 equiv. total) was added and the reaction was left to stir overnight. The next day, TLC showed full consumption of 15. The crude was concentrated in vacuo onto silica and was purified via flash column chromatography (dry load on silica, 0 to 1% EtOAc / Hex, 1% increments) to yield SI-2 as a yellow oil (7.39 g, 18.2 mmol, 92% yield). The product is very non-polar, so it is best to load SI-2 onto the column using a dry load technique. It is also challenging to accurately weigh out the CsF onto weigh paper without it absorbing water. As such, it is best to measure several spatula tips worth of CsF to get a rough estimate of what the desired amount looked like, and then added a similar amount to the flask.

[0131] H NMR (600 MHz, Acetone) δ 7.66 (d, J = 7.9 Hz, 1H), 7.42 – 7.33 (m, 2H), 7.32 – 7.27 (m, 2H), 7.21 (t, J = 7.4 Hz, 1H), 7.13 (dd, J = 7.6, 1.8 Hz, 1H), 7.09 (d, J = 7.2 Hz, 2H), 0.17 (s, 9H).

[0132] C NMR (151 MHz, Acetone) δ 142.9, 142.8, 134.8, 130.7, 130.5, 130.2, 129.2, 128.4, 127.0, 126.8, 124.2 (q, J = 291.4 Hz), 83.7 (hept, J = 29.4 Hz), 1.4.

[0133] F NMR (565 MHz, Acetone) δ -71.5.

[0134] HR-EI-MS m / z for C H OSiF [M] : 406.1182. Calculated: 406.1188.Attorney docket No.00012-089WO1

[0135] 2-(2-benzylphenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (13)

[0136] In a 250 mL flask, g, 17.9 mmol, 1.0 equiv.) was dissolved in anhydrous THFM to SXII), then the reaction was cooled to 0 °C. TBAF (1 M in THF, 35.8 mL, 35.8 mmol, 2.0 equiv.) was added to the reaction, and it was allowed to stir at 0 °C for 10 min. The ice bath was then removed, and the reaction was stirred at room temperature for 2 hours. When TLC showed full consumption of 2, the crude was concentrated in vacuo and was purified via flash column chromatography (0 to 7.5% EtOAc / Hex, 2.5% increments) to yield 13 as a yellow oil (5.35 g, 16.0 mmol, 89% yield).

[0137] H NMR (600 MHz, CDCl) δ 7.66 (d, J = 8.2 Hz, 1H), 7.42 (td, J = 7.5, 1.3 Hz, 1H), 7.34 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.31 – 7.26 (m, 3H), 7.21 (t, J = 7.4 Hz, 1H), 7.09 (d, J = 7.1 Hz,

[0138] C NMR (151 MHz, CDCl) δ 142.0, 140.9, 134.9, 130.3, 128.8, 128.5, 128.3, 128.3, 128.2, 128.2, 128.2, 127.7, 126.7, 126.4, 125.9, 124.0, 120.2, 81.1, 80.9, 80.7, 80.5, 80.33, 40.82.

[0139] F NMR (565 MHz, CDCl) δ -74.5.

[0140] HR-ESI-MS m / z for C H OF [M-H] : 333.0720. Calculated: 333.0720.

[0141] 9,9-bis(trifluoromethyl)-9,10-dihydroanthracene (11)

[0142] A flame dried 500charged with KH (30 wt% in mineral oil, 1.01 g, 25.3 mmol, 1.6 equiv.), then evacuated and backfilled with nitrogen three times. The KH dispersion was stirred with hexanes (15 mL) for 5 min at room temperature, allowed to settle for 5 min, and then the hexanes was syringed off andAttorney docket No.00012-089WO1 quenched in cold 9:1 hexanes:iPrOH. The KH dispersion was washed with hexanes (15 mL) an additional two times via this method. Anhydrous EtO (90 mL) was added to the washed KH and the reaction was cooled to 0 °C. 13 (5.28, 15.8 mmol, 1.0 equiv.) was dissolved in EtO (15 mL, 105 mL total, 0.15 M to 13) in a separate flame dried flask. The solution of 13 was added dropwise to the reaction flask, and the reaction was stirred for 1 hr at 0 °C. Then, TfO (2.93 mL, 17.4 mmol, 1.1 equiv.) was added dropwise to the yellow reaction mixture at 0 °C. After stirring for 10 min at 0 °C, the ice bath was removed, and the reaction mixture was gradually brought up to room temperature and stirred overnight. The next day, the reaction was slowly quenched with HO (6 mL). The ether solution was washed with sat. NaHCO three times. The aqueous wash layers were extracted with EtO, and the organics were combined. The combined organics were washed with brine, dried with NaSO, and concentrated in vacuo into a 250 mL flask to yield a brown oil that was used without further purification. TFA (160 mL, 0.1 M to 13) was added to the reaction flask at room temperature and the reaction was stirred for 4 hr. A white solid should be seen crashing out after ~5 min of stirring. After 4 hr, the TFA was removed on a rotary evaporator, the solid crude was brought up in EtO. The organic layer was washed with sat. NaHCO twice and brine once, dried with NaSO, and concentrated in vacuo. The crude oil was purified via flash column chromatography (0 to 2% EtOAc / Hex, 1% increments) to yield 11 as a fluffy, white solid (4.55 g, 14.4 mmol, 91% yield).

[0143] It is important to remove the mineral oil from the KH in order to facilitate purification. The product is extremely non- polar and will coelute with the mineral oil. Due to the extreme non-polar nature of 11, it is best to load the column using a dry load technique.

[0144] It was found this procedure using KH to be more robust rigorously reproducible than those with KOMe. Attempts to purify the intermediate triflate by column chromatography instead led to the isolation of trace amounts of 11.

[0145] H NMR (600 MHz, CDCl) δ 8.04 (d, J = 8.4 Hz, 2H), 7.48 – 7.42 (m,7.40 – 7.33 (m, 4H), 4.27 (s, 2H).Attorney docket No.00012-089WO1

[0146] C NMR (151 MHz, CDCl) δ 136.3, 130.3 (heptet, J = 4.3 Hz), 129.6, 129.0, 126.8, 125.3, 124.6 (q, J = 288.1 Hz), 56.7 (heptet, J = 26.2 Hz), 34.7.

[0147] F decoupled C NMR (151 MHz, CDCl) δ 124.6, 56.7.

[0148] F NMR (565 MHz, CDCl) δ -63.9.

[0149] HR-EI-MS m / z for C H F [M]: 316.0685. Calculated: 316.0687.

[0150] 2,7-dinitro-9,9-bis(trifluoromethyl)-9,10- dihydroanthracene (10)

[0151] A 500 mL HNO (167 mL, 0.1M to 11) and cooled to 0 (5.30 g, 16.8 mmol, 1.0 equiv.) was added in small scoopfuls over the course of 5 min. The reaction was stirred at 0 °C for 1 hr. After, the reaction was quenched into a flask with ice (~200 g). The slurry was filtered after the ice had just barely completely melted, and the filtrate was washed an additional 4x with ice cold water (500 ml total). The crude material was dissolved in CHCl and washed with brine. The combined organics were dried with NaSO, and the solvent was removed. The crude material was purified by column chromatography (0 to 50% EtOAc / Hex, 10% increments). Two undesired nitration isomers (with nitration occurring ortho to the methylene carbon in varying amounts) coelute first at approximately 10 to 20% EtOAc, and desired isomer comes off at 30 to 50% EtOAc. The desired 2,7- dinitro isomer 10 was isolated as a white solid (3.27 g, 8.05 mmol, 48% yield). The use of fuming HNO was useful in this reaction. It was discovered that using more traditional nitration conditions (HNO / HSO) the selectivity of the reaction flipped and the desired 2,7-dinitro isomer was the minor product. Over time any column fractions containing 10 will gradually turn blue. This was attributed to the faint color shift to a tautomer of 10 as the methylene C-H bonds are likely weakly acidic. Once the solvent is removed it will revert to a white solid.

[0152] H NMR (600 MHz, CDCl) δ 9.01 (s, 2H), 8.37 (dd, J = 8.5, 2.2 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H), 4.49 (s, 2H).Attorney docket No.00012-089WO1

[0153] C NMR (151 MHz, CDCl) δ 147.4, 142.0, 130.2, 126.0 (p, J = 4.5 Hz), 125.7, 125.0, 123.7 (q, J = 287.6 Hz), 56.9 (hept, J = 27.0 Hz), 34.8.

[0154] F NMR (565 MHz, CDCl) δ -64.3.

[0155] HR-ESI-MS m / z = Calculated. for C HFNO [M+H] 407.0467, found 407.0462.

[0156] 3,6-diamino-10,10-bis(trifluoromethyl)anthracen-9(10H)- one (9)

[0157] A 500 mL g, 7.93 mmol,1.0 equiv.) and AcOH , adding CrO (4.77 g, 47.6 mmol, 6.0 equiv.) and equipping a reflux condenser. The reaction was refluxed under nitrogen overnight. The next day, the reaction was cooled to room temperature and the solvent was removed in vacuo. The resulting green residue was dissolved in CHCl and sat. NaHCO. The aqueous layer was extracted with CHCl three times. The combined organic layers were washed with sat. NaHCO and then brine. The organic layer was dried with NaSO and the solvent wash removed to yield a white solid with a green / yellow tint, this crude was used without further purification. A 250 mL flask was charged with the crude from the previous step, AcOH (53 mL, 0.15 of 10), and EtOH (53 mL, 0.15 M of 10). Iron mesh (4.44 g, 79.3 mmol, 10.0 equiv.) was then added and the reaction was equipped with a reflux condenser (a large, football stir bar should be used, as the reaction mixture is heterogenous and vigorous stirring should be used). The reaction was refluxed at 100 °C overnight. The next morning, the reaction was cooled to room temperature, then quenched with NaOH (29 g) in water, then finally with sat. NaHCO until pH ≈ 10. This solution was extracted with EtOAc three times. The combined organic layers were washed with water and then brine, dried with NaSO, and concentrated in vacuo onto celite. The crude was purified via flash column chromatography (dry load on celite, 0 to 50% EtOAc / Hex, 10% steps) to yield 9 as a yellow solid (2.63 g, 7.30 mmol, 92% yield). The solubility of theAttorney docket No.00012-089WO1 crude mixture containing 9 could be challenging, requiring more solvent to load 9 onto the column than would be preferred. To simplify loading and improve purification, it was helpful to use a dry load technique.

[0158] H NMR (600 MHz, CDCl) δ 8.33 (d, J = 8.5 Hz, 1H), 7.25 (qd, J = 3.7, 1.9 Hz, 1H), 6.89 (dd, J = 8.5, 2.2 Hz, 1H), 4.27 (s, 2H).

[0159] C NMR (151 MHz, CDCl) δ 179.1, 150.8, 132.0, 130.9, 123. 8 (q, J = 286.1 Hz), 123.6, 116.5, 115.1 (heptet, J = 4.4 Hz), 55.0 (heptet, J = 26.0 Hz).

[0160] F NMR (565 MHz, CDCl) δ -64.3.

[0161] HR-ESI-MS m / z = C H O NF [M+H]: 361.0770. Calculated: 361.0770.

[0162] 3,6-bis(dimethylamino)-10,10-bis(trifluoromethyl) anthracen-9(10H)-one (16)

[0163] A 75 9 (1.01 g, 2.78mmol, 1.0 equiv.), KCO (2.31 g, 16.7 mmol, 6.0 equiv.), and anhydrous DMF (28.0 mL, 0.1 M to 9). MeI (1.04 mL, 16.7 mmol, 6.0 equiv.) was added, the flask was capped, and the reaction vessel was submerged into a 100 °C oil bath with vigorous stirring. After 75 minutes, an additional batch of MeI (1.00 mL, 16.1 mmol, 5.8 equiv.) was added. After an additional 75 min of stirring at 100 °C, additional MeI (1.00 mL, 16.1 mmol, 5.8 equiv.) was added. After stirring for an additional 75 min of stirring at 100 °C, a fourth addition of MeI (0.50 mL, 8.0 mmol, 2.9 equiv.) was made. After stirring for an additional 75 min of stirring at 100 °C, LCMS confirmed complete conversion of the intermediate tri-methyl product to the desired 16. The solution was diluted with CHCl and quenched with water. The aqueous layer was extracted three times with CHCl. The combined organics were dried with NaSO and the solvent was removed. The crude material was purified via flash column chromatography (dry load on silica, 0 to 50% EtOAc / CHCl, 10% steps) to yield 16 as a yellow solid (1.09 g, 2.62 mmol, 94%Attorney docket No.00012-089WO1 yield). Due to the low boiling point of methyl iodide, it was useful to perform this reaction in a bomb flask. Even with this precaution, it was helfpul to periodically add additional equivalents of methyl iodide as the reaction was sluggish and would stall out. Presumably this is due to the loss of the volatile methyl iodide despite the use of a bomb flask. More reactive electrophiles (dimethyl sulfate) would result in over alkylation to the trimethyl ammonium cation. Reductive amination conditions (formaldehyde, NaBHCN, AcOH) were less efficient in the experiments. As such, the portion-wise addition of methyl iodide was used. The reaction was easily followed by LCMS as the various alkylation states are easily separated on a C-18 analytical column.

[0164] H NMR (600 MHz, CDCl) δ 8.37 (d, J = 8.9 Hz, 2H), 7.28 (s, 2H), 6.92 (dd, J = 8.9, 2.4 Hz, 2H), 3.13 (s, 12H).

[0165] C NMR (151 MHz, CDCl) δ 179.3, 152.8, 131.6, 130.2, 121.4, 113.3, 112.4 (heptet, J = 4.5 Hz), 40.3.

[0166] F decoupled C NMR (151 MHz, CDCl) δ 124.1, 55.1.

[0167] F NMR (565 MHz, CDCl) δ -64.3.

[0168] HR-ESI-MS m / z for C H O NF [M+H]: 417.1398. Calculated: 417.1396.

[0169] 3,6-dibromo-10,10-bis(trifluoromethyl)anthracen-9(10H)- one (18)

[0170] A flame9 (0.592 g, 1.64 mmol, 1.0 equiv.) and CuBr (0.922 g, 4.11 mmol, 2.5 equiv.), the reagents were then vac cycled three times. Anhydrous acetonitrile (16.5 mL, 0.1 M to 9) was added to the flask, followed by a dropwise addition of t-BuONO (0.59 mL, 4.9 mmol, 3.0 equiv.) at room temperature. The flask was fitted with a reflux condenser and the flask was submerged into a 65 °C oil bath, where it was stirred for 3 hr. After TLC showed full consumption of 9, the reaction was quenched into 1 M HCl. The aqueous layer was extracted with CHCl three times. The combined organics were dried withAttorney docket No.00012-089WO1 NaSO and concentrated in vacuo onto silica. The crude was purified via flash column chromatography (dry load on silica, 0 to 5% toluene / hexanes, 1% increments) to yield 18 as a white solid (0.607 g, 1.24 mmol, 76% yield).

[0171] H NMR (600 MHz, CDCl) δ 8.38 (d, J = 8.4 Hz, 2H), 8.30 – 8.27 (m, 2H), 7.88 (dd, J = 8.4, 1.7 Hz, 2H).

[0172] C NMR (151 MHz, CDCl) δ 179.9, 134.5, 133.5 (heptet, J = 4.6 Hz), 131.3, 130.7, 130.4, 129.7, 123.3 (q, J = 287.2 Hz), 54.9 (heptet, J = 27.0 Hz).

[0173] F decoupled C NMR (151 MHz, CDCl) δ 123.3, 54.9.

[0174] F NMR (565 MHz, CDCl) δ -64.1.

[0175] HR-EI-MS m / z for C HOF Br Br [M] : 487.8677. Calculated: 487.8669.

[0176] 3,6-dibromo-9-(o-tolyl)-10,10-bis(trifluoromethyl)-9,10- dihydroanthracen-9-ol (19)

[0177] mmol) was subjectedto General Procedure (1). The reaction yielded 19 as a white, foamy product (138 mg, 0.238 mmol, 81% yield).

[0178] H NMR (600 MHz, CDCl) δ 8.31 (d, J = 7.8 Hz, 1H), 8.22 (s, 2H), 7.51 (dd, J = 8.5, 1.8 Hz, 2H), 7.44 (t, J = 7.7 Hz, 1H), 7.32 (t, J = 7.4 Hz, 1H), 7.07 – 7.01 (m, 3H), 2.17 (s, 1H), 1.39 (s, 3H).

[0179] C NMR (151 MHz, CDCl) δ 143.1, 140.9, 134.97, 134.4, 133.0 (qq, J = 6.5, 3.1 Hz), 132.4, 131.2, 128.6, 126.8, 125.8, 125.7, 124.3 (q, J = 287.5 Hz), 123.5 (q, J = 285.6 Hz), 122.7, 73.0, 55.6 (heptet, J = 26.5 Hz), 20.7.

[0180] F NMR (565 MHz, CDCl) δ -61.3 (q, J = 7.4 Hz), -66.0 (q, J = 7.3 Hz).

[0181] HR-ESI-MS m / z for C H OF Br [M-H] : 576.9247. Calculated: 576.9243.

[0182] 3,6-dibromo-9-(2,5-dimethylphenyl)-10,10- bis(trifluoromethyl)-9,10-dihydroanthracen-9-ol (SI-3)Attorney docket No.00012-089WO1

[0183] mmol) was subjected to General ProcedureSI-3 as a white, foamy product (219 mg, 0.369 mmol, 90% yield).

[0184] H NMR (600 MHz, CDCl) δ 8.24 – 8.17 (m, 2H), 8.14 – 8.08 (m, 1H), 7.50 (dd, J = 8.5, 1.9 Hz, 2H), 7.12 (dd, J = 7.6, 1.9 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 7.5 Hz, 1H), 2.50 (s, 3H), 2.14 (s, 1H), 1.33 (s, 3H).

[0185] C NMR (151 MHz, CDCl) δ 142.8, 141.0, 135.3, 134.4, 132.9 (qq, J = 6.6, 3.3 Hz), 132.3, 131.8, 131.2, 129.1, 127.5, 125.7, 124.3 (q, 288.1 Hz), 123.6 (q, 285.3 Hz), 122.7, 72.9, 55.7 (heptet, J = 26.6 Hz), 21.6, 20.2.

[0186] F NMR (565 MHz, CDCl) δ -61.3 (q, J = 7.5 Hz), -66.0 (q, J = 7.5 Hz).

[0187] HR-ESI-MS m / z for C H OF Br [M-H] : 590.9406. Calculated: 590.9399.

[0188] (SI-1) 3,6-dibromo-10,10-bis(trifluoromethyl)-3'H,10H- spiro[anthracene-9,1'-isobenzofuran]-3'-one

[0189] 18 (61.0was added to a flask and then dissolved in glacial AcOH (0.80 mL, 0.13 M to 18). At room temperature conc. HSO (0.15 mL, 15 equiv.) was added slowly, dropwise creating a blood red solution (this color is assumed to come from generating a trityl carbocation) that was stirred for ~5 min. CrO (67.0 mg, 0.631 mmol, 6.0 equiv.) was then added to the reaction as a solid. The reaction was stirred at room temperature overnight. The next day, the reaction had changed from blood red to pale green. The reaction was quenched into water,Attorney docket No.00012-089WO1 extracted with CHCl three times. The combined organics were washed with brine, dried over NaSO, and concentrated in vacuo onto silica. The crude was purified via flash column chromatography (dry load on silica, 0 to 10% EtOAc / Hex, 2.5% increments) to yield SI-1 as a white solid (46.0 mg, 0.0777 mmol, 74% yield).

[0190] H NMR (600 MHz, CDCl) δ 8.24 (s, 2H), 8.06 (d, J = 7.6 Hz, 1H), 7.62 – 7.54 (m, 4H), 6.86 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 7.5 Hz, 1H).

[0191] C NMR (151 MHz, CDCl) δ 170.0, 155.2, 136.0, 134.4, 133.6, 133.3 (heptet, J = 4.7 Hz), 130.4, 130.3, 126.2, 126.0, 124.5, 123.9, 123.5 (q, J = 285.8 Hz), 123.4 (q, J = 286.9 Hz), 122.9, 82.7, 55.3 (heptet, J = 26.9 Hz).

[0192] F NMR (565 MHz, CDCl) δ -63.7 (q, J = 7.3 Hz), -64.4 (q, J = 7.5 Hz).

[0193] HR-ESI-MS m / z for C H OF Br [M+H] : 590.9031. Calculated: 590.9024.

[0194] (21) tert-butyl 3,6-dibromo-3'-oxo-10,10- bis(trifluoromethyl)-3'H,10H-spiro[anthracene-9,1'-isobenzofuran]- 6'-carboxylate

[0195] A bar was charged withSI-3 (219 mg, 0.369 mmol, 1.0 equiv.) and AcOH (2.80 mL, 0.13 M of SI-3). This was placed in an ice bath and concentrated HSO (0.53 mL, 0.70 M of SI-3) was added dropwise. To the resulting purple solution was added CrO (233 mg, 2.33 mmol, 6.3 equiv.), and the reaction was allowed to stir at room temperature. After 16 hours additional AcOH (1.40 mL, 0.26 M of SI-3), concentrated HSO (0.26 mL, 1.40 M of SI-3) and CrO (71.2 mg, 0.712 mmol, 1.9 equiv.) were added to the reaction. After 4 hours the reaction was poured into 100 mL of water, and the resulting aqueous layer was extracted 5x with CHCl (300 mL total). The organic solution was dried with NaSO, and the solvent was removed. The isolated material wasAttorney docket No.00012-089WO1 transferred to a round bottom and dissolved in toluene (9.0 mL, 0.041 M of SI-3). The flask was heated in an 80 °C oil bath and the turbid solution was vigorously stirred. N,N-Dimethylformamide di- tert-butyl acetal (0.35 mL, 1.5 mmol, 4.1 equiv.) was dissolved in toluene (1.50 mL) and the resulting solution was added dropwise to the benzoic acid over 20 min. The turbid solution became clear during the first quarter of the addition. After stirring for 3 hours, the reaction was cooled to room temperature and poured into 50 mL of water. The aqueous solution was extracted three times with CHCl. The combined organics were dried with NaSO and the solvent was removed. The crude residue was purified by column chromatography (loaded onto the column with toluene, 0 to 12% EtOAc / Hexanes, 2% increments). The tert-butyl ester 21 was isolated as a white solid (152 mg, 0.220 mmol, 60% yield over two steps).

[0196] A benefit of telescoping this reaction sequence is the ability to avoid purifying the intermediate benzoic acid, which was found to require the use of acetic acid. Acetic acid is challenging to completely remove from the product and would consume N,N- Dimethylformamide di-tert-butyl acetal in the subsequent reaction. This procedure alleviates that issue.

[0197] H NMR (500 MHz, CDCl) δ 8.29 – 8.23 (m, 2H), 8.19 (dd, J = 8.0, 1.3 Hz, 1H), 8.08 (dd, J = 8.1, 0.7 Hz, 1H), 7.57 (dd, J = 8.6, 1.9 Hz, 2H), 7.40 (t, J = 1.0 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 1.50 (s, 9H).

[0198] C NMR (126 MHz, CDCl) δ 169.23, 163.50, 155.16, 139.17, 134.48, 133.53, 133.07, 131.35, 130.20, 127.19, 126.22, 125.95, 124.08, 123.88, 83.00, 28.04.

[0199] F decoupled C NMR (126 MHz, CDCl) δ 123.42, 123.39, 55.31.

[0200] F NMR (471 MHz, CDCl) δ -63.6 (q, J = 7.4 Hz), -64.6 (q, J = 7.4 Hz).

[0201] HR-ESI-MS m / z for C H OF BrNa [M+Na] : 712.9384. Calculated: 712.9368.

[0202] (4a) N-(7-(dimethylamino)-10-(o-tolyl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)-N-methylmethanaminiumAttorney docket No.00012-089WO1

[0203] Synthesis round bottom flask with a stir bar was vac(22 uL, 0.018 mmol, 4.0 equiv.) and THF (1.40 mL, 0.13 M of 2- bromotoluene). The flask was cooled to -78 °C in a dry ice bath before adding t-BuLi (0.21 mL, 1.7 M in pentanes, 0.36 mmol, 8.0 equiv.) dropwise over 5 minutes. After 30 minutes, the flask was warmed up to 0 °C in an ice bath. After 10 min at 0 °C, the flask was then cooled back down to -78 °C. After 15 minutes at -78 °C, a solution of 16 (19 mg, 0.046 mmol, 1.0 equiv.) in THF (0.70 mL, 0.066 M of 16) was added dropwise to the flask over 2-3 minutes. The solution became a bright red. After 10 minutes at -78 °C, the flask was allowed to warm to room temperature. An aliquot showed complete consumption after 1 hour, at which point the reaction was quenched with AcOH (0.77 mL). The solvent was removed from the brilliant blue solution in vacuo. The blue residue was dissolved with CHCl and loaded onto a silica pipette column, which was first eluted with CHCl to remove any non-polar impurities and then 10% MeOH / CHCl to collect the blue rhodamine dye. The solvent was removed in vacuo and the resulting blue material was purified by prep-TLC (5 to 10% MeOH / CHCl, 2.5% increments). The silica was extracted with 30% MeOH / CHCl which was then pushed through a PTFE filter. The solvent was removed in vacuo, the residue was dissolved CHCl and filtered through a second PTFE filter. After removing the solvent in vacuo and sonicating the blue film with hexanes, the presumed bromide salt of 4a was isolated as a blue powder (23 mg, 0.040 mmol, 87% yield).

[0204] Synthesis 2: Dibromo trityl alcohol 19 (11 mg, 0.019 mmol) was subjected to General Procedure (2) using dimethylamine solution (2.0 M in THF) as the amine. The reaction yielded 4a as a blue, waxy solid product (8.0 mg, 0.013 mmol, 68% yield).

[0205] H NMR (600 MHz, CDCl) δ 7.57 (s, 1H), 7.45 (td, J = 7.5, 1.4 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.15 (d, J = 9.5 Hz,Attorney docket No.00012-089WO1 1H), 7.08 (d, J = 7.2 Hz, 0H), 6.97 (dd, J = 9.5, 2.4 Hz, 1H), 3.46 (s, 12H), 1.95 (s, 3H).

[0206] C NMR (151 MHz, CDCl) δ 164.9, 156.1, 139.0, 135.8, 134.2, 133.4, 130.9, 130.0, 128.8, 126.2, 123.1 (q, J = 285.8 Hz), 123.0 (q, J = 286.4 Hz), 121.2, 117.8, 115.1, 56.5 (hept, J = 26.3 Hz), 41.9, 19.3.

[0207] F NMR (565 MHz, CDCl) δ -65.9 (q, J = 7.7 Hz), -67.1 (q, J = 7.3 Hz).

[0208] HR-ESI-MS m / z for C H FN [M+H]: 491.1914. Calculated: 491.1916.

[0209] (4b) N-(7-(diethylamino)-10-(o-tolyl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)-N-ethylethanaminium

[0210] 0.019 mmol) wassubjected to General Procedure (2) using diethylamine as the amine. The reaction yielded 4b as a blue, waxy solid product (9.0 mg, 0.014 mmol, 74% yield).

[0211] H NMR (600 MHz, CDCl) δ 7.56 (s, 2H), 7.47 (td, J = 7.6, 1.4 Hz, 2H), 7.38 (d, J = 7.9 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 9.5 Hz, 2H), 7.08 (dd, J = 7.5, 1.3 Hz, 1H), 6.99 (dd, J = 9.5, 2.4 Hz, 2H), 3.77 (q, J = 7.2 Hz, 8H), 2.01 (s, 3H), 1.37 (t, J = 7.2 Hz, 12H).

[0212] C NMR (151 MHz, CDCl) δ 164.1, 154.5, 139.2, 135.9, 134.2, 133.5, 130.9, 130.1, 128.9, 126.3, 121.1, 117.7, 115.0, 47.2, 19.4, 13.2.

[0213] F decoupled C NMR (151 MHz, CDCl) δ 123.4, 123.3, 56.5.

[0214] F NMR (565 MHz, CDCl) δ -65.7 (q, J = 6.9 Hz), -66.8 (q, J = 7.2 Hz).

[0215] HR-ESI-MS m / z for C H NF [M] : 547.2540. Calculated: 547.2542.Attorney docket No.00012-089WO1

[0216] (4c) 1-(7-(azetidin-1-yl)-10-(o-tolyl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)azetidin-1-ium

[0217] 0.026 mmol) was subjected toas the amine. The reaction yielded 4c as a blue, waxy solid product (8.0 mg, 0.013 mmol, 50% yield).

[0218] H NMR (600 MHz, CDCl) δ 7.46 (td, J = 7.6, 1.4 Hz, 1H), 7.36 (dd, J = 8.2, 6.0 Hz, 2H), 7.12 (s, 1H), 7.11 – 7.06 (m, 3H), 6.49 (dd, J = 9.2, 2.2 Hz, 2H), 4.42 (t, J = 7.7 Hz, 8H), 2.64 (p, J = 7.7 Hz, 3H), 1.96 (s, 3H).

[0219] C NMR (151 MHz, CDCl) δ 164.5, 154.9, 138.8, 135.9, 134.5, 133.4, 130.9, 130.0, 128.9, 126.2, 121.2, 115.8, 112.7, 52.4, 19.2, 16.0.

[0220] F decoupled C NMR (151 MHz, CDCl) δ 123.1, 123.0, 56.4.

[0221] F NMR (565 MHz, CDCl) δ -66.0 (q, J = 7.5 Hz), -67.22 (q, J = 7.5 Hz).

[0222] HR-ESI-MS m / z for C H NF [M] : 515.1915. Calculated: 515.1916.

[0223] (4d) 1-(7-(3,3-difluoroazetidin-1-yl)-10-(o-tolyl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)-3,3-difluoroazetidin- 1-ium F F F F

[0224] 0.026 mmol) was subjected to General Procedure (2) using 3,3-difluoroazetidine HCl as the amine. The reaction yielded 4d as a blue, waxy solid product (12 mg, 0.017 mmol, 65% yield).Attorney docket No.00012-089WO1

[0225] H NMR (600 MHz, CDCN) δ 7.55 (td, J = 7.6, 1.4 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.44 (t, J = 7.4 Hz, 1H), 7.25 (s, 2H), 7.23 (d, J = 9.2 Hz, 2H), 7.20 (dd, J = 7.6, 1.6 Hz, 1H), 6.66 (dd,135.2, 134.4, 131.6, 131.0, 129.8, 127.0, 122.8, 118.1, 115.9 (t, J = 271.4 Hz), 115.4, 64.7 (t, J = 29.6 Hz), 19.4.

[0227] F decoupled C NMR (151 MHz, CDCN) δ 124.0, 123.9, 57.2.

[0228] F NMR (565 MHz, CDCN) δ -66.2 (q, J = 8.0 Hz), -67.6 (q, J = 7.2 Hz), -102.0 (p, J = 11.8 Hz).

[0229] HR-ESI-MS m / z for C H NF [M] : 587.1540. Calculated: 587.1540.

[0230] (4e) 7-(7-(7-azabicyclo[2.2.1]heptan-7-yl)-10-(o- tolyl)-9,9-bis(trifluoromethyl)anthracen-2(9H)-ylidene)-7- azabicyclo[2.2.1]heptan-7-ium

[0231] mmol) wassubjected to General Procedure (2) using 7-azabicyclo[2.2.1]heptane HCl as the amine. The reaction yielded 4e as a blue, waxy solid product (15 mg, 0.021 mmol, 81% yield).

[0232] H NMR (600 MHz, CDCl) δ 7.51 (s, 2H), 7.47 (td, J = 7.6, 1.3 Hz, 1H), 7.39 – 7.34 (m, 2H), 7.11 (d, J = 9.3 Hz, 2H), 7.06 (dd, J = 7.5, 1.3 Hz, 1H), 6.90 (dd, J = 9.4, 2.2 Hz, 2H), 4.64 (s, 4H), 1.99 (s, 3H), 1.97 – 1.87 (m, 9H), 1.79 – 1.68 (m, 8H).

[0233] C NMR (151 MHz, CDCl) δ 163.2, 152.2, 139.6, 135.9, 134.2, 134.0, 130.9, 130.1, 128.8, 126.3, 123.3 (d, J = 286.9 Hz), 123.1 (d, J = 285.3 Hz), 121.9, 116.1, 58.2, 56.4 (heptet, J = 26.3 Hz), 28.7, 18.8.

[0234] F NMR (565 MHz, CDCl) δ -65.6 (q, J = 7.6 Hz), -66.8 (q, J = 7.9 Hz).Attorney docket No.00012-089WO1

[0235] HR-ESI-MS m / z for C H NF [M] : 595.2541. Calculated: 595.2542.

[0236] (4f) 4-(7-morpholino-10-(o-tolyl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)morpholin-4-ium

[0237] mmol) was subjected tothe amine. The reaction yielded 4f as a blue, waxy solid product (4.0 mg, 0.0058 mmol, 21% yield).

[0238] H NMR (600 MHz, CDCN) δ 7.72 (s, 2H), 7.54 (td, J = 7.6, 1.4 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.21 – 7.16 (m, 3H), 7.04 (dd, J = 9.6, 2.5 Hz, 2H), 3.82 (dd, J = 5.7, 4.0 Hz, 8H), 3.76 (dd, J = 5.7, 4.0 Hz, 8H), 1.98 (s, 3H).

[0239] C NMR (151 MHz, CDCN) δ 165.1, 156.9, 139.9, 136.8, 135.2, 134.3, 131.5, 130.8, 129.8, 127.0, 122.5, 119.0, 116.3, 66.8, 48.7, 19.4.

[0240] C NMR (151 MHz, CDCN) δ 124.2, 124.1, 57.2.

[0241] F NMR (565 MHz, CDCN) δ -66.2 (q, J = 6.8 Hz), -67.4 (t, J = 7.8 Hz).

[0242] HR-ESI-MS m / z for C H ONF [M] : 575.2125. Calculated: 575.2125.

[0243] (4g) 1-(7-(piperazin-1-ium-1-yl)-10-(o-tolyl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)piperazine-1,4-diium

[0244] A 1-dried and cooled under vacuum. The vial was then charged with starting material (19, 26 mg, 0.045 mmol, 1.0 equiv.), Palladacycle XPhos G4Attorney docket No.00012-089WO1 (7.7 mg, 0.0089 mmol, 0.20 equiv.), XPhos (4.4 mg, 0.0092 mmol, 0.20 equiv.), CsCO (146 mg, 0.45 mmol, 10 equiv.), and N-Boc- piperazine (67 mg, 0.36 mmol, 8.0 equiv.), then vac cycled three times. Anhydrous toluene (0.90 mL, 0.03 M to 19) was added, and the cap was quickly swapped for a non-septa Teflon screw top. The reaction was brought up to 110 °C under pressure and refluxed overnight. The next day, the reaction would appear metallic gray, indicating complete consumption of the starting material. The crude was then diluted with CHCl and filtered through a celite plug to remove palladium and concentrated under reduced pressure. Crude was suspended in a 3:1 CHCl:TFA mix (2 mL) and stirred at room temperature for 30 min. Reaction was concentrated in vacuo, then purified via preparative HPLC (following the general HPLC method above), where pure fractions were concentrated in vacuo. Product was then drawn up in MeOH and passed through a short C18 silica plug to remove any grease from the compound, then concentrated in vacuo to yield the tri-cation 4g as a crystalline, blue solid (18 mg, 0.020 mmol, 44% yield).

[0245] H NMR (600 MHz, MeOD) δ 7.85 (s, 2H), 7.58 (td, J = 7.6, 1.4 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 9.5 Hz, 2H), 7.30 (dd, J = 9.6, 2.4 Hz, 2H), 7.21 (dd, J = 7.5, 1.3 Hz, 1H), 4.18 – 4.07 (m, 8H), 3.51 (dd, J = 6.3, 4.5 Hz, 8H), 2.01 (s, 3H).

[0246] C NMR (151 MHz, MeOD) δ 168.5, 157.7, 141.2, 137.1, 135.6, 135.2, 131.9, 131.5, 130.0, 127.3, 123.9, 119.6, 117.4, 45.6, 43.9, 19.3.

[0247] C NMR (151 MHz, MeOD) δ 124.5, 124.4, 57.8.

[0248] F NMR (565 MHz, MeOD) δ -66.7 (q, J = 7.7 Hz), -67.9 (q, J = 7.5 Hz).

[0249] HR-ESI-MS m / z for C H NF [M] : 573.2454. Calculated: 573.2447.

[0250] (4h) 1-(10-(o-tolyl)-7-(4-tosylpiperazin-1-yl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)-4-tosylpiperazin-1-iumAttorney docket No.00012-089WO1

[0251] mmol) was subjected toas the amine. The reaction yielded 4h as a blue, waxy solid product (10 mg, 0.010 mmol, 59% yield).

[0252] H NMR (600 MHz, MeOD) δ 7.71 (s, 2H), 7.69 (d, J = 8.4 Hz, 4H), 7.52 (td, J = 7.6, 1.3 Hz, 1H), 7.46 – 7.39 (dd, J = 11.1,7.9 Hz, , (d, J = 9.6 Hz, 2H), 7.16 – 7.11 (m, 3H), 3.97 – 3.87 (m, 8H), 3.24 (t, J = 5.2 Hz, 8H), 2.42 (s, 6H), 1.95 (s, 3H).

[0253] C NMR (151 MHz, MeOD) δ 166.7, 157.3, 145.9, 140.6, 137.1, 135.4, 135.1, 134.0, 131.8, 131.3, 131.1, 130.0, 129.0, 127.2, 123.2, 119.5, 117.0, 48.4, 46.8, 21.5, 19.3.

[0254] F decoupled C NMR (151 MHz, MeOD) δ 124.5, 124.4, 57.6.

[0255] F NMR (565 MHz, MeOD) δ -66.9 (q, J = 7.2 Hz), -68.1 (q, J = 7.1 Hz).

[0256] HR-ESI-MS m / z for C H OSNF [M] : 881.2626. Calculated: 881.2624.

[0257] (4i) 4-(7-thiomorpholino-10-(o-tolyl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)thiomorpholin-4-ium

[0258] mmol) was subjected to General Procedure (2) using thiomorpholine as the amine. The reaction yielded 4i as a blue, waxy solid product (24 mg, 0.033 mmol, 63% yield).Attorney docket No.00012-089WO1

[0259] H NMR (600 MHz, CDCl) δ 7.65 (s, 2H), 7.49 (td, J = 7.6, 1.4 Hz, 1H), 7.41 – 7.36 (m, 2H), 7.24 (d, J = 9.5 Hz, 2H), 7.10 (dd, J = 7.6, 1.3 Hz, 1H), 7.06 (dd, J = 9.6, 2.5 Hz, 2H), 4.20 – 4.13 (m, 8H), 2.90 – 2.82 (m, 8H), 2.00 (s, 3H).

[0260] C NMR (151 MHz, CDCl) δ 165.9, 155.1, 140.1, 135.8, 134.3, 133.7, 131.0, 130.4, 128.8, 126.3, 122.2, 118.1, 115.9, 51.7, 27.7, 19.4.

[0261] F decoupled C NMR (151 MHz, CDCl) δ 123.2, 123.1, 56.5.

[0262] F NMR (565 MHz, CDCl) δ -65.5 (q, J = 7.2 Hz), -66.7 (q, J = 7.7 Hz).

[0263] HR-ESI-MS m / z for C H SNF [M] : 607.1666. Calculated: 607.1671.

[0264] (4j) 4-(7-(1,1-dioxidothiomorpholino)-10-(o-tolyl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)thiomorpholin-4-ium 1,1-dioxide

[0265] mmol) wassubjected to General Procedure (2) using thiomorpholine 1,1-dioxide as the amine. The reaction yielded 4j as a blue, waxy solid product (7.0 mg, 0.0089 mmol, 47% yield).

[0266] H NMR (600 MHz, MeOD) δ 7.88 (s, 2H), 7.58 (td, J = 7.6, 1.4 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 9.5 Hz, 2H), 7.36 (dd, J = 9.6, 2.2 Hz, 2H), 7.21 (dd, J = 7.5, 1.3 Hz, 1H), 4.41 – 4.32 (m, 8H), 3.41 – 3.35 (m, 8H), 2.02 (s, 3H).

[0267] C NMR (151 MHz, MeOD) δ 167.5, 155.9, 140.1, 135.7, 134.4, 133.8, 130.5, 130.1, 128.6, 125.9, 122.5, 118.4, 116.2, 51.0, 46.4, 17.9.

[0268] C NMR (151 MHz, MeOD) δ 124.5, 124.4, 57.8.Attorney docket No.00012-089WO1

[0269] F NMR (565 MHz, MeOD) δ -66.7 (q, J = 7.2 Hz), -67.8 (q, J = 7.7 Hz).

[0270] HR-ESI-MS m / z for C H OSNF [M] : 671.1466. Calculated: 671.1467.

[0271] (4k) 7-amino-10-(o-tolyl)-9,9-bis(trifluoromethyl) anthracen-2(9H)-iminium

[0272] flame-dried 5 mL flask,mmol, 1.0 equiv.), bis(trimethylsilyl)amine (0.417 mL, 1.99 mmol, 2.0 equiv.), and tetra-n-butylammonium fluoride solution (1.0 M in THF, 0.100 mL, 0.100 mmol, 0.1 equiv.). The reaction was sealed and stirred overnight at room temperature. Volatiles were removed in vacuo, and the reagent was left on high vac for ~2 hr. Assuming full conversion, the compound was dissolved in 1 mL of toluene to produce a 1.0 M solution of benzophenone imine.

[0273] A 1-dram vial with a septa top was flame-dried and cooled under vacuum. The vial was then charged with starting material (19, 25 mg, 0.043 mmol, 1.0 equiv.), Palladacycle XPhos G4 (7.5 mg, 0.0087 mmol, 0.20 equiv.), XPhos (4.2 mg, 0.0088 mmol, 0.20 equiv.), and CsCO (142 mg, 0.44 mmol, 10 equiv.), then vac cycled three times. Anhydrous toluene (0.90 mL, 0.03 M to 19) and benzophenone imine solution (1.0M, 0.35 mL, 0.35 mmol, 8.8 equiv.) were added, and the cap was quickly swapped for a non-septa Teflon screw top. The reaction was brought up to 110 °C under pressure and refluxed overnight. The next day, the reaction would appear metallic gray, indicating complete consumption of the starting material. The crude was quenched, and the product was deprotected by adding DI HO with a few drops of TFA to the vial, then allowed to stir vigorously at room temperature for 30 min. The crude was concentrated under reduced pressure and run through a short silica column (0 to 2% MeOH / CHCl, 1% increments) collecting all blue fractions, to remove excess benzophenone. Blue fractions wereAttorney docket No.00012-089WO1 concentrated in vacuo, then purified via preparative HPLC (following the general HPLC method above), where pure fractions were concentrated in vacuo. Product was then drawn up in MeOH and passed through a short C18 silica plug to remove any grease from the compound, then concentrated in vacuo to yield 4k as a crystalline, purple solid (7.0 mg, 0.013 mmol, 30% yield).

[0274] H NMR (600 MHz, MeOD) δ 7.69 (s, 1H), 7.52 (td, J = 7.6, 1.4 Hz, 1H), 7.45 (d, J = 7.8 Hz, 0H), 7.43 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 9.2 Hz, 2H), 6.80 (dd, J = 9.2, 2.1 Hz, 2H), 2.02 (s, 3H).

[0275] C NMR (151 MHz, MeOD) δ 166.6, 160.8, 141.3, 137.1, 135.9, 135.6, 131.7, 131.0, 130.0, 127.2, 124.6 (q, J = 285.8 Hz), 124.6 (q, J = 283.6 Hz), 121.9, 121.5, 117.4, 57.5 (heptet, J = 26.5 Hz), 19.3.

[0276] F NMR (565 MHz, MeOD) δ -67.2 (q, J = 7.5 Hz), -68.3 (q, J = 7.3 Hz).

[0277] HR-ESI-MS m / z for C H NF [M] : 435.1292. Calculated: 435.1290.

[0278] (4l) (E)-N-(7-(ethylamino)-10-(o-tolyl)-9,9- bis(trifluoromethyl)anthracen-2(9H)-ylidene)ethanaminium

[0279] mmol) wassubjected to General Procedure (2) using ethylamine as the amine. The reaction yielded 4l as a blueish, purple solid product (5.7 mg, 0.012 mmol, 74% yield).

[0280] H NMR (600 MHz, CDCl) δ 7.65 (s, 2H), 7.45 (td, J = 7.6, 1.4 Hz, 1H), 7.39 – 7.32 (m, 2H), 7.12 – 7.08 (m, 1H), 7.06 (d, J = 9.3 Hz, 2H), 6.73 – 6.62 (m, 2H), 5.60 (s, 2H), 3.44 (q, J = 7.3 Hz, 4H), 1.99 (s, 3H), 1.35 (t, J = 7.3 Hz, 6H).

[0281] C NMR (151 MHz, CDCl) δ 163.1, 156.8, 139.5, 136.1, 134.9, 133.5, 130.7, 129.7, 129.0, 126.0, 121.2, 38.8, 19.3, 13.8.Attorney docket No.00012-089WO1

[0282] F decoupled C NMR (151 MHz, CDCl) δ 123.2, 123.1, 56.3.

[0283] F NMR (565 MHz, CDCl) δ -66.1 (bs), -67.3 (bs).

[0284] HR-ESI-MS m / z for C H NF [M] : 491.1916. Calculated: 491.1916.

[0285] Note: Two of the carbon peaks are missing in the C spectra, and some appear to broaden. It is hypothesized that these peaks are not visible due to rotamers around either the aryl C-N or alkyl C-C bonds, which complicates the spectra. When performing NMR experiments at elevated temperatures in order to resolve these peaks, decomposition was observed.

[0286] (4m) (E)-2,2,2-trifluoro-N-(10-(o-tolyl)-7-((2,2,2- trifluoroethyl)amino)-9,9-bis(trifluoromethyl)anthracen-2(9H)- ylidene)ethan-1-aminium

[0287] 0.033 mmol) wassubjected to General Procedure (2) using 2,2,2-trifluoroethylamine HCl as the amine. The reaction yielded 4m as a metallic, blue solid product (18.9 mg, 0.0263 mmol, 81% yield).

[0288] H NMR (600 MHz, CDCN) δ 8.38 (s, 2H), 7.79 (s, 2H), 7.55 (td, J = 7.6, 1.4 Hz, 1H), 7.49 – 7.41 (m, 2H), 7.31 (d, J = 9.2 Hz, 2H), 7.21 (dd, J = 7.5, 1.4 Hz, 1H), 7.01 (dd, J = 9.3, 2.2 Hz, 2H), 4.23 (q, J = 8.9 Hz, 4H), 1.97 (s, 3H).

[0289] C NMR (151 MHz, CDCN) δ 169.7, 159.1, 142.4, 136.9, 135.0, 131.6, 131.1, 129.8, 126.9, 125.4 (q, J = 279.5 Hz), 123.7, 45.2 (q, J = 34.3 Hz), 19.4.

[0290] F decoupled C NMR (151 MHz, CDCN) δ 124.1, 124.0, 57.1.

[0291] F NMR (565 MHz, CDCN) δ -66.20 (d, J = 7.9 Hz), - 67.52, -71.79 (t, J = 8.8 Hz), -76.36.

[0292] HR-ESI-MS m / z for C H NF [M] : 599.1348. Calculated: 599.1351.Attorney docket No.00012-089WO1

[0293] Similar to compound 4l, broadened peaks were observed in the carbon NMR in addition to missing peaks. It was hypothesized that these are due to rotamers around either the aryl C-N bond or the C-C or C-N bonds on the trifluoroethyl group.

[0294] (17a)3,6-bis(dimethylamino)-10,10-bis(trifluoromethyl)- 3'H,10H-spiro[anthracene-9,1'-isobenzofuran]-3'-one

[0295] Synthesis with stir bar wasvac cycled before -4,4-dimethyl-4,5- dihydrooxazole (52 mg, 0.20 mmol, 4.0 equiv.). The flask was again vac cycled before adding THF (1.6 mL, 0.13 M of oxazole) and cooling to -78 °C. After 15 minutes, t-BuLi (0.23 mL, 1.7 M in pentanes, 0.39 mmol, 7.8 equiv.) was added dropwise over 2-3 minutes. The reaction was warmed to 0 °C in an ice bath after 20 minutes. After 10 minutes at 0 °C, the flask was cooled back down to -78 °C. After 15 minutes at -78 °C, a solution of 16 (21 mg, 0.050 mmol, 1.0 equiv.) in THF (0.77 mL, 0.065 M of 16) was added dropwise over 2-3 minutes. The reaction was allowed to warm to room temperature after 10 minutes, and after an additional 2 hours the reaction was quenched with AcOH (0.84 mL). The solvent was removed in vacuo, and the crude material was dissolved in 6M HCl (3.0 mL, 0.017 M of 16) and heated to 80 °C. After 15 hours, the flask was cooled to room temperature and poured into sat. NaHCO. The aqueous solution was extracted three times with CHCl. The combined organics were dried with NaSO and the solvent was removed in vacuo. The crude material was purified by column chromatography (0 to 24% EtOAc / Hex, 4% increments). The lactone 17a was isolated as a faint yellow solid (18 mg, 0.035 mmol, 70% yield).

[0296] Synthesis 2: Dibromo lactone SI-1 (11 mg, 0.019 mmol) was subjected to General Procedure (3) using dimethylamine solution (2.0 M in THF) as the amine. The reaction yielded 17a as a light blue, solid product (5.0 mg, 0.0096 mmol, 51% yield).Attorney docket No.00012-089WO1

[0297] H NMR (600 MHz, CDCl) δ 8.02 – 7.97 (m, 1H), 7.54 – 7.48 (m, 2H), 7.34 (s, 2H), 6.88 (dd, J = 5.5, 2.3 Hz, 1H), 6.77 (d, J = 8.9 Hz, 2H), 6.74 (dd, J = 8.9, 2.4 Hz, 2H), 2.99 (s, 12H).

[0298] C NMR (151 MHz, CDCl) δ 171.1, 156.5, 150.1, 135.2, 129.7, 129.1, 126.2, 125.8, 125.1, 124.4 (q, J = 285.6 Hz), 124.3 (q, J = 285.3 Hz), 123.1, 121.8, 114.9, 113.1 (heptet, J = 4.8 Hz), 85.6, 55.5 (heptet, J = 25.9 Hz), 40.3.

[0299] F NMR (565 MHz, CDCl) δ -63.4 (q, J = 7.1 Hz), -64.0 (q, J = 7.1 Hz).

[0300] HR-ESI-MS m / z for C H ONF [M+H] : 521.1659. Calculated: 521.1658.

[0301] (17b)3,6-di(azetidin-1-yl)-10,10-bis(trifluoromethyl)- 3'H,10H-spiro[anthracene-9,1'-isobenzofuran]-3'-one

[0302] was subjectedto General Procedure (3) using azetidine as the amine. The reaction yielded 17b as a white solid (9.0 mg, 0.017 mmol, 53% yield).

[0303] H NMR (500 MHz, CDCl) δ 8.01 – 7.95 (m, 1H), 7.54 – 7.47 (m, 2H), 6.99 (p, J = 2.2 Hz, 2H), 6.90 – 6.83 (m, 1H), 6.72 (d, J = 8.7 Hz, 2H), 6.44 (dd, J = 8.7, 2.3 Hz, 2H), 3.98 – 3.85 (m, 8H), 2.39 (p, J = 7.2 Hz, 4H).

[0304] C NMR (126 MHz, CDCl) δ 171.0, 156.5, 151.6, 135.2, 129.7, 129.1, 126.0, 125.7, 125.1, 123.1, 122.5, 113.9, 111.9 (heptet, J = 4.5 Hz), 85.6, 52.1, 16.9.

[0305] F decoupled C NMR (126 MHz, CDCl) δ 124.1, 124.1, 55.3.

[0306] F NMR (471 MHz, CDCl) δ -63.5 (q, J = 7.2 Hz), -64.2 (q, J = 7.3 Hz).

[0307] HR-ESI-MS m / z for C H ONF [M+H] : 545.1657. Calculated: 545.1658.

[0308] (BF ) 3,6-bis(dimethylamino)-3'-oxo-10,10- bis(trifluoromethyl)-3'H,10H-spiro[anthracene-9,1'-isobenzofuran]- 6'-carboxylic acidAttorney docket No.00012-089WO1

[0309] A stir bar was flame dried and vac cycled.21 (15 mg, 0.022 mmol, 1.0 equiv.), Palladacycle XPhos G4 (3.7 mg, 0.0043 mmol, 0.20 equiv.), XPhos (2.0 mg, 0.0042 mmol, 0.19 equiv.) and CsCO (74 mg, 0.23 mmol, 10.5 equiv.). The vial was vac cycled three times before adding dimethylamine (0.09 mL, 2.0 M in THF, 0.18 mmol, 8.2 equiv.) and toluene (0.72 mL, 0.03 M of 21). The septa top was quickly replaced with a Teflon top, and the reaction was heated to 110 °C for 16 hours. After cooling to room temperature, the reaction was filtered through a pipette silica plug using toluene and then EtOAc to completely elute the product. The solvent was removed in vacuo, and the crude residue was purified by column chromatography (0 to 3% EtOAc / toluene, 0.5% increments). The fractions containing the tetramethyl rhodamine were collected and the solvent was removed in vacuo. The residue was then dissolved in CHCl (1.0 mL, 0.022 M of 21) and TFA (1.0 mL, 0.022 M of 21). The deprotection was complete by LCMS within 90 min, and the solvent was removed in vacuo. The blue residue was purified by prep-TLC (2.5% MeOH / CHCl to 5% MeOH / CHCl). The desired band was extracted with MeOH, and the residue was filtered through a PTFE filter, yielding BF (9.0 mg, 0.016 mmol, 73% yield) as a light blue solid. The material isolated after the initial EtOAc / toluene column will contain multiple products in addition to the desired rhodamine. Although you could run the TFA deprotection after the amide coupling without purification, it is believed that running this intermediate column simplified purification of BF . The initial filtration through the silica plugs was to remove the palladium and enable the removal of the solvent before the column purification. Considerable streaking due to small amounts of THF was present when the crude reaction was purified directly without the initial silica plug.Attorney docket No.00012-089WO1

[0310] When moderately scaling this reaction up, the initial Buchwald-Hartwig coupling is performed in multiple vials before combining the material before the EtOAc / toluene column. On even larger scales, it is anticipated that heavy walled pressure vessels would prove adequate. An exemplary scaled up comprises: Two separate dram vials with a septa top and stir bars were flame dried and vac cycled. The first vial was charged with 21 (30.5 mg, 0.0441 mmol, 1.0 equiv.), Palladacycle XPhos G4 (7.8 mg, 0.0091 mmol, 0.21 equiv.), XPhos (4.2 mg, 0.0088 mmol, 0.20 equiv.) and CsCO (149.5 mg, 0.459 mmol, 10.4 equiv.). The vial was vac cycled three times before adding dimethylamine (0.17 mL, 2.0 M in THF, 0.34 mmol, 7.7 equiv.) and toluene (1.50 mL, 0.03 M of 21). The second vial was charged with 21 (30.9 mg, 0.0446 mmol, 1.0 equiv.), Palladacycle XPhos G4 (8.0 mg, 0.0093 mmol, 0.21 equiv.), XPhos (4.1 mg, 0.0086 mmol, 0.20 equiv.) and CsCO (146.6 mg, 0.450 mmol, 10.2 equiv.). The second vial was vac cycled three times before adding dimethylamine (0.17 mL, 2.0 M in THF, 0.34 mmol, 7.7 equiv.) and toluene (1.50 mL, 0.03 M to 21). Both septa tops were quickly replaced with Teflon caps, and the reactions were heated to 110 °C for 16 hours. After cooling to room temperature, each reaction was filtered through a pipette silica plug using toluene and then EtOAc to completely elute the product. The two coupling reactions were combined at this stage (61.4 mg total, 0.0887 mmol scale). The solvent was removed in vacuo, and the crude residue was purified by column chromatography (0 to 3.5% EtOAc / toluene, 0.5% increments). The fractions containing the tetramethyl rhodamine were collected and the solvent was removed in vacuo. The residue was then dissolved in CHCl (10.0 mL, 0.009 M of 21) and TFA (2.0 mL, 0.044 M of 21). After 5.5 hours, the reaction was diluted with 3.0 mL of toluene, and the solvent was removed in vacuo. The blue residue was purified by prep-TLC (2.5% MeOH / CHCl to 5% MeOH / CHCl). The desired band was extracted with MeOH, and the residue was filtered through a PTFE filter, yielding BF (38.4 mg, 0.0680 mmol, 77% yield) as a blue solid.

[0311] H NMR (600 MHz, CDCN) δ 8.27 (dd, J = 8.2, 1.4 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H), 7.69 (s, 1H), 7.58 (s, 1H), 6.99 (d, J = 9.3 Hz, 2H), 6.91 (dd, J = 9.3, 2.5 Hz, 2H), 3.21 (s, 12H).Attorney docket No.00012-089WO1

[0312] C NMR (151 MHz, CDCN) δ 167.5, 166.5, 154.5, 136.1, 133.1, 131.8, 131.2, 130.6, 129.4, 125.5, 123.6, 122.7, 117.4, 116.4, 41.7, 30.9, 30.4.

[0313] F decoupled C NMR (151 MHz, CDCN) δ 124.5, 124.5, 56.9.

[0314] F NMR (565 MHz, CDCN) δ -65.9 (s), -66.1 (s).

[0315] HR-ESI-MS m / z for C H ONF [M+H] : 565.1562. Calculated: 565.1557.

[0316] (BF ) 3,6-di(azetidin-1-yl)-3'-oxo-10,10- bis(trifluoromethyl)-3'H,10H-spiro[anthracene-9,1'-isobenzofuran]- 6'-carboxylic acid

[0317] A bar was flamedried and vac cycled. The vial was then charged with 21 (15 mg, 0.022 mmol, 1.0 equiv.), Palladacycle XPhos G4 (3.9 mg, 0.0045 mmol, 0.20 equiv.), XPhos (2.2 mg, 0.0046 mmol, 0.21 equiv.) and CsCO (70 mg, 0.21 mmol, 9.5 equiv.). The vial was vac cycled three times before adding azetidine (12.0 μL, 0.18 mmol, 8.1 equiv.) and toluene (0.72 mL, 0.03 M of 21). The septa top was quickly replaced with a Teflon top, and the reaction was heated to 110 °C for 16 hours. After cooling to room temperature, the reaction was purified directly by column chromatography (0 to 3.5% EtOAc / toluene, 0.5% increments). The fractions containing the desired rhodamine were collected and the solvent was removed in vacuo. The residue was then dissolved in CHCl (2.50 mL, 0.009 M of 21) and TFA (0.50 mL, 0.043 M of 21). After 5 hours the reaction was diluted with 3.0 mL toluene, and the solvent was removed in vacuo. The crude material was azeotroped with MeOH before further purification. The blue residue was purified by prep-TLC (2.5% MeOH / CHCl to 5% MeOH / CHCl). The desired band was extracted with MeOH, and the residue was filtered through a PTFE filter, yielding BF (8.0 mg, 0.014 mmol, 64% yield) as a blue solid.Attorney docket No.00012-089WO1

[0318] H NMR (600 MHz, MeOD) δ 8.14 (dd, J = 8.0, 1.2 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.44 (s, 1H), 7.00 (s, 2H), 6.72 (d, J = 8.7 Hz, 2H), 6.56 (dd, J = 8.7, 2.3 Hz, 2H), 3.94 – 3.88 (m, 8H),

[0319] C NMR (151 MHz, MeOD) δ 172.5, 172.4, 157.2, 153.4, 146.3, 131.4, 130.9, 128.1, 127.1, 125.3, 124.8, 123.7, 115.3, 112.8, 87.3, 53.2, 17.6.

[0320] F decoupled C NMR (151 MHz, MeOD) δ 125.5, 125.4, 56.5.

[0321] F NMR (565 MHz, MeOD) δ -65.1 (q, J = 7.2 Hz), -65.2 (q, J = 7.7 Hz).

[0322] HR-ESI-MS m / z for C H ONF [M+H] : 589.1560. Calculated: 589.1557.

[0323] (BF ) TMR HaloTag Ligandchlorohexyl)oxy)ethoxy)ethyl)carbamate (HTL, 23.1 mg, 0.0713 mmol) was dissolved in CHCl (0.60 mL, 0.11 M of HTL) and TFA (0.60 mL, 0.11 M of HTL). After 30 min, toluene (1.0 mL) was then added, and the solvent was removed in vacuo. The resulting residue was azeotroped an additional two times with toluene (1.0 mL), and then stored in vacuo for 12 hours. In preparation for the amide coupling, the residue was dissolved in DMSO (1.49 mL, 0.048 M of HTL) in preparation for the amide coupling.

[0325] A vial with BF (10 mg, 0.018 mmol, 1.0 equiv.) and a stir bar was charged with DMSO (0.56 mL, 0.032 M of BF ) and i- PrNEt (0.03 mL, 0.17 mmol, 9.4 equiv.). Upon stirring the solution became clear and colorless. TSTU (7.3 mg, 0.024 mmol, 1.3 equiv.) was added, with the solution becoming a faint yellow. After 5 min, the solution of HTL (0.55 mL, 0.026 mmol, 1.4 equiv.) was added.Attorney docket No.00012-089WO1 After 2.5 hours, additional TSTU (6.1 mg, 0.020 mmol, 1.1 equiv.) was added. This was followed 15 min later with additional HTL (0.55 mL, 0.026 mmol, 1.4 equiv.). After 30 min, the reaction was quenched with MeCN / HO with 0.05% TFA. The solution was purified directly by preparative HPLC (10 to 100% MeCN / HO, 0.05% TFA). The solvent in the fractions containing BF was removed in vacuo, and the residue was dissolved in CHCl to transfer to a smaller vial. After removing the solvent, the blue residue was dissolved in MeOH and filtered through a C-18 pipette column with MeOH. After removing the solvent, BF (9.0 mg, 0.012 mmol, 67% yield) was isolated as a light blue film.

[0326] H NMR (600 MHz, CDCl) δ 8.04 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.36 (s, 2H), 7.21 (s, 1H), 6.80 – 6.71 (m, 4H), 6.66 (t, J = 5.5 Hz, 1H), 3.61-3.54 (m, 6H), 3.52 – 3.50 (m, 4H), 3.37 (t, J = 6.6 Hz, 2H), 3.01 (s, 12H), 1.74 (p, J = 6.8 Hz, 2H), 1.51 (p, J = 6.9 Hz, 2H), 1.41 (p, J = 7.5 Hz, 2H), 1.31 (p, J = 7.8 Hz, 2H).

[0327] C NMR (151 MHz, CDCl) δ 170.0, 166.2, 156.5, 150.2, 141.3, 129.8, 128.4, 128.2, 126.4, 125.5, 121.9, 121.3, 115.2, 113.4, 71.3, 70.4, 70.1, 69.7, 45.1, 40.4, 40.1, 32.6, 29.5, 26.8, 25.5.

[0328] F decoupled C NMR (151 MHz, CDCl) δ 124.3, 124.1, 55.5.

[0329] F NMR (565 MHz, CDCl) δ -63.4 – -63.5 (m), -63.8 (q, J = 7.4 Hz).

[0330] HR-ESI-MS m / z for C H ONF Cl [M+H] : 770.2791. Calculated: 770.2790.

[0331] BF (Azetidine HaloTag Ligand)Attorney docket No.00012-089WO1

[0332] A vial with tert-butyl (2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)carbamate (HTL, 29.8 mg, 0.0920 mmol) was dissolved in CHCl (0.80 mL, 0.11 M of HTL) and TFA (0.60 mL, 0.11 M of HTL). After 30 min, toluene (1.0 mL) was then added, and the solvent was removed in vacuo. The resulting residue was azeotroped an additional two times with toluene (1.0 mL), and then stored in vacuo for 12 hours. In preparation for the amide coupling, the residue was dissolved in DMSO (1.90 mL, 0.048 M of HTL) in preparation for the amide coupling.

[0333] A vial with BF (12 mg, 0.020 mmol, 1.0 equiv.) and a stir bar was charged with DMSO (0.57 mL, 0.035 M of BF ) and i- PrNEt (0.06 mL, 0.34 mmol, 17 equiv.). Upon stirring the solution became clear and colorless. TSTU (7.8 mg, 0.026 mmol, 1.3 equiv.) was added. After 20 min, the solution of HTL (0.66 mL, 0.032 mmol, 1.6 equiv.) was added. After an hour, complete conversion was observed by LCMS. The reaction was quenched with MeCN / HO with 0.05% TFA. The solution was purified directly by preparative HPLC (10 to 100% MeCN / HO, 0.05% TFA). The solvent in the fractions containing BF were diluted with CHCl (HPLC grade) and quenched with sat. NaHCO. The resulting aqueous layer was extracted three times with CHCl. The combined organics were dried with NaSO, and the solvent was removed. The thin film was dissolved MeOH and filtered through a C-18 silica plug with MeOH to remove any grease. Removal of the solvent resulted in the isolation of BF (5.8 mg, 0.0073 mmol, 37% yield) as a transparent film. Unlike with the tetramethyl derivatives, decomposition of the azetidine dyes were observed when removing the MeCN / HO solvent directly after the prep-HPLC. To avoid this, the acid was neutralized and extracted the mixture with chloroform in order to isolate BF . HPLC grade chloroform was used in an attempt to minimize the amount of grease introduced into the system relative to typical bulk dichloromethane.

[0334] H NMR (600 MHz, CDCl) δ 8.03 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 6.99 (s, 2H), 6.69 (d, J = 8.7 Hz, 2H), 6.65 (t, J = 5.1 Hz, 1H), 6.43 (dd, J = 8.7, 2.0 Hz, 2H), 3.96 – 3.88 (m, 8H), 3.61 – 3.54 (m, 6H), 3.53 – 3.50 (m, 4H), 3.38 (t, J = 6.6 Hz, 2H), 2.40 (p, J = 7.2 Hz, 4H), 1.74 (p, J = 6.8 Hz,Attorney docket No.00012-089WO1 2H), 1.52 (p, J = 6.8 Hz, 2H), 1.42 (p, J = 7.5 Hz, 2H), 1.35 – 1.29 (m, 2H).

[0335] C NMR (151 MHz, CDCl) δ 170.0, 166.1, 156.6, 151.8, 141.4, 129.7, 128.3, 128.0, 126.2, 125.4, 121.9, 121.8, 113.9, 112.0, 86.0, 71.3, 70.4, 70.1, 69.7, 52.1, 51.0, 45.1, 40.1, 32.6, 29.5, 26.8, 25.5, 16.9.

[0336] F decoupled C NMR (151 MHz, CDCl) δ 124.1, 124.0, 55.4.

[0337] F NMR (565 MHz, CDCl) δ -63.6 (q, J = 7.7 Hz), -63.9 (q, J = 8.2 Hz).

[0338] HR-ESI-MS m / z for C H ONF Cl [M+H] : 794.2774. Calculated: 794.2795.

[0339] A number of embodiments have been described herein. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

Attorney docket No.00012-089WO1 WHAT IS CLAIMED IS:

1. A xanthene-based fluorophore comprising a structure of Formula I or Formula II:or a pharmaceutically acceptable salt or solvate thereof, wherein, X-X are independently selected from N or C, wherein when an X group is an N, then the R group is absent; and R-R and R-R are independently selected from H, D, optionally substituted functional group (FG), optionally substituted (C-C )alkyl, optionally substituted (C-C )heteroalkyl, optionally substituted (C-C )alkenyl, optionally substituted (C-C )heteroalkenyl, optionally substituted (C-C )alkynyl, optionally substituted (C- C )heteroalkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted mixed ring system, wherein one or more adjacent R groups can be linked together to form one or more substituted rings selected from the group comprising cycloalkyl, cycloalkenyl, heterocycle, aryl, and mixed ring system;Attorney docket No.00012-089WO1 R16 1F3C CF3R8or a X is selected from O, S, NH or CH; X is selected from O, S, NH, or CH; X is a halo; R and R are selected from an optionally substituted (C-C)alkyl, or can be joined together to form a heterocycle comprising 3, 4, or 5 carbon atoms; R and R are selected from an optionally substituted (C-C)alkyl, or can be joined together to form a heterocycle comprising 3, 4, or 5 carbonatom; R is ; R -R are each individuallyselected (C-C) alkyl, optionallysubstituted (C-C) alkenyl, optionally substituted (C-C) alkynyl, alkoxy, hydroxyl, ketone, ester, aldehyde, amino, azide, cyano, halo, thiol; v is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; y is an integer selected from 0, 1, 2, 3, 4, 5, and 6. Z is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

2. The xanthene-based fluorophore of claim 1, wherein the xanthene-based fluorophore has a structure of Formula I(a):Attorney docket No.00012-089WO1or a pharmaceutically acceptable salt or solvate thereof, wherein, R-R are independently selected from H, D, optionally substituted FG, optionally substituted (C-C)alkyl, optionally substituted (C-C)heteroalkyl, optionally substituted (C- C)alkenyl, optionally substituted (C-C)heteroalkenyl, optionally substituted (C-C)alkynyl, optionally substituted (C- C)heteroalkynyl, optionally substituted (C-C)cycloalkyl, optionally substituted (C-C)cycloalkenyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted mixed ring system, wherein one or more adjacent R groups can be linked together to form one or more substituted rings selected from the group comprising cycloalkyl, cycloalkenyl, heterocycle, aryl, and mixed ring system.

3. The xanthene-based fluorophore of claim 1, wherein the compound has a structure selected from Formula IIa, IIb, IIc and IId: ,Attorney docket No.00012-089WO14. A construct comprising a biomolecule tethered to a xanthene- based fluorophore of any one of the preceding claims.

5. The construct of claim 4, wherein the biomolecule is selected from an antigen, a receptor, a steroid, a protein, an antibody, a scFV, and a peptide.

6. The construct of claim 5, wherein the biomolecule is an antibody or a scFV.

7. An imaging or diagnostic assay comprising the xanthene-based fluorophore of any one of claims 1 to 3 or the construct of any one of claims 4 to 6.

8. The imaging or diagnostic assay of clam 7, wherein the imaging or diagnostic assay is an in vitro assay.

9. A method to image cells, comprising: contacting a cell with a xanthene-based fluorophore of any one of claim 1 to 3; illuminating the cells with light having a first wavelength; imaging the cells by detecting light having a second wavelength, wherein the first wavelength and second wavelength of light have different wavelengths, and wherein the light having the second wavelength is in the far red to near infrared region.Attorney docket No.00012-089WO1 10. The method of claim 9, where the cell is contacted to measure a biological process.

11. The method of claim 10, wherein the biological process is an enzymatic reaction.

12. A kit comprising: a plurality of aliquots which comprise a compound of any one of claims 1 to 3 in a buffered solution, or a concentrated solution.

13. A method to produce a compound of any one of claims 1 to 3 comprising methods and schemes as shown and described in the specification and figures.

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