Azabicyclic-substituted rhodamines

Abstract

Fluorophores incorporating a 2‑azabicyclo[2.1.1]hexane substituent are disclosed. This compact bicyclic motif increases fluorescence quantum yield, photostability, and effective brightness. Methods of synthesis and use in advanced microscopy applications are also provided.

Description

AZABICYCLIC-SUBSTITUTED RHODAMINESbyLuke D. Lavis, Jonathan B. Grimm, Sarah E. Plutkis, and Brian P. EnglishAssignee: Howard Hughes Medical InstituteAttorney Docket No.: 18074N / 25010WORELATED APPLICATIONS

[0001] This application claims priority from U. S. Provisional Application Serial No.63 / 755,353 filed February 7, 2025, the entire disclosure of which is incorporated herein by this reference.TECHNICAL FIELD

[0002] The presently disclosed subject matter relates to fluorescent compounds. In particular, the presently disclosed subject matter relates to chemical fluorophores with azabicyclic substituents as well as methods for making and using the same.INTRODUCTION

[0003] Fluorescence microscopy relies on molecules that can absorb one wavelength of light and emit another color. A key consideration in any fluorescence imaging experiment is the brightness of the fluorophore, which is defined as how many photons are emitted by a fluorophore at a given illumination intensity. Another important attribute is the photostability of the fluorophore, which is defined as how many total photons emitted by the fluorophore before irreversible photobleaching. These two properties depend on many factors including the fluorescence quantum efficiency or fluorescence quantum yield (Φf), the triplet lifetime (τT), andthe resistance to photochemical pathways that lead to fluorophore degradation. Design of new fluorophores with improved brightness and photostability is important for advanced microscopy experiments that require large photon outputs such as imaging individual molecules in living cells.

[0004] A particularly useful class of fluorophores are the rhodamines, exemplified by tetramethylrhodamine (TMR, 1; FIG. 1). This dye, first described in 1887, has been widely used in a variety of fluorescence imaging experiments. A limitation of TMR is its relatively low fluorescence quantum yield (Φf= 41%), which stems from an efficient nonradiative decay pathway called twisted internal charge transfer (TICT). This process starts with absorption, giving an excited state. Electron transfer from a nitrogen atom in one of the A( / V-dimethylamino groups to the xanthene system then occurs with a concomitant twist around the carbon-nitrogen bond (FIG. 1). This lower energy TICT intermediate relaxes back down to the ground state without emission of a photon, thereby competing with fluorescence and lowering Φf.

[0005] Tetramethylrhodamine (1) also exhibits relatively poor photostability. One photobleaching pathway involves intersystem crossing (ISC) to the first triplet excited state (Ti). This long-lived state can relax back down to the ground state through formation of singlet oxygen (1O2) from3O2. The singlet oxygen can oxidize the aniline nitrogen to the radical cation, which can undergo H-atom abstraction to form an iminium species. This can be hydrolyzed to give the dealkylated trimethylrhodamine (2). This dealkylation results in an undesirable hypsochromic (blue) shift in spectral properties and ultimately leads to complete photobleaching through a poorly understood pathway.

[0006] Both of these unwanted processes involve electron transfer from the aniline nitrogen; this occurs in the excited state in the TICT process or the ground state in the dealkylation / photobleaching process. The A'-methyl groups in the dye structure cause a relatively low ionization potential of the nitrogen atom, making the unwanted TICT and photobleaching processes more favorable. However, these methyl substituents in TMR are also responsible for the desirable spectral properties of the dye, giving an absorption maximum (λabs) of 548 nm and a fluorescence emission maximum (λem) of 570 nm. Thus, the N-methyl groups in TMR andanalogous fluorophores play opposing roles, decreasing brightness and photostability but improving spectral properties.

[0007] These issues demonstrate the need for new strategies to create rhodamine dyes with N-alkyl groups that preserve favorable spectral properties such as shifting λabsand λem, but improve fluorophore brightness and photostability through structural modification with unique substituents.SUMMARY

[0008] The presently disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0009] This Summary describes several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

[0010] The presently disclosed subject matter includes a class of rhodamine-based fluorophores incorporating a 2-azabicyclo[2. l.l]hexane substituent, a structural motif that reshapes both the electronic and conformational behavior of the dye scaffold.

[0011] The presently disclosed includes compounds having the following structure:

[0012] In embodiments of the compound, Q can be selected from the group consisting of C(alkyl), C(alkyl)2, NH, N(alkyl), O, S, SO2, Si(alkyl)2, P(O)(aryl), P(O)(alkyl), PO2H, PO2(alkyl), Se, and replaced with two H atoms. Ri, R2, R3, and R4 can be independently selected from the group consisting of H, D, halogen, alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CHO, C(O)(alkyl), C(O)(aryl), CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NH2, NH(alkyl), N(alkyl)2, NH(aryl), N(aryl)2, NHC(O)alkyl, NO2, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), SO3H, and PO3H2. X1can be independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl substituted alkenyl, alkynyl substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), N3, NH2, NH(alkyl), N(alkyl)2, OH, and O(alkyl). X2can be independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, substituted alkyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, and C(O)NH(aryl).

[0013] In embodiments of the compound, when Y is not present in the structure, Z is selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl, and substituted alkenyl, CH2OH, CO2, CO2H, CO2(alkyl), CO2(aryl), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, OH, O(alkyl), O(aryl), SO3−, SO3H, SO2NH(alkyl), SO2N(alkyl)2, SO2NH(aryl), SO2N(aryl)2, PO32−, PO3H−, and PO3H2. In embodiments of the compound, when Y is not present in the structure and when Z is a monoanionic substituent then the compound is optionally prepared as a net-neutral inner salt form. In embodiments of the compound, when Y is not present in the structure and when Z is not a monoanionic substituent then the compound is optionally prepared in a salt form with an appropriate counterion. In embodiments of the compound, when Y is present in the structure, Z is selected from the group consisting of C(O), SO2, PO2H, and CR2where each R is independently selected from the group consisting of H,alkyl, and substituted alkyl; and Y is selected from the group consisting of O, S, C(O), C(N2), NH, N(alkyl), N(aryl), N(SO2R) where R can be alkyl, substituted alkyl, and CN; and

[0014] R5, R6, R7, and R8are independently selected from the group consisting of H, D, halogen, alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NH2, NHC(O)(alkyl), NH(alkyl), N(alkyl)2, NH(aryl), N(aryl)2, NO2, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), SO3H, SO2NH2, SO2NH(alkyl), SO2N(alkyl)2, SO2NH(aryl), SO2N(aryl)2, and PO3H2.

[0015] The presently disclosed subject matter also includes methods of detecting target substances using the compounds disclosed herein. In these methods, a sample is contacted with a disclosed compound, which upon excitation emits detectable fluorescence indicative of the presence of a target analyte such as a protein, carbohydrate, nucleic acid, receptor, antigen, or other biomolecule. These methods are compatible with both in vitro analyses and live-cell or live-organism imaging.

[0016] The features and embodiments described herein establish a versatile andhigh-performance compound that advances beyond prior rhodamine technologies. By leveraging a substituent that simultaneously enhances electronic properties, supports extensive tunability, and maintains synthetic accessibility, the presently disclosed subject matter provides compounds capable of delivering substantially improved brightness, stability, and functional adaptability for improved biological imaging.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:

[0018] FIG. 1: The process of absorption and fluorescence emission versus TICT or ISC for tetramethylrhodamine (TMR, 1) and the process of dealkylation to yield trimethylrhodamine (2).

[0019] FIG. 2: Rhodamines 3–14 that show increased quantum yield relative to TMR (1) and improved photostability.

[0020] FIG.3: Spectral properties of fluorophores 1, 8, 11, 12, and 15-28.

[0021] FIG. 4: Spectral properties of fluorophores 30-34.

[0022] FIG. 5: Structure of 2,4-methanoproline (35).

[0023] FIG. 6: Synthesis of rhodamines 38, 40, 42, 44, 46, 47, 49, and 50.

[0024] FIG. 7: Synthesis of thio-rhodamines 55, and 57-59.

[0025] FIG. 8: Synthesis of carborhodamines 61 and 62.

[0026] FIG. 9: Synthesis of carborhodamines 67 and 68.

[0027] FIG. 10: Synthesis of Si-rhodamines 70 and 71.

[0028] FIG. 11: Synthesis of Si-rhodamines 75 and 76.

[0029] FIG. 12: Spectral properties of fluorophores 38, 40, 42, 44, 46, 47, 49, 50, 55, 57-59, 61, 62, 67, 68, 70, 71, and 75.

[0030] FIG. 13: Synthesis of fluorinated rhodamines 78 and 79.

[0031] FIG. 14: Spectral properties of fluorophores 79-81.

[0032] FIG. 15: Synthesis of rhodamine ligands 86, 88, 91, and 92.

[0033] FIG. 16: Synthesis of thio-rhodamine ligand 96.

[0034] FIG. 17: Synthesis of carborhodamine ligands 99, 100, 102, and 105-107.

[0035] FIG. 18: Synthesis of carborhodamine ligands 111 and 112.

[0036] FIG. 19: Synthesis of Si-rhodamine ligands 116, 117, 120, and 121.

[0037] FIG. 20: Synthesis of Si-rhodamine ligands 124 and 125.

[0038] FIG. 21: Synthesis of carboxy-substituted Si-rhodamine ligand 129.

[0039] FIG. 22: Synthesis of hydroxymethyl-containing Si-rhodamine 131 andHaloTag® ligand 135.

[0040] FIG. 23: Synthesis of biotin-containing multifunctional rhodamine 142.

[0041] FIG.24: Structures of HaloTag® and SNAP-Tag® ligands 86, 91, 99, 102, 105, 107, 116, 117, 120, 121, 135, and 143-150

[0042] FIG. 25: Airy scan images of cells expressing vimentin-HaloTag® fusion proteins and labeled with HaloTag® ligands 86 (left) or 91 (right).

[0043] FIG. 26: Loading curves for HaloTag® ligands 86, 91, 143, and 144.

[0044] FIG.27: Loading curves for HaloTag® ligands 99, 105, 116, 120, 145, and 146.

[0045] FIG. 28: Loading curves for SNAP-tag® ligands 102, 107, 117, 121, 147, and 148.

[0046] FIG. 29: Bleaching curves for HaloTag® ligands 86, 91, 143, and 144.

[0047] FIG.30: Bleaching curves for HaloTag® ligands 99, 105, 116, 120, 145, and 146.

[0048] FIG.31: Bleaching curves for SNAP-Tag® ligands 102, 107, 117, 121, 147, and 148.

[0049] FIG.32: Images of live U2OS cells expressing histone H2B-HaloTag® fusion proteins labeled with JF549-HaloTag® ligand (143) or JFSsei-HaloTag® ligand (86) before and after 300 s bleaching cycle; scale bars: 5 μm.

[0050] FIG.33: Bleaching curves from experiment shown in FIG. 32; error bars show ± SEM.

[0051] FIG.34: Comparison of fluorescence intensity per frame for ligands 86 and 143 in single-particle tracking experiments in live U2OS cells expressing histone H2B-HaloTag® fusion proteins; error bars show ± SEM.

[0052] FIG. 35: Comparison of track lengths (frames) for ligands 86 and 143 in single-particle tracking experiments in live U2OS cells expressing histone H2B–HaloTag® fusion proteins; error bars show ± SEM.

[0053] FIG.36: FCS curves showing a decreased triplet state population of HaloTag-bound JFS561 (86-HT) vs. HaloTag® bound JF549 (143-HT).

[0054] FIG.37: STED microscopy image (A) and image inset (B) of live U2OS cells expressing TOMM20-HaloTag® fusion proteins and labeled with JFS561-HaloTag® ligand (86);775-nm depletion; scale bars: 10 and 2 μm.

[0055] FIG.38: Bleaching curves from STED microscopy experiment in fixed cells expressing TOMM20-HaloTag® fusion proteins and labeled with JFS561-HaloTag® ligand (86), JF549-HaloTag® ligand (143), or BD566-HaloTag® ligand (149); 660-nm depletion.

[0056] FIG.39: Comparison of the shift in KL–Zupon fluorine incorporation with azetidine-containing dyes 18 and 25 and 2-azabicyclo[2.1.1]hexane-containing dyes 61 and 62.

[0057] FIG. 40: Absolute absorbance of Si-rhodamine ligands JF646 -HaloTag® ligand (145), JF635-HaloTag® ligand (150), JFS657-HaloTag® ligand (116), and JFS645-HaloTag® ligand (120) in the absence (-HT) or presence (+HT) of excess HaloTag® protein.

[0058] FIG. 41: Absolute fluorescence intensity of Si-rhodamine dyes ligands JF646-HaloTag® ligand (145), JF635-HaloTag® ligand (150), JFS657-HaloTag® ligand (116), and JFS645-HaloTag® ligand (120) incubated with purified Ca2+indicator HaloCaMP1a in the absence (open circles) and presence (closed circles) of excess Ca2+; error bars show ± SEM.

[0059] FIG. 42: Absolute fluorescence intensity of Si-rhodamine dyes ligands JF646-HaloTag® ligand (145), JF635-HaloTag® ligand (150), JFS657-HaloTag® ligand (116), and JFS645-HaloTag® ligand (120) incubated with purified Ca2+indicator HaloCaMP1b in the absence (open circles) and presence (closed circles) of excess Ca2+; error bars show ± SEM.

[0060] FIG. 43: Image of live U2OS cells expressing HaloTag-ensconsin and labeled with JFS645b-HaloTag® ligand (140) rendered as a SOFI image (A; from 1000 frames) or SMLM image (B; from 5000 frames); 100 Hz; scale bars: 10 μm.

[0061] FIG. 44: Schematic showing the JFS fluorophore system, highlighting the properties afforded by the different substituents on the molecule including the azabicyclo[2.1.1]hexane motif.DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0062] The details of one or more embodiments of the presently disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0063] Fluorescent dyes are important tools for biological research. A key property of fluorescence dyes is fluorescence quantum efficiency or fluorescence quantum yield (Φf) of the fluorophore, which dictates how many photons are emitted versus how many photons are absorbed. It was previously discovered that incorporating azabicyclic structures with two bridgehead carbon atoms next to the nitrogen atom could improve the Φfof rhodamine dyes. As disclosed herein, the 2-azabicyclo[2.1.1]hexane system, an azabicyclic structure with only one bridgehead carbon next to the nitrogen atom, can increase the Φfof rhodamine dyes. This small, symmetrical motif allows fine-tuning of fluorophore properties using 2-azabicyclo[2.1.1]hexane groups substituted at the 4-position with fluorine atoms. These new bright rhodamine dyes will enable new imaging experiments where large photon yields are required, such as single-molecule tracking in living cells.

[0064] The presently disclosed subject matter includes fluorophores containing 2-azabicyclo[2.1.1 ]hexane groups as well as methods for making and using the same.

[0065] In some embodiments the compound has the following formula:

[0066] In the compound, Q is selected from the group consisting of C(alkyl). C(alkyl)2, NH, N(alkyl), O, S, SO2, Si(alkyl)2, P(O)(aryl), P(O)(alkyl), PO2H, PO2(alkyl), Se, and replaced with two H atoms.

[0067] Ri, R2, R3, and R4 are independently selected from the group consisting of H, D, halogen, alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CHO, C(O)(alkyl), C(O)(aryl), CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NH2, NH(alkyl), N(alkyl)2, NH(aryl), N(aryl)2, NHC(O)alkyl, NO2, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), SO3H, and PO3H2.

[0068] X1is selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), N3, NH2, NH(alkyl), N(alkyl)2, OH, and O(alkyl).

[0069] X2is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, substituted alkyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, and C(O)NH(aryl).

[0070] When Y is not present in the structure, Z is selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, CH2OH, CO2−, CO2H, CO2(alkyl), CO2(aryl), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, OH, O(alkyl), O(aryl), SO3−, SO3H, SO2NH(alkyl), SO2N(alkyl)2, SO2NH(aryl), SO2N(aryl)2, PO32−, PO3H−, and PO3H2.

[0071] When Y is not present in the structure and when Z is a monoanionic substituent, then the compound can be prepared in a net-neutral, inner salt form. A monoanionic substituent refers to a group carrying a single negative charge, such as, for example, a carboxylate group (-CO2 ) or a sulfonate group (-SO3 ). A net-neutral, inner salt form refers to a zwitterionic species in which a positive charge and a negative charge are both present within the same molecule such that the overall formal charge of the compound is zero. In this context, the monoanionic substituent provides the internal negative charge, and the cationic portion of the dye structure provides the internal positive charge.

[0072] When Y is not present in the structure and when Z is not a monoanionic substituent, then the compound can be prepared in a net-neutral salt form with an appropriate counterion. A net-neutral salt form refers to a composition in which the compound carries a formal positive charge that is balanced by an external counterion, resulting in an overall neutral species; the counterion is not covalently bound but ionically associated. For example, if Y is not present in the structure and Z is a carboxylic acid (-CO2H) then the compound could be prepared as a salt with acetate ion (CH3CO2 ), trifluoroacetate ion (CF3CO2 ), or an another anionic counterion. Such appropriate counterions are known to and can be readily selected by one or ordinary skill in the art upon consideration of this document.

[0073] When Y is present in the structure, Z is selected from the group consisting of C(O), SO2, PO2H, or CR2 where each R is independently selected from the group consisting of H, alkyl, and substituted alkyl; and Y is selected from the group consisting of O, S, C(O), C(N2), NH, N(alkyl), N(aryl), N(SO2R) where R can be alkyl, substituted alkyl, and CN.

[0074] R5, R6, R7, and R8are independently selected from the group consisting of H, D, halogen, alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NH2, NHC(O)(alkyl), NH(alkyl), N(alkyl)2, NH(aryl), N(aryl)2, NO2, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), SO3H, SO2NH2, SO2NH(alkyl), SO2N(alkyl)2, SO2NH(aryl), SO2N(aryl)2, and PO3H2.

[0075] In some embodiments of the compound, R6is selected from the group consisting of alkyl, substituted alkyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl),CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, and NHC(O)(alkyl).In some embodiments of the compound, R6is selected from the group consisting of

[0076] In some embodiments of the compound, Q is selected from the group consisting of \ / \ / 9, P?'Sup PHP,PhV^X, X°X, X X, X" 'Z, -Y V', X X, X'PxX, X"'PX, and replaced with two H atoms. In some embodiments of the compound, Q is selected from the group consisting of X'°X, X"&X,X^X, and XS’X.

[0077] In some embodiments of the compound, X1is selected from the group consisting of o< H,, H,. f \fY H o 7 *2^ V IIC o sH, F, OCH3, N3, NHBoc, NH2, CO2CH3, CO2H, and0 0

[0078] In some embodiments of the compound, each R1, R2, R3, R4, R5, R7, and R8is independently selected from the group consisting of H and F.

[0079] In some embodiments, the compound has a structure chosen from the following,Owhere Q is selected from the group consisting of X X, X,, and X' X; X1is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl substituted alkenyl, alkynyl substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), N3, NH2, NH(alkyl), N(alkyl)2, OH, and O(alkyl); R5, R7, and R8are selected from H or F; R6is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NHC(O)(alkyl), NH(alkyl), and N(alkyl)2:

[0080] In some embodiments, the compound has a structure chosen from the following:

[0081] In some embodiments, the compound has a structure chosen from the following:

[0082] The presently disclosed subject matter also includes a method for detecting a target substance. In some embodiments, the method involves contacting a sample with a compound asdisclosed herein, and detecting an emission light from the compound, the emission light indicating the presence of the target substance. The detection can be performed, for example, using a microscope. The target substance can be, for example, a protein, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a substrate, a metabolite, an inhibitor, a drug, a nutrient, a growth factor, a lipoprotein, and a combination thereof.

[0083] In some embodiments of the method the contacting step and the detecting step are performed in a live cell.

[0084] In some embodiments of the method, the compound includes a first compound and a second compound; the first compound being selective for a first target substance and capable of emitting a first emission light; the second compound being selective for a second target substance and capable of emitting a second emission light, and the detecting step includes detecting the first emission light that indicates the presence of the first target substance and the second emission light that indicates the presence of the second target substance.

[0085] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0086] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0087] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0088] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0089] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (See, iubmb.qmul.ac.uk / ).

[0090] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are described herein.

[0091] The present application can “comprise” (open ended) or “consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0092] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0093] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0094] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0095] As used herein, ranges can be expressed as from “about” one particular value, and / or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0096] The term “absorption wavelength” as used herein refers to the wavelength of light capable of being absorbed by a compound in order to excite the compound to emit a light. The light emitted from a compound that has been excited with an absorption light will have an “emission wavelength.”

[0097] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compounds disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds.

[0098] As used herein, the term “protein” means any polymer comprising any of the 20 proteogenic amino acids, regardless of its size. Although “polypeptide” is often used in reference to relatively large proteins, and “peptide” is often used in reference to small proteins, usage of these terms in the art overlaps and varies. The term “protein” as used herein refers to peptides, polypeptides and proteins, unless otherwise noted.

[0099] The term “selectively bind” is used herein to refer to the property of an atom, moiety, and / or molecule preferentially being drawn to or binding a particular compound. In some instances, the atom, moiety, and / or molecule selectively binds to a particular site on a compound, such as an active site on a protein molecule.

[0100] The term “detect” is used herein to refer to the act of viewing, imagining, indicating the presence of, measuring, and the like a target substance based on the light emitted from the present compounds. More specifically, in some instances the present compounds can bebound to a target substance, and, upon being exposed to an absorption light, will emit an emission light. The presence of an emission light can indicate the presence of a target substance, whereas the quantification of the light intensity can be used to measure the concentration of a target substance.

[0101] A “self-labeling protein” is a protein that can covalently attach to a specific chemical substrate, referred to as a ligand or self-labeling protein ligand. A “self-labeling protein ligand” is a chemical compound that specifically binds to a self-labeling protein, forming a stable covalent bond. These ligands can be linked to chemicals or functional groups. Together, the selflabeling protein and the self-labeling protein ligand provide a system for facilitating the specific attachment of a compound or functional group to a protein within a living cell or in vitro. The term “self-labeling” indicates that the protein is capable of catalyzing the attachment to the compound without the need for additional enzymes or co-factors. A self-labeling protein / ligand system includes the self-labeling protein (SLP) (sometimes referred to in the art as a self-labeling protein tag) and the SLP ligand. The SLP and the SLP ligand form a specific bond. In this regard, when the SLP ligand is attached to a compound or functional group, the SLP forms a bond with the compound or functional group via the SLP ligand. This bond formation ensures a stable and irreversible attachment of the compound or functional group to the protein, allowing for various applications such as visualization, purification, and interaction studies.

[0102] Examples of SLPs and their ligands will be known to those of ordinary skill in the art. One such example is HaloTag® which is a modified bacterial enzyme that binds covalently to synthetic ligands containing a chloroalkane (CLA) moiety, including the following structures:Additional details about HaloTag® Protein and Ligands can be found in the Technical Manual entitled “HaloTag® Technology: Aqueous-Soluble Ligands” published by Promega Corporation (Madison, WI) and available at: www.promega.com / resources / protocols / technical-manuals / 500 / halotag-technology-aqueous-soluble-ligands-protocol / .

[0103] Another example is SNAP -tag®, which is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT), and binds covalently to derivatives of its substrate O6-benzylguanine (BG). Another example is CLIP-tag®, which is similar to SNAP-tag®, but it binds to O2-benzylcytosine (BC) derivatives, allowing for orthogonal labeling in combination with SNAP -tag®. Another example is TMP-tag®, which is a self-labeling protein that binds to trimethoprim (TMP) derivatives. Another example is a tetracysteine tag, which is a peptide sequence that binds to biarsenical dyes like fluorescein arsenical helix binder (FlAsH) and Resorufin Arsenical Helix binder (ReAsH), which are useful for fluorescent labeling.Another example is βLac-tag, which is a self-labeling protein derived from β-lactamase, which is often used with a ligand that is a [3-lactam antibiotic, such as cephalosporin or penicillin derivatives.

[0104] The term “target substance” refers to a substance that is selectively bound directly by the presently disclosed compounds and / or indirectly by a molecule that is bound to the present compound. A target substance can include, but is not limited to, a protein, carbohydrates, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, inhibitor, drug, nutrient, growth factor, and the like. In some embodiments the target substance refers to an entire molecule, and in other embodiments the target substances refer to a site on a molecule, such as a binding site on a particular protein.

[0105] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In one aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Absent a specific indication to the contrary, this disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds.

[0106] Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Unless stated otherwise, all chemical groups described herein include both unsubstituted and substituted varieties.

[0107] Where substituent groups are specified by their conventional chemical formula written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left. For instance, — CH2O — also encompasses recite — OCH2 —.

[0108] It should be understood that the bond types and locations in the chemical structures provided herein may adapt depending on the substituents in the compound, even if not specifically recited. For instance, — X — where X can be either C or N can refer to, respectively, — CH2 — or — NH —, where the lone pair of electrons on N is not illustrated. Thus, even if not specifically illustrated, the chemical compounds described herein include any hydrogen atoms, lone pair of electrons, and the like necessary for completing a chemical structure.

[0109] The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, / / -propyl, isopropyl, / / -butyl, isobutyl,.s-butyl, / -butyl, / / -pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also refer to both substituted or unsubstituted alkyls. For example, the alkyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms.

[0110] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” specifically refers to an alkyl group that is substitutedwith one or more halide, e.g., fluorine, chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.

[0111] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term. The term “alkyl” is inclusive of “cycloalkyl.”

[0112] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0113] In this regard, the term “heterocycle,” as used herein refers to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2,3-oxadiazole, 1,2,5-oxadi azole and 1, 3, 4-oxadi azole, thiadi azole, including, 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1.3.4-thiadiazole, triazole, including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including 1, 2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, triazine, including 1.2.4-triazine and 1,3,5-triazine, tetrazine, including 1,2,4,5-tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like.

[0114] The terms “alkoxy” and “alkoxy!” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as — OA1where A1is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as — OA1— OA2or — OA1— (OA2)a— OA3, where “a” is an integer of from 1 to 200 and A1, A2, and A3are alkyl and / or cycloalkyl groups.

[0115] The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. The term is inclusive of linear and ring-forming (i.e., cycloakenyl) groups. Asymmetric structures such as (A1A2)C=C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0116] The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxy benzene, and the like. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0117] The term “ring” as used herein refers to a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. A ring includes fused ring moieties, referred to as a fused ring system wherein a ring may be fused to one or more rings selected from a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl in any combination. The number of atoms in a ring is typically defined by the number of members in the ring. For example, a “5- to 8-membered ring” means there are 5 to 8 atoms in the encircling arrangement. A ring can optionally include a heteroatom. The term “ring” further includes a ring system comprising more than one “ring”, wherein each “ring” is independently defined as above.

[0118] Some of the unsaturated structures described herein, such as ring structures including cycloalkyl and aryl, are illustrated with dashed bonds to signify the potential existence of a resonance structure. Structures having dashed bonds are intended to reflect every possible configuration of the structure, but does not necessarily imply that all possible structures are in existence. It should be understood that the types of bonds (e.g., single bond, double bond) in such structures will vary depending on the atoms in the structure as well as whether the structures are substituted with one or more additional atoms or moieties.

[0119] The term “aldehyde” as used herein is represented by a formula — C(O)H.Throughout this specification “C(O)” is a shorthand notation for a carbonyl group, i.e., C=O.

[0120] The terms “amine” or “amino” as used herein are represented by a formula NA1A2A3, where A1, A2, and A3can be, independently, hydrogen or optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. In specific embodiments amine refers to any of NH2, NH(alkyl), NH(aryl), N(alkyl)2, and N(aryl)2.

[0121] The term “carboxylic acid” as used herein is represented by a formula CO2H.

[0122] The term “imide” as used herein is represented by a formula A1C(O)NC(O)A2where A1and A2can be, independently, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein, or A1and A2, together with the atoms they are connected to, form a unsubstituted or substituted 4, 5, 6, 7, or 8-membered ring.

[0123] The term “halide” or “halogen” refers to at least one of the halogens selected from fluorine, chlorine, bromine, and iodine.

[0124] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0125] The presently disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.EXAMPLES

[0126] Example 1; Fluorophore-optimization. Several strategies have been utilized to improve the brightness and photostability of fluorophores by modifying the substitution patterns on the nitrogen atoms (FIG. 2). These include rigidifying the system through fused rings, such as in the julolidine-containing rhodamine 101 (3), the tetrahydroquinoline-containing Q-rhodamine (4) or ATTO 550 (5), and the dihydroquinoline-containing ATTO 590 (6). In this strategy, the fused ring systems prevent twisting of the nitrogen substituent, thereby minimizing TICT and increasing < Pf. The cyclic ring systems also raise the energy of the iminium intermediate that results from H-atom abstraction, thereby increasing photostability. Finally, the -dimethyl groups adjacent to the nitrogen and ATTO 550 (5) and ATTO 590 (6) eliminates the presence ofthe alpha-hydrogen, which prevents the formation of the iminium species that leads to dealkylation and photobleaching.

[0127] Since both TICT and dealkylation start with electron transfer from the aniline nitrogen, a second strategy to improve fluorophore properties is to increase the ionization potential of this group. This suppresses TICT by making electron transfer from the nitrogen atom to the xanthene system of the dye less favorable. This also decreases oxidation of the nitrogen by1C>2. The, V, A'-dimethylamino group in TMR (1) has a relatively low ionization potential leading to efficient TICT and a lower quantum yield. Replacing this group with azacyclic groups such as pyrrole (7), azetidine (8), A, A-dimethylpiperazine (9), and 1,1 -dioxothiomorpholine (10), however, improves fluorescence quantum yield relative to TMR (1) to varying degrees (FIG.2).

[0128] A third strategy to improve dye brightness and photostability is to deuterate the N-alkyl groups; examples include such as the deuterated azetidine-containing rhodamine JFX549 (11) and the deuterated pyrrolidine-containing rhodamine JFX554 (12; FIG.2). Deuteration increases the ionization potential through a secondary isotope effect; this destabilizes the radical cation that results after electron transfer thereby raising the energy of the TICT intermediate and increasing fluorescence quantum yield (f / ’i). The carbon-deuterium bond is stronger than a carbon-hydrogen bond, leading to slower dealkylation and improving photostability.

[0129] A fourth method to improve fluorophore properties is to use amine-containing bicyclic systems where the bridgehead carbons are alpha to the nitrogen substituent. Examples include 7-azabicyclo[2.2.1]heptane (12), and 3-oxa-8-azabicyclo[3.2.1]octane (13; FIG. 2). The higher ionization potentials of these substituents cause an increase in ^ relative to TMR. These dyes also show improved photostability due to the higher energy of the iminium intermediate, which slows dealkylation and subsequent photobleaching.

[0130] The azetidine-containing Janelia Fluor 549 (JF549; 8) is a unique member of this collection of improved fluorophores 3-14 (FIG. 2) because it exhibits a high quantum yield ( i = 0.88), and because the spectral and chemical properties of this scaffold can be tuned over a broad spectral range by straightforward structural modifications. The spectral properties of rhodamine dyes can be coarsely tuned by replacing the xanthene oxygen with a gem-dimethylcarbon moiety to yield tetramethylcarborhodamine (CTMR, 15, FIG.3; ~60 nm red-shift) or em-dimethylsilicon moiety to yield tetramethyl-Si-rhodamine (SiTMR, 16; —100 nm red-shift). These strategies were also applied to the azetidine-containing dyes, yielding the red-shifted analogs of JF549 (8): Thio-rhodamine JF570 (17), carborhodamine JFeos (18) and Si-rhodamine JF646 (19). Dyes 17 and 18 show higher Rvalues compared to the corresponding tetramethylrhodamine analogs 15 and 16. This strategy has also been used to shift the wavelengths of the deuterated azetidine-containing 11 to yield JFXeos (20) and JFX646 (21) and the deuterated pyrrolidine-containing dye 12 to give JFXsi2 (22) and JFXeso (23; FIG.3)

[0131] Use of substituted azetidines can enable further fine-tuning of spectral properties. This is due to the small size and symmetry of the azetidine functional group, which allows the substituents on the azetidine to exert a substantial effect on fluorophore properties without introducing any unwanted stereochemistry. For example, incorporating 3 -fluoroazetidine into the rhodamine structure can shift the absorption and fluorescence emission wavelengths of the dye ~11-13 nanometers into the blue, yielding JF536 (24). This strategy also works for the carbo- and Si-rhodamine systems, giving JF595 (25) and JF635 (26). Use of 3,3-difluoroazetidine elicits a larger, ~25 nm blue shift, yielding the rhodamine JF525 (27), carborhodamine JFsss (28), and Si-rhodamine JF614 (29). Introducing fluorine atoms into the azetidine also modestly improves quantum yield (FIG.3). The spectral properties of rhodamine dyes can be tuned in the opposite direction by installing fluorine atoms on the pendant phenyl ring system (FIG.4). This elicits a ~23 nm bathochromic shift in z;,bs and zcmrelative to nonfluorinated dyes, with a modest decrease in < Pf. This can be observed in the azetidine-containing JF593 (30), JF632 (31), and JF669 (32) and the deuterated pyrrolidine-containing JFX612 (33) and JFX673 (34).

[0132] In addition to shifting absorption and fluorescence emission wavelengths to shorter values, incorporating azetidines with electron-withdrawing fluorine substituents can also modify chemical properties including the equilibrium between the colorless, nonfluorescent lactone form and the colored, fluorescent zwitterionic form. This equilibrium, with the equilibrium constant XL-Z, is a key determinant in the performance of dyes in biological systems, the fluorogenicity of these dyes upon binding a biomolecular target, which is a useful property shown by ligands based on Si-rhodamines such as JF646 (19) and JF635 (26). Incorporating fluorine substituents on the pendant phenyl ring has the opposite effect, increasing XL Z, leading to higher absorptivity, especially for the Si-rhodamine dyes. For example, JF646 (19) has anextinction coefficient (E) of 5,600 M 'em1whereas JF669 (32) shows s = 112,000 M 'em1(FIG.3, FIG. 4)

[0133] Example 2; Introduction of azabicyclo[2.1.1]hexane. The strategy of fine-tuning spectral and chemical properties using substituted azetidine substituents is difficult to apply to other classes of improved rhodamines. Installing substituents beyond methyl groups in the fused ring-containing rhodamines such as 3-6 (FIG. 2) is synthetically challenging. The deuterated analogs 11, 12, and 20-23 (FIG.2, FIG. 3) require deuterated building blocks which are difficult to further modify with substituents. The dyes containing bicyclic systems, such as 13 and 14 (FIG.2), are also difficult to fine-tune since incorporating substituents into these structures is synthetically challenging and introduces undesirable chiral centers into the molecule. In contrast, the azetidine building blocks used in many Janelia Fluor dyes, can be obtained with many different chemical substitutions including fluorine atoms at the 3-position of the azetidine, resulting in a symmetrical substituent that does not complicate stereochemistry (e.g, 24-29, FIG.3).[00134J Example 3: Oxygen-containing rhodamines. To date, rhodamine dyes containing azabicyclic structures have utilized structures where both bridgehead carbons are directly attached to the nitrogen atom (e.g., 12 and 13, FIG. 2). As disclosed herein, studies were conducted to determine whether a smaller azabicyclic structure with only one bridgehead carbon would preserve brightness and photostability but allow fine-tuning of fluorophore properties through structural modification without introducing unwanted stereochemistry. The 2-azabicyclo[2.1.1]hexane system was considered, which is found in 2,4-methanoproline (35; FIG.5), a natural product from the legume Ateleia herbert-smithii Pittier where it serves as an antifeedant in the plant’s seeds. This bicyclic structure is compact, has a plane of symmetry along the two bridgehead carbon atoms, and has been widely used in medicinal chemistry, resulting in availability of various commercial derivatives. These properties made the 2-azabicyclo[2.1.1]hexane system a promising functionality to incorporate into rhodamine dye structures.

[0135] A comprehensive collection of dyes containing 2-azabicyclo[2.1.1]hexane motifs were prepared starting with the classic oxygen-containing rhodamines (FIG. 6). Fluoresceinditrifl ate (36) reacted with unsubstituted 2-azabicyclo[2.1.1 ]hexane hydrochloride (37) to yield rhodamine 38. Reaction of 36 with 4-fluoro-2-azabicyclo[2.1. l]hexane hydrochloride (39) gave rhodamine 40. Compound 36 was coupled to 4-methoxy-2-azabicyclo[2.1.1]hexane hydrochloride (41) to yield rhodamine 42 and 4-azido-2-azabicyclo[2.1.1]hexane hydrochloride (43) to yield rhodamine 44. Pd-catalyzed coupling of 36 and Boc-protected 4-amino-2-azabicyclo[2.1.1]hexane (45) gave rhodamine 46, which could be deprotected with acid to yield diamino-rhodamine 47. Ditriflate 36 and methyl ester 48 combined to give rhodamine diester 49, which was hydrolyzed to diacid 50.

[0136] Example 4: Thio-rhodamines. Thio-rhodamines were then prepared using condensation with lactol intermediates (FIG. 7). Dibromide 51 was coupled with either 2-azabicyclo[2.1.1]hexane hydrochloride (37) or 4-fhioro-2-azabicyclo[2.1.1]hexane hydrochloride (39) to give thioether derivatives 52 and 53. Compound 52 reacted with lactol 54 to yield thiorhodamine 55 or fluorinated lactol 56 to give fluorinated thio-rhodamine 57. Likewise, fluorinated 53 reacted with either 54 or 56 to yield rhodamines 58 and 59, respectively.[00137J Example 5: Carborhodamines. Carborhodamines were then prepared using different synthetic strategies. Ditriflate 60 was coupled to bicyclic compounds 37 or 39 to give dyes 61 and 62 (FIG. 8). Anthrone 63 underwent Pd-catalyzed cross-coupling with 37 or 39 to give 64 and 65, which were reacted with deprotonated 3,4,5,6-tetrafluorobenzoic acid (66) to yield fluorinated carborhodamines 67 and 68 (FIG. 9).

[0138] Example 6; Si-rhodamines. A series of Si-rhodamines was then synthesized. Ditriflate 69 underwent Pd-catalyzed cross-coupling with bicyclic amines 37 or 39 to yield dyes 70 and 71, respectively (FIG. 10). Dibromide 72 underwent cross-coupling with 37 or 39 to yield the silanes 73 and 74. Oxidative condensation of these intermediates with tetrafluorol actol 56 in 2,2,3,3,4,4,4-heptafluoro-l-butanol afforded fluorinated Si-rhodamines 75 and 76 (FIG. 11)

[0139] Example 7: Characterization. Evaluation of the spectral properties of these new dyes revealed that incorporation of this bicyclic structure improved the quantum yield relative to TMR (1; f = 0.41; FIG. 1, FIG. 3). The quantum yields of the azabicyclic-containing rhodamines (38, 40, 42, 44, 46, 47, 49, and 50) ranged from 0.75 to 0.84 (FIG. 12). Likewise,the carborhodamines 61 and 62 exhibit devalues of 0.65 and 0.72 (FTG. 12), higher than the carbon-containing tetramethylrhodamine analog 15 ( f = 0.52; FIG. 3). The Si-rhodamine 70 shows a quantum yield value of 0.47 (FIG. 12), higher than SiTMR ( f = 0.41; FIG. 3). The quantum yield of Si-rhodamine 71 was not measured due to low absorptivity. The quantum yield values of the dyes containing the 2-azabicyclo[2.1. l]hexane motif were similar to deuterated pyrrolidine analogs 12 (d>f= 0.80), 22 (< Pf= 0.70), and 23 ( >f= 0.53; FIG.3, FIG. 12). Dyes with fluorinated pendant rings typically show lower quantum yields than their nonfluorinated counterparts and this trend was pronounced in dyes with the 2-azabicyclo[2.1.1]hexane substituent. Carborhodamine 67 ( f = 0.50) and Si-rhodamines 75 ( f = 0.32) were lower than JFX637 (33; < Z>f= 0.70) and JFX673 (34; d>f= 0.42; FIG.3, FIG. 4, FIG. 12).

[0140] Introduction of this bicyclic motif also increasedabs andby ^- 1 12 nm relative to the tetramethylrhodamine dyes 1,15-16 and the Janelia Fluor® dyes 1,17-19. Similar to using 3 -fluoroazetidine as in dyes 24-26 (FIG. 3), introduction of the 4-fluoro-2-azabicyclo[2.1.1]hexane group in compounds 40, 62, 68, 71, and 76 elicits a blue-shift in spectra compared to the unsubstituted azabicyclo[2.1.1]hexane-containing compounds (38, 61, 67, 70, and 75). This results in a precise, 1-nm shorter z;lbs in compounds 40, 62, and 71 compared to the corresponding JF dyes 8, 18-19 with similar 2emvalues. Use of 4-fluoro-2-azabicyclo[2.1.1]hexane also modestly improves quantum yields compared the nonfluorinated analogs as seen in compounds 40, 62, 68, and 76 (FIG. 12).

[0141] The thio-rhodamine 55 shows the same 12-nm red-shift in2abs and corresponding red-shift 2em compared to azetidine-containing JF570 (17; FIG.3). The thio-rhodamines 55 and 57 show quantum yield values of 0.61 and 0.63, respectively (FIG. 12), which is similar to JF570 (17; f = 0.63; FIG.3). Fluorination of the pendant ring to give 58 and 59 causes the expected decrease in f = 0.54-0.55.

[0142] Dyes with fluorine substituents at the 2' and 7' positions of the rhodamine were also prepared starting from diiodofluoran 77. Pd-catalyzed cross-coupling with 37 or 39 gave 78 and 79 (FIG. 13). The spectral properties of these dyes were compared to 2 ',7 '-difluoro- JF549 (z.e., JF552; 80; < Z>f= 0.83) and 2',7'-difluoro-JFX554(81; < Z>f= 0.37; FIG. 14). Compound 78 containing the azabicyclo[2.1. l]hexane structure showed a low < Pf = 0.27 that was partiallyrescued by fluorination of the bicyclic structure in 79 (< J>f = 0.59). This is consistent with the TICT mechanism. Incorporation of fluorine substituents on the xanthene ring structure makes it a better electron acceptor. This promotes TICT leading to a decrease in quantum yield. The azetidine substituent is a poor electron donor, so the decrease in quantum yield is modest; 74 = 0.88 for JF549 (8) vs. I> = 0.83 for JF552 (80). The decrease observed for 81 and 78 is more substantial since pyrrolidine and azabicyclo[2.1.1 Jhexane are better electron donors than azetidine. The 4-fluoro-azabicyclo[2.1.1 Jhexane motif is a poorer electron donor due to the electronegative fluorine atom, resulting in a substantial increase in / 4.

[0143] These compounds were given the name “Janelia Fluor S” or “JFS” dyes with designations that reflect the zabs of the free dyes in aqueous solution; for example, dye 38 was called “JFS561”

[0144] Example 8: Self-labeling protein ligands. HaloTag® and SNAP -tag® ligand derivatives of these JFS dyes were then synthesized and evaluated (FIG. 15). The / -butyl ester of 6-carboxyfluorescein ditriflate (82) underwent Pd-catalyzed cross-coupling with 37 to afford the rhodamine dye ester 83. Deprotection with TFA yielded acid 84, which could be coupled with either HaloTag® amine 85 to yield the JFS561 -HaloTag® ligand (86) or chloropyrimidine (cp) SNAP-Tag® ligand 87 to give and JFSsei-cp SNAP-Tag® ligand (86). Likewise, cross-coupling of 82 and fluorine-containing 39 gave the rhodamine dye ester 89. Deprotection with TFA yielded 90 and coupling with 85 or 87 gave the JFS548-HaloTag® ligand (91) and JFS548-cpSNAP-Tag® ligand (92).

[0145] A thio-rhodamine derivative was synthesized in a similar manner. Thioether 52 underwent condensation with carboxylactol 93 to give acid 94. Coupling with HaloTag® amine 85 afforded JFS582-HaloTag® ligand (95; FIG. 16).

[0146] The synthesis of the HaloTag® and SNAP-Tag® ligand derivatives of carborhodamine dyes started with the / -butyl ester of 6-carboxycarbofluorescein ditriflate (96; FIG. 17). Pd-catalyzed cross-coupling with 37 afforded the rhodamine dye ester 97; deprotection with TFA yielded acid 98. This could be coupled with HaloTag® amine 85 to yield the JFS619-HaloTag® ligand (99), the chloropyrimidine (cp) SNAP-Tag® ligand 87 to give JFSei9-cpSNAP-Tag® ligand (100), or the SNAP-Tag® ligand 101 to afford JFSe 19- SNAP-Tag® ligand(102). Likewise, cross-coupling of 96 and fluorine-containing 39 gave the rhodamine dye ester 103. Deprotection with TFA yielded 104 and coupling with 85, 87, or 101 gave JFSei9-HaloTag® ligand (105), JFS6i9-cp SNAP-Tag® ligand (106), and JFS6i9-SNAP-Tag® ligand (107). Carborhodamine-based ligands with fluorinated pendant phenyl rings started with JFS643 (67) and JFS632 (68). Reaction with MAC reagent 108 gave derivatives 109 and 110. Treatment with acid, and then addition of 85 and excess base gave JFS643-HaloTag® ligand (111) and JFS632-HaloTag® ligand (112; FIG. 18).

[0147] Synthesis of HaloTag® and SNAP-Tag® ligand derivatives of Si-rhodamine dyes began with 6-carboxy-Si-fluorescein ditriflate / -butyl ester (113; FIG. 19). Pd-catalyzed crosscoupling with 37 afforded ester 114 and deprotection with TFA yielded acid 115. Amide coupling with HaloTag® amine 85 SNAP-Tag® amine 101 afforded JFSesr-HaloTag® ligand (116) and JFSei9-SNAP-Tag® ligand (117). Likewise, cross-coupling of 113 and fluorine-containing 39 gave ester 118. Deprotection with TFA yielded 119 and coupling with 85 or 101 gave J F SMS -HaloTag® ligand (120) and JFS645-SNAP-Tag® ligand (121). Si-rhodamine-based ligands with fluorinated pendant phenyl rings started with JFS678 (75) and JFS670 (76). Reaction with MAC reagent 108 gave 122 and 123. Treatment with acid followed by addition of 85 and 7V, A-diisopropylethylamine (DIEA) gave JFSe78-HaloTag® ligand (124) and JFS67o-HaloTag® ligand (125; FIG. 20).

[0148] Advanced derivatives of the JFS dyes were prepared starting with a JFSes? derivative with additional carboxyl groups to decrease membrane permeability. The 6-carboxy-Si -fluorescein ditriflate / -butyl ester (113) was coupled with methyl ester 48 to yield Si-rhodamine 126. Selective deprotection of the / -butyl ester under acidic conditions gave 127, which was coupled with HaloTag® amine 85 to give 128. Saponification of the methyl esters afforded polar ligand 129 (FIG. 21).

[0149] To investigate replacement of the or / Ao-carboxyl group on the pendant phenyl ring with a different functionality, the spirocyclic ether 130 was coupled with 39 to give compound 131. To prepare a HaloTag® ligand derivative, the / -butyl ester 132 underwent Pd-catalyzed cross-coupling with 39 to give 133. Deprotection with trifluoroacetic acid (TFA) gave 134 and coupling with HaloTag® amine 85 produced HaloTag® ligand 135 (FIG.22).

[0150] To explore adding functionality on the azabicyclo[2.1.1 ]hexane portion of the molecule, a biotin derivative was prepared (FIG. 23). The / -butyl ester of 6-carboxyfluorescein ditriflate (82) underwent Pd-catalyzed cross-coupling with a single equivalent of 37 to afford the intermediate 136 followed by coupling with an equivalent of the methyl ester-containing amine 48 to give rhodamine ester 137. The asymmetric dye was selectively deprotected with TFA to give acid 138. Amide coupling with 85 gave 139; hydrolysis of the methyl ester gave acid 140, which reacted with biotin-amine 141 containing a short polyethylene glycol (PEG) linker to give multifunctional HaloTag® ligand 142.

[0151] Example 9: Biological evaluation and imaging. A series of HaloTag® and SNAP-Tag® ligands were evaluated in vitro and in cellular experiments. These include ligands based on known dyes: JFs49-HaloTag® ligand (143), JFXs54-HaloTag® ligand (144), JF646-HaloTag® ligand (145), JFX65o-HaloTag® ligand (146), JF646-SNAP-Tag® ligand (147), JFXeso-SNAP-Tag® ligand (148), BDsee-HaloTag® ligand (149), and JFess-HaloTag® ligand (150; FIG. 24). These also include JFS derivatives: JF S 561 -HaloTag® ligand (86), JFSs48-HaloTag® ligand (91), JFSei9-HaloTag® ligand (99), JFSeov-HaloTag® ligand (105), JFS657-HaloTag® ligand (116), JFS645-HaloTag® ligand (120), JFS6i9-SNAP-Tag® ligand (102), JFS607-SNAP-Tag® ligand (107), JFS657- SNAP-Tag® ligand (117), and JFS645-SNAP-Tag® ligand (121; FIG.24).

[0152] JFSsei-HaloTag® ligand (86) and JFS548-HaloTag® ligand (91) were evaluated in live-cell experiments in cells expressing HaloTag® fused to the intermediate fdament protein vimentin. Upon incubation, both dye ligands entered live cells with JFSsei -HaloTag® ligand (86) showing higher effective brightness than JFSs48-HaloTag® ligand (91), evidenced by the lower laser power needed to achieve comparable brightness (FIG. 25).

[0153] The rate of loading was evaluated in live U2OS cells expressing HaloTag® protein or SNAP-Tag® protein fused to histone H2B. JF S 61 -HaloTag® ligand (86), JFS548-HaloTag® ligand (91), JF549-HaloTag® ligand (143), and JFX554-HaloTag® ligand (144) showed comparable loading rates (FIG. 26). Likewise, JF646 -HaloTag® ligand (145), JFXeso-HaloTag® ligand (146), JFSei9-HaloTag® ligand (99), JFSeo7-HaloTag® ligand (105), JFS657-HaloTag® ligand (116), and JFS645 -HaloTag® ligand (120) exhibited similar labeling kinetics(FIG. 27). The SNAP-Tag® ligands showed different behavior with JFXsso-SNAP-Tag® ligand (148), JFS6i9-SNAP-Tag® ligand (102), JFS607- SNAP-Tag® ligand (107), and JFS657-SNAP-Tag® ligand (117) showing efficient labeling kinetics but JFS645-SNAP-Tag® ligand (121) and JF646-SNAP-Tag® ligand (147) labeling substantially slower under these conditions (FIG. 28).

[0154] Photostability in a cellular context was then evaluated by labeling fixed cells expressing HaloTag® protein or SNAP-Tag® protein fused to histone H2B and then subjecting these cells to repeated high illumination intensity bleach cycles. Under these conditions JFS548-HaloTag® ligand (91) showed slower photobleaching compared to JFSsei-HaloTag® ligand (86), JF549-HaloTag® ligand (143), and JFX554-HaloTag® ligand (144; FIG.30). JFS657-HaloTag® ligand (116) showed the slowest photobleaching compared to JF646-HaloTag® ligand (145), JFXeso-HaloTag® ligand (146), JFSei9-HaloTag® ligand (99), JFSeov-HaloTag® ligand (105), and JFS645-HaloTag® ligand (120; FIG. 30). For the SNAP-Tag® ligands, JFS6i9-SNAP-Tag® ligand (102) showed the slowest bleaching compared to JF646-SNAP-Tag® ligand (147), JFX65o-SNAP-Tag® ligand (148), JFS607-SNAP-Tag® ligand (107), JFS657- SNAP-Tag® ligand (117), and JFS645-SNAP-Tag® ligand (121; FIG.31). Photostability was also assessed in live cells expressing Hal oTag-hi stone H2B. JFs49-HaloTag® ligand (143) bleached faster than JFSsei-HaloTag® ligand (86) under equivalent imaging conditions (FIG.32, FIG.33)

[0155] FFSsei-HaloTag® ligand (86) was then evaluated in single-particle tracking (SPT) experiments, comparing to the structural and spectral analog JFs49-HaloTag® ligand (143). The JFS561 ligand 86 showed a ~3-fold improvement in photons / s under equivalent imaging conditions in cells expressing Hal oTag-hi stone H2B fusions indicating higher brightness (FIG.34). JFS561 -HaloTag® ligand (86) also shows a ~2-fold longer track length demonstrating improved photostability (FIG. 35). These data demonstrate the higher brightness and photostability of the JFS561 ligand 86 compared to known JF549 derivative 143.

[0156] The higher effective brightness observed for JFSsei-HaloTag® ligand (86) in the SPT experiments (FIG.34, FIG.35) cannot be attributed to differences in molecular brightness.JFS561 (38) exhibits a e = 109,000 M 'em1and f = 0.77 (FIG. 12) whereas JF549 (8) shows e = 101,000 M cm1and = 0.88 (FIG.3) so the expected brightness should be similar. A possible explanation of this unexpected behavior is a shorter triplet lifetime (n). Dyes canundergo intersystem crossing to the long-lived triplet state (FIG. 1) Under high illumination conditions, such as those used in SPT experiments, a substantial fraction of fluorophores can be shelved in the triplet state, leading to lower effective brightness. To investigate this further, fluorescence correlation spectroscopy (FCS) experiments were performed on JFSsei-HaloTag® ligand (86) or JFs49-HaloTag® ligand (143) bound to purified HaloTag® protein. The resulting autocorrelation curves were compared to curves resulting from a pure diffusion model (FIG. 36).Deviation from a pure diffusion model at the short lag times indicates a substantial fraction of molecules in the triplet-state. The protein conjugate of JFs49-HaloTag® ligand (143) shows a significant difference in short lag times but the HaloTag® protein labeled with JF S 561 -HaloTag® ligand (86) shows a near-optimal fit to the pure diffusion model. This allowed estimation of both the triplet lifetime (TT) and the fraction of molecules in the triplet state (T) under these conditions. The HaloTag-JFs49 conjugate from 143 showed T = 2.9 ps, consistent with previous measurements of rhodamine triplet states, and T = 9.4%. The HaloTag: JFSs6i conjugate from 86 showed no measurable triplet lifetime (TT = 0 ps), and T = 0%. These data indicate that incorporation of the azabicyclo[2.1.1]hexane significantly decreases the triplet state lifetime, which leads to higher effective brightness, especially under high intensity illumination, and improved photostability, likely due to decreasedJO2 production and subsequent slower dealkylation (FIG. 1) and other bleaching pathways.

[0157] Select HaloTag® ligands were then testing in stimulated emission depletion (STED) microscopy using live and fixed cells expressing TOMM20-HaloTag® fusion proteins. STED microscopy uses far-red illumination to deplete the outer rim of a diffraction-limited spot, allowing super-resolution imaging. This trading of excited states for resolution makes the use of photostable dyes particularly important in STED microscopy. Cells labeled with JFSsei-HaloTag® ligand (86) and imaged using STED using a 775-nm depletion beam showed no evidence of mitochondrial fragmentation (FIG.37) even after several minutes of continuous imaging. Fixed cells were labeled with JFSsei-HaloTag® ligand (86), JFs49-HaloTag® ligand (143), or BDsee-HaloTag® ligand (149), and the photostability in STED imaging was evaluated using 660-nm depletion. The JFS ligand 86 showed the best photostability, JF549 ligand 143 showed intermediate bleaching rate, and the BD566 ligand 149 showed the fastest bleaching (FIG. 38). This further demonstrates the improvement in photostability afforded by theazabicyclo[2.1,l]hexane motif, especially compared to BD566, which contains a bicyclic structure with two bridgehead carbon atoms alpha to the nitrogen.

[0158] The effect of the azabicyclo[2.1.1]hexane motif on equilibrium constant between the lipophilic, nonfluorescent lactone and fluorescent zwitterion ( TL-Z) was then investigated. The K -Z of JFS561 (38) was 4.31, which was higher than the value for JF549 (8; K -Z = 3.47). Incorporating a fluorine atom into the azabicyclo[2.1.1]hexane or azetidine substituents caused a decrease in this value: KL-Z = 1.60 for JFS548 (40) and KL-Z = 1.01 for JF536 (25). Examining the values for the carborhodamine system confirmed that the bicyclic substituent increases this value relative to the azetidine functionality; KL-Z = 1.19 for JFSei9 (61) and K - = 0.0911 for JF608 (18) was found. Fluorination of the azabicyclo[2.1.1]hexane motif had a dramatic effect on this dye scaffold with JFSeo? (62) exhibiting KL-Z = 0.0194, a decrease of ~100-fold. In contrast, fluorination of the azetidine substituent only decreased this value ~10-fold with JF595 (62) exhibiting KL-Z = 0.00776 (FIG. 39). This demonstrates that the azabicyclo[2.1.1]hexane system can magnify the effect of fluorine substitution on the KL-Z for certain dye scaffolds.[00159J The KL-Z. value also affects the fluorogenic effect that is observed when certain ligands bind their biomolecular target. Dye-ligands can adopt the nonfluorescent lactone form when free in solution but switch to the fluorescent zwitterionic form upon binding due to the change in environment. The HaloTag® ligands of the Si-rhodamine dyes JF646 (145) and IF 635 (150) were compared to the analogous compounds containing the azabicyclo[2.1.1]hexane system: JFS657-HaloTag® ligand (116) and JFS645-HaloTag® ligand (120; FIG. 40). JF646-HaloTag® ligand (145) shows a 22-fold increase in absorption upon binding the HaloTag® protein and the absorptivity of the resulting conjugate is high (s = 148,000 'em '). The JF635-HaloTag® ligand (150) shows a higher increase in absorption of 114-fold, but the absorptivity of the resulting conjugate is relatively low (s = 91,100 M 'em I) The JFSesv-HaloTag® ligand (116) exhibited a lower turn-on compared to JF646-HaloTag® ligand (145) due to higher absorption of the free ligand; the absorptivity of the HaloTag® conjugate is comparable (e = 147,000 M 'em '). JFS645-HaloTag® ligand (120) shows both a high turn on (109-fold) and a high absorptivity of the resulting HaloTag® conjugate (e = 137,000 M 'em '), showing that substituted azabicyclo[2.1.1]hexane allows fine-tuning of dyes to yield a fluorogenic dye with both high turn-on and high absorptivity.

[0160] These dyes were then tested with the chemigenetic calcium ion indicator HaloCaMP (FIG. 41, FIG. 42), which uses Ca2+-driven conformational changes in the protein to modulate the fluorescence of fluorogenic HaloTag® ligands bound to the protein. JF646-HaloTag® ligand (145) and JFeas-HaloTag® ligand (150) showed lower fluorescence intensity in the Ca2-free state compared to the analogous JFS657-HaloTag® ligand (116) or JFS645-HaloTag® ligand (120) using both HaloCaMPla (FIG. 41) and HaloCaMP lb (FIG. 42). The JF646 ligand 145, JFeas ligand 150, and JFSes? ligand 116 showed modest increases in absolute fluorescence intensity, but the JFS645 ligand 120 showed a substantially larger changes in fluorescence intensity. These results demonstrate the JFS dyes, which contain the azabicyclo[2.1.1]hexane motif, maintain the fluorogenicity observed for other Si-rhodamines and this effect can be tuned by installing electron-withdrawing group fluorine groups on the azabicyclo[2.1.1]hexane moiety to yield indicator systems with unexpectedly large changes in fluorescence intensity.

[0161] The utility of the hydroxymethyl analog of JFS645 was then evaluated in superresolution microscopy experiments. Live U2OS cells expressing HaloTag-ensconsin were labeled with HaloTag® ligand 135 (FIG. 22) and imaged at 100 Hz. The dye exhibited spontaneous blinking due to transient protonation / deprotonation. The first 1000 frames were analyzed using super-resolution optical microscopy (SOFI) algorithm, and the first 5000 frames were analyzed a single-molecule localization microscopy (SMLM) algorithm, yielding high-resolution images of the cytoskeletal protein (FIG. 43). Due to this blinking behavior and the utility in super-resolution experiments, compound 135 was given the name “JFS645b-HaloTag® ligand” where the “b” indicates the blinking character of the molecule.

[0162] Example 10: Beneficial features. In summary, a unique substituent, azabicyclo[2.1.1]hexane, was introduced into rhodamine dyes yielding a highly modular system (FIG. 44) This small, symmetrical bicyclic structure increases the brightness and photostability of rhodamines by decreasing both TICT and — unexpectedly — the triplet lifetime (TT; FIG. 1, FIG. 36). This motif also increases the T / of rhodamine dyes relative to azetidine-containing analogs, but surprisingly magnifies the effect of the fluorine substituents on the lactone-zwitterion equilibrium (FIG. 39). Unlike other rhodamines containing bicyclic systems, the azabicyclo[2.1.1]hexane motif allows fine-tuning of the spectral and chemical properties withoutintroducing unwanted stereochemistry. This results in the bright and highly fluorogenic JFS645-HaloTag® ligand (120) that shows improved properties with both the HaloTag® protein (FIG.40) and the HaloCaMP indicator system (FIG. 41, FIG. 42). More generally, the azabicyclo[2.1.1]hexane is compatible with other methods to modify rhodamine properties: addition of substituents on the xanthene system to modulate Φf; fluorination of the pendant phenyl ring to modify λabs, λem, and KL–Z; and changing the ortho-substituent on the pendant phenyl ring to introduce blinking (FIG. 22, FIG. 43). The JFS dyes can be used with existing labeling strategies (FIG. 24-31) using standard synthetic approaches. Additional groups can be introduced using this substituent yielding multifunctional fluorophores (FIG. 23). This results in dyes that enable advanced microscopy experiments such as SPT (FIG. 34, FIG. 35), STED (FIG. 37), SOFI, and SMLM (FIG. 43) that are brighter and more photostable than other optimized fluorophores such as the JF, JFX, and BD dyes. The azabicyclo[2.1.1]hexane functionality improves and adds flexibility to the rhodamine scaffold and the resulting JFS compounds will enable new imaging experiments in biological systems.

[0163] Example 11: General Experimental Information for Synthesis. Commercial reagents were obtained from reputable suppliers and used as received. All solvents were purchased in septum-sealed bottles stored under an inert atmosphere. All reactions were sealed with septa through which a nitrogen atmosphere was introduced unless otherwise noted.Reactions were conducted in round-bottomed flasks or septum-capped crimp-top vials containing Teflon-coated magnetic stir bars. Heating of reactions was accomplished with a silicon oil bath or an aluminum reaction block on top of a stirring hotplate equipped with an electronic contact thermometer to maintain the indicated temperatures.

[0164] Reactions were monitored by thin layer chromatography (TLC) on precoated TLC glass plates (silica gel 60 F254, 250 μm thickness) or by LC / MS (Phenomenex Kinetex 2.1 mm x 30 mm 2.6 pm C18 column; 5 μL injection; 5–98% MeCN / H2O, linear gradient, with constant 0.1% v / v HCO2H additive; 6 min run; 0.5 mL / min flow; ESI; positive ion mode). TLC chromatograms were visualized by UV illumination or developed with p-anisaldehyde, ceric ammonium molybdate, or KMnO4stain. Reaction products were purified by flash chromatography on an automated purification system using pre-packed silica gel columns or by preparative HPLC (Phenomenex Gemini–NX 30 × 150 mm 5 μm C18 column). AnalyticalHPLC analysis was performed with an Agilent Eclipse XDB 4.6 × 150 mm 5 μm C18 column under the indicated conditions. High-resolution mass spectrometry was performed by HHMI Janelia Mass Spectrometry.

[0165] NMR spectra were recorded on a 400 MHz spectrometer. ’ll and13C chemical shifts were referenced to TMS or residual solvent peaks, and19F chemical shifts were referenced to CFCl3. Data for1H NMR spectra are reported as follows: chemical shift (8 ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet; b = broad), coupling constant (Hz), integration. Data for13C NMR spectra are reported by chemical shift (8 ppm) with hydrogen multiplicity (C, CH, CH2, CH3) information obtained from DEPT spectra. The13C NMR spectra are not reported for compounds containing trifluoro- or tetrafluoro-substituted aryl rings, as the numerous distinct fluorine couplings confounded interpretation of the spectra.

[0166] Example 12: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (JFS548, 40)

[0167] A vial was charged with fluorescein ditriflate (36; Grimm, J. B.; Lavis, L. D. Org. Lett. 2011, 13, 6354-6357; 300 mg, 0.503 mmol), 4-fluoro-2-azabicyclo[2.1.1]hexane hydrochloride (39; 166 mg, 1.21 mmol, 2.4 eq), Pd2dba3 (46.1 mg, 50.3 pmol, 0.1 eq), XPhos (71.9 mg, 0.151 mmol, 0.3 eq), and Cs2CO3(787 mg, 2.41 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x). Dioxane (3.5 mL) was added, and the reaction was flushed again with nitrogen (3x). The reaction was then stirred at 100 °C for 4 h. It was subsequently cooled to room temperature, diluted with MeOH, deposited onto Celite, and concentrated to dryness. Purification by silica gel chromatography (0-10% MeOH (2 M NH3) / CH2Cl2, linear gradient; dry load with Celite) afforded the title compound (209 mg, 83%) as a pink solid.1H NMR (CD3OD, 400 MHz) δ 8.10 - 8.05 (m, 1H), 7.69 - 7.61 (m, 2H), 7.24 -7.19 (m, 1H), 7.09 (d, J= 9.0 Hz, 2H), 6.80 (dd, J= 9.0, 2.4 Hz, 2H), 6.78 (d, J= 2.3 Hz, 2H),4.71 (dt, JHF = 21.0 Hz, J= 2.5 Hz, 2H), 3.58 (bs, 4H), 2.40 - 2.27 (m, 4H), 2.16 - 2.05 (m, 4H);19F NMR (CD3OD, 376 MHz) δ -165.46 (bd, JFH= 20.9 Hz);13C NMR (CD3OD, 101 MHz) δ 172.8 (C), 157.9 (C), 155.4 (C), 143.4 (C), 139.3 (C), 138.2 (C), 132.5 (CH), 132.1 (CH), 130.9 (CH), 129.6 (CH), 129.0 (CH), 114.0 (C), 113.6 (CH), 97.7 (CH), 91.4 (d,1JCF= 263.6 Hz, CF), 53.0 (d,3JCF= 23.1 Hz, CH), 52.4 (d,2JCF= 28.4 Hz, CH2), 46.3 (d,2JCF= 19.3 Hz, CH2);Analytical HPLC: tR= 12.1 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C30H25F2N2O3[M+H]+499.1828, found 499.1829.

[0168] Example 13: 2-(2,7-Difluoro-3,6-bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)benzoate (JFS551, 79)

[0169] The title compound (72%, off-white solid) was prepared from 2',7'-difluoro-3',6'-diiodofluoran (77; Grimm, J. B. et al. JACS Au 2021, 1, 690-696) and 4-fluoro-2-azabicyclo[2.1.1]hexane hydrochloride (39) according to the procedure described for Example 1.1H NMR (CDCl3, 400 MHz) δ 8.03 (dt, J= 7.3, 1.1 Hz, 1H), 7.70 (td, J= 7.4, 1.3 Hz, 1H), 7.65 (td, J= 7.4, 1.1 Hz, 1H), 7.19 (dt, J= 7.6, 1.0 Hz, 1H), 6.55 (d,4JHF= 7.4 Hz, 2H), 6.35 (d,3JHF= 12.6 Hz, 2H), 4.14 (dt, JHF= 21.1 Hz, J= 2.4 Hz, 2H), 3.41 - 3.33 (m, 4H), 2.17 - 2.04 (m, 8H);19F NMR (CDCh, 376 MHz) 8 -130.93 (ddt, JFH = 11.4, 7.1, 3.4 Hz, 2F), -165.07 (bd, JFH= 21.1 Hz, 2F);13C NMR (CDCl3, 101 MHz) δ 169.1 (C), 152.3 (C), 150.0 (d,1JCF= 240.8 Hz, CF), 148.4 (d,4JCF= 1.5 Hz, C), 141.4 (d,2JCF= 11.7 Hz, C), 135.3 (CH), 130.2 (CH), 127.0 (C), 125.4 (CH), 124.0 (CH), 114.2 (d, CF = 22.8 Hz, CH), 108.5 (d, VCF = 6.9 Hz, C), 102.7 (d, VCF = 3.9 Hz, CH), 91.0 (dd,1JCF= 264.7 Hz,5JCF= 3.0 Hz, CF), 83.4 (C), 54.6 (dd,2JCF= 26.0 Hz,4JCF= 4.8 Hz, CH2), 51.3 (dd,3JCF= 24.9 Hz,4JCF= 2.6 Hz, CH), 43.9 (d,2JCF= 18.9 Hz, CH2), 43.8 (d,2JCF= 18.9 Hz, CH2); Analytical HPLC: tR= 12.2 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 555 nm); HRMS (ESI) calcd for C30H23F4N2O3[M+H]+535.1640, found 535.1641.

[0170] Example 14: 2-(3,6-Bis(4-methoxy-2-azabicyclo[2.1.1 ]hexan-2-yl)xanthylium-9-yl)benzoate (JFS553, 41)

[0171] The title compound (65%, red-purple solid) was prepared from fluorescein ditriflate (36) and 4-methoxy-2-azabicyclo[2.1.1]hexane hydrochloride (41) according to the procedure described for Example 1.1H NMR (CDCl3, 400 MHz) δ 8.02 – 7.97 (m, 1H), 7.64 (td, J = 7.4, 1.3 Hz, 1H), 7.58 (td, J = 7.4, 1.1 Hz, 1H), 7.19 (dt, J= 7.6, 1.0 Hz, 1H), 6.57 (d, J= 8.7 Hz, 2H), 6.47 (d, J= 2.4 Hz, 2H), 6.34 (dd, J= 8.7, 2.4 Hz, 2H), 4.15 (t, J = 2.4 Hz, 2H), 3.43 (s, 6H), 3.29 - 3.22 (m, 4H), 2.08 - 1.99 (m, 4H), 1.81 - 1.73 (m, 4H);13C NMR (CDCl3, 101 MHz) δ 169.9 (C), 153.1 (C), 153.0 (C), 152.2 (C), 134.7 (CH), 129.5 (CH), 129.1 (CH), 127.7 (C), 125.0 (CH), 124.3 (CH), 110.0 (CH), 108.2 (C), 99.8 (CH), 85.8 (C), 82.1 (C), 54.1 (CH2), 54.0 (CH3), 52.6 (CH), 42.1 (CH2); Analytical HPLC: tR= 11.4 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 560 nm); HRMS (ESI) calcd for C32H31N2O5[M+H]+523.2228, found 523.2227.

[0172] Example 15: 2-(3,6-Bis(4-((ter / -butoxycarbonyl)amino)-2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)benzoate (46)

[0173] The title compound (79%, red-purple solid) was prepared from fluorescein ditriflate (36) and / ert-butyl A-(2-azabicyclo[2.1.1 ]hexan-4-yl)carbamate (45) according to the procedure described for Example 1.1H NMR (CD3OD, 400 MHz) δ 8.12 – 8.08 (m, 1H), 7.66 (td, J = 7.5, 1.5 Hz, 1H), 7.61 (td, J = 7.4, 1.6 Hz, 1H), 7.24 – 7.20 (m, 1H), 7.21 (d, J = 9.3 Hz, 2H), 6.86 (dd, J= 9.4, 2.3 Hz, 2H), 6.77 (d, J= 2.3 Hz, 2H), 4.77 (t, J= 2.0 Hz, 2H), 3.60 (bs,4H), 2.42 - 2.30 (m, 4H), 1.95 - 1.86 (m, 4H), 1.49 (s, 18H);13C NMR (CD3OD, 101 MHz) 8 173.4 (C), 160.4 (C), 159.1 (C), 157.1 (C), 155.9 (C), 141.4 (C), 134.4 (C), 133.2 (CH), 130.8 (CH), 130.7 (CH), 130.6 (CH), 130.3 (CH), 115.0 (C), 114.5 (CH), 96.6 (CH), 80.5 (C), 59.1 (C), 58.4 (CH), 54.2 (CH2), 45.7 (CH2), 28.7 (CH3); Analytical HPLC: tR= 13.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 560 nm); HRMS (ESI) calcd for C40H45N4O7 [M+H]+693.3283, found 693.3284.

[0174] Example 16: 2-(3,6-Bis(4-amino-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (JFS555, 47)If "j 7 j". X,ctyO '

[0175] 2-(3,6-Bis(4-((to -butoxycarbonyl)amino)-2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)benzoate (Example 6; 46; 175 mg, 0.253 mmol) was taken up in CH2Cl2(5 mL), and trifluoroacetic acid (1 mL) was added. The reaction was stirred at room temperature for 2 h. Toluene (6 mL) was added; the reaction mixture was concentrated to dryness and then azeotroped with MeOH three times to provide the title compound as a dark red powder (204 mg, 97%, 3 TFA salt). Analytical HPLC and NMR indicated that the material was >99% pure and did not require further purification.1H NMR (CD3OD, 400 MHz, 325 K) δ 8.36 – 8.30 (m, 1H), 7.85 (td, J= 7.4, 1.5 Hz, 1H), 7.80 (td, J= 7.6, 1.5 Hz, 1H), 7.43 - 7.36 (m, 1H), 7.16 (d, J= 9.2 Hz, 2H), 6.98 (dd, J= 9.3, 2.3 Hz, 2H), 6.95 (d, J= 2.3 Hz, 2H), 5.00 (t, J= 2.1 Hz, 2H), 3.77 (bs, 4H), 2.50 - 2.39 (m, 4H), 2.10 - 1.99 (m, 4H);13C NMR (CD3OD, 101 MHz, 325 K) δ 168.1 (C), 161.8 (C), 159.4 (C), 156.4 (C), 135.2 (C), 133.9 (CH), 132.8 (CH), 132.5 (CH), 132.4 (C), 131.5 (CH), 131.4 (CH), 115.8 (C), 115.5 (CH), 97.2 (CH), 58.9 (CH), 56.9 (C), 53.0 (CH2), 44.9 (CH2); Analytical HPLC: tR= 9.1 min, >99% purity (10-40% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C30H29N4O3[M+H]+493.2235, found 493.2234.

[0176] Example 17: 2-(3,6-Di(2-azabicyclo[2.1.1 ]hexan-2-yl)xanthylium-9-yl)benzoate (JFS561, 38)

[0177] The title compound (77%, dark red-purple solid) was prepared from fluorescein ditriflate (36) and 2-azabicyclo[2.1.1]hexane hydrochloride (37) according to the procedure described for Example 1.1H NMR (CD3OD, 400 MHz) δ 8.09 – 8.04 (m, 1H), 7.63 (td, J = 7.5, 1.6 Hz, 1H), 7.59 (td, J = 7.4, 1.7 Hz, 1H), 7.25 – 7.21 (m, 1H), 7.20 (d, J = 9.4 Hz, 2H), 6.87 (dd, J = 9.3, 2.3 Hz, 2H), 6.78 (d, J= 2.3 Hz, 2H), 4.86 (dt, J= 6.9, 1.8 Hz, 2H), 3.57 (bs, 4H), 3.12 - 3.04 (m, 2H), 2.28 - 2.17 (m, 4H), 1.59 - 1.50 (m, 4H);13C NMR (CD3OD, 101 MHz) δ 173.5 (C), 161.7 (C), 159.2 (C), 156.4 (C), 141.8 (C), 133.8 (C), 133.2 (CH), 130.9 (CH), 130.6 (CH), 130.5 (CH), 130.4 (CH), 114.9 (CH), 96.6 (CH), 63.9 (CH), 53.1 (CH2), 41.8 (CH2), 39.8 (CH); Analytical HPLC: tR = 12.4 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 575 nm); HRMS (ESI) calcd for C30H27N2O3[M+H]+463.2017, found 463.2017.

[0178] Example 18: 2-(3,6-Bis(4-azido-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (44)

[0179] The title compound (27%, red solid) was prepared from fluorescein ditriflate (36) and 4-azido-2-azabicyclo[2.1. l]hexane hydrochloride (43) according to the procedure described for Example 1.1H NMR (CDCl3, 400 MHz) δ 8.02 – 7.98 (m, 1H), 7.65 (td, J= 7.4, 1.3 Hz, 1H), 7.59 (td, J= 7.4, 1.1 Hz, 1H), 7.18 (dt, J= 7.4, 1.0 Hz, 1H), 6.59 (d, J= 8.7 Hz, 2H), 6.48 (d, J = 2.4 Hz, 2H), 6.34 (dd, J = 8.7, 2.4 Hz, 2H), 4.29 (t, J = 2.3 Hz, 2H), 3.33 - 3.23 (m, 4H), 2.15 -2.06 (m, 4H), 1.92- 1.82 (m, 4H);13C NMR (CDCl3, 101 MHz) δ 169.8 (C), 153.1 (C), 152.9(C), 151.9 (C), 134.8 (CH), 129.6 (CH), 129.2 (CH), 127.5 (C), 125.0 (CH), 124.2 (CH), 110.2 (CH), 108.8 (C), 100.2 (CH), 84.8 (C), 64.7 (C), 55.5 (CH2), 54.5 (CH), 43.5 (CH2); Analytical HPLC: tR= 12.3 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 555 nm); HRMS (ESI) calcd for C30H25N8O3[M+H]+545.2045, found 545.2045.

[0180] Example 19: 2-(3,6-Bis(4-(methoxycarbonyl)-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (49)O. XJ~ u X X,

[0181] The title compound (64%, red-purple solid) was prepared from fluorescein ditriflate (36) and methyl 2-azabicyclo[2.1.1]hexane-4-carboxylate hydrochloride (48) according to the procedure described for Example 1.1H NMR (CD3OD, 400 MHz) δ 8.12 – 8.05 (m, 1H), 7.65 (td, J= 7.5, 1.6Hz, 1H), 7.61 (td, J = 7.4, 1.7 Hz, 1H), 7.23 -7.19 (m, 1H), 7.16 (d, J = 9.3 Hz, 2H), 6.86 (dd, J= 9.3, 2.3 Hz, 2H), 6.80 (d, J= 2.3 Hz, 2H), 4.82 (t, J= 1.7 Hz, 2H), 3.78 (s, 6H), 3.75 (bs, 4H), 2.50 - 2.39 (m, 4H), 1.91 - 1.82 (m, 4H);13C NMR (CD3OD, 101 MHz) δ 173.2 (C), 171.2 (C), 158.7 (C), 156.0 (C), 155.3 (C), 140.4 (C), 135.9 (C), 133.0 (CH), 131.1 (CH), 130.8 (CH), 130.4 (CH), 129.9 (C), 114.7 (C), 114.6 (CH), 97.3 (CH), 61.3 (CH), 54.0 (CH2), 52.7 (CH3), 52.5 (C), 44.6 (CH2); Analytical HPLC: tR= 11.4 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 560 nm); HRMS (ESI) calcd for C34H31N2O7[M+H]+579.2126, found 579.2126.

[0182] Example 20: 2-(3,6-Bis(4-carboxy-2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)benzoate (JFS561i, 50)

[0183] 2-(3,6-Bis(4-(methoxycarbonyl)-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (Example 9; 49; 60 mg, 0.104 mmol) was dissolved in MeOH (4 mL), and 1 M NaOH (415 pL, 0.415 mmol, 4 eq) was added. After stirring the reaction at room temperature for 18 h, it was acidified with 1 M HC1 (500 pL) and directly purified by reverse phase HPLC (10-75% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive) to provide 64.0 mg (93%, TFA salt) of the title compound as a dark red solid.1H NMR (CD3OD, 400 MHz, 325 K) δ 8.34 – 8.30 (m, 1H), 7.84 (td, J= 7.5, 1.5 Hz, 1H), 7.78 (td, J= 7.6, 1.5 Hz, 1H), 7.42 - 7.37 (m, 1H), 7.10 (d, J= 9.3 Hz, 2H), 6.94 (bd, J= 9.3 Hz, 2H), 6.89 (bs, 2H), 4.89 (t, J= 1.8 Hz, 2H), 3.80 (bs, 4H), 2.53 - 2.43 (m, 4H), 1.95 - 1.86 (m, 4H);13C NMR (CD3OD, 101 MHz, 325 K) δ 172.4 (C), 168.1 (C), 160.7 (C), 159.3 (C), 156.3 (C), 135.3 (C), 133.8 (CH), 132.6 (CH), 132.5 (CH), 132.4 (C), 131.5 (CH), 131.4 (CH), 115.5 (CH), 115.2 (C), 97.0 (CH), 61.8 (CH), 53.9 (CH2), 52.7 (C), 44.8 (CH2); Analytical HPLC: tR= 9.9 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 560 nm); HRMS (ESI) calcd for C32H27N2O7[M+H]+551.1813, found 551.1811.

[0184] Example 21: 2-(3,6-Di(2-azabicyclo[2.1.1]hexan-2-yl)-2,7-difluoroxanthylium-9-yl)benzoate (JFS565, 78)

[0185] The title compound (80%, pink solid) was prepared from 2',7'-difluoro-3',6'-diiodofluoran (77) and 2-azabicyclo[2.1.1]hexane hydrochloride (37) according to the procedure described for Example 1.1H NMR (CDCl3, 400 MHz) δ 8.02 (dt, J = 7.5, 1.1 Hz, 1H), 7.69 (td, J = 7.5, 1.3 Hz, 1H), 7.63 (td, J= 7.4, 1.1 Hz, 1H), 7.21 (dt, J= 7.6, 1.0 Hz, 1H), 6.58 (d,4JHF= 7.5 Hz, 2H), 6.33 (d,3JHF= 12.7 Hz, 2H), 4.27 (dt, J= 6.7, 1.8 Hz, 2H), 3.39 (ddt, J= 8.1 Hz, JHF = 3.7 Hz, J= 1.2 Hz, 2H), 3.32 (ddt, J= 8.1 Hz, JHF = 3.7 Hz, J = 1.2 Hz, 2H), 2.96 - 2.89 (m, 2H), 1.99 - 1.89 (m, 4H), 1.51 - 1.42 (m, 4H);19F NMR (CDCl3, 376 MHz) δ -130.76 (ddt, JFH= 11.3, 7.4, 3.6 Hz);13C NMR (CDCl3, 101 MHz) δ 169.3 (C), 152.4 (C), 150.2 (d, VCF = 240.1 Hz, CF), 148.6 (d, CF = 1.4 Hz, C), 143.0 (d, CF = 11.8 Hz, C), 135.1 (CH), 130.0 (CH),127.2 (C), 125.2 (CH), 124.1 (CH), 113.9 (d,2JCF = 22.9 Hz, CH), 107.4 (d,3JCF = 6.8 Hz, C), 102.5 (d,3JCF= 4.3 Hz, CH), 84.2 (C), 61.4 (d,5JCF= 1.9 Hz, CH), 56.3 (d,4JCF= 4.9 Hz, CH2), 40.1 (d,4JCF= 2.5 Hz, CH), 38.9 (CH2), 38.7 (CH2); Analytical HPLC: tR= 12.3 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 570 nm); HRMS (ESI) calcd for C3oH25F2N203 [M+H]+499.1828, found 499.1829.

[0186] Example 22: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)thioxanthylium-9-yl)benzoate (JFS568, 58)

[0187] Step I: A crimp-top vial was charged with bis(3-bromophenyl)sulfane (51;Grimm, J. B. et al. Nat. Methods 2020, 77, 815-821; 1.00 g, 2.91 mmol), 4-fluoro-2-azabicyclo[2.1.1]hexane hydrochloride (39; 960 mg, 6.98 mmol, 2.4 eq), Pd2dba3 (266 mg, 0.291 mmol, 0.1 eq), XPhos (416 mg, 0.872 mmol, 0.3 eq), and Cs2CO3(4.55 g, 13.95 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x). Dioxane (14 mb) was added; after flushing the reaction again with nitrogen (3x), it was stirred at 100 °C for 18 h. It was then cooled to room temperature, filtered through Celite with CH2C12, and evaporated. The residue was purified by silica gel chromatography (0-50% Et2O / hexanes, linear gradient) to afford bis(3-(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)phenyl)sulfane (53; 860 mg, 77%) as pale yellow, viscous gum.1H NMR (CDCl3, 400 MHz) δ 7.14 (t, J= 7.9 Hz, 2H), 6.74 (ddd, J= 7.6, 1.7, 0.9 Hz, 2H), 6.69 (dd, J= 2.4, 1.7 Hz, 2H), 6.55 (ddd, J= 8.2, 2.4, 0.9 Hz, 2H), 4.10 (dt, J= 21.4, 2.5 Hz, 2H), 3.30 (bs, 4H), 2.10 - 2.00 (m, 8H);13C NMR (CDCl3, 101 MHz) δ 150.8 (C), 136.7 (C), 130.0 (CH), 120.9 (CH), 116.0 (CH), 112.7 (CH), 91.2 (d, VCF = 264.9 Hz, CF), 53.8 (d,2JCF= 25.7 Hz, CH2), 50.4 (d,3JCF= 25.3 Hz, CH), 44.4 (d,2JCF= 19.0 Hz, CH2); HRMS (ESI) calcd for C22H23F2N2S [M+H]+385.1545, found 385.1544.

[0188] Step 2: The product from Step 1 (53; 250 mg, 0.650 mmol) and phthalaldehydic acid (54; 97.6 mg, 0.650 mmol, 1 eq) were combined in 2,2,2-trifluoroethanol (12 mL) in around-bottom flask. The reaction mixture was sparged with O2 from a balloon for 10 min, then stirred at 80 °C under the O2 balloon for 48 h. The reaction was cooled to room temperature, concentrated in vacuo, and purified by silica gel chromatography (0-10% MeOH (2 M NH3) / CH2C12, linear gradient) to provide the title compound 2-(3,6-bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)thioxanthylium-9-yl)benzoate as a light purple solid (44.8 mg, 13%).1H NMR (CDCl3, 400 MHz) δ 7.95 (dt, J= 7.5, 1.1 Hz, 1H), 7.64 (ddd, J= 8.0, 7.0, 1.2 Hz, 1H), 7.60 - 7.54 (m, 2H), 6.87 (d, J= 8.7 Hz, 2H), 6.71 (d, J= 2.5 Hz, 2H), 6.41 (dd, J = 8.8, 2.5 Hz, 2H), 4.15 (dt, JHF = 21.3 Hz, J= 2.6 Hz, 2H), 3.35 - 3.30 (m, 4H), 2.13 -2.07 (m, 4H), 2.05 - 1.98 (m, 4H);19F NMR (CDCl3, 376 MHz) δ -164.56 – -164.68 (m);13C NMR (CDCl3, 101 MHz) δ 170.2 (C), 153.5 (C), 149.9 (C), 134.5 (CH), 133.4 (C), 129.6 (CH), 128.2 (CH), 126.1 (C), 125.8 (CH), 124.1 (CH), 121.9 (C), 112.1 (CH), 110.1 (CH), 91.08 (d,1JCF = 264.9 Hz, CF), 87.8 (C), 53.3 (d,2JCF = 26.0 Hz, CH2), 50.6 (d,3JCF = 24.9 Hz, CH), 44.7 (d,2JCF= 19.1 Hz, CH2); Analytical HPLC: tR= 11.9 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 575 nm); HRMS (ESI) calcd for C30H25F2N2O2S [M+H]+515.1600, found 515.1600.

[0189] Example 23: 2-(3,6-Di(2-azabicyclo[2.1. l]hexan-2-yl)thioxanthylium-9-yl)benzoate (JFS582, 55)

[0190] Step 1: A crimp-top vial was charged with bis(3-bromophenyl)sulfane (51; 1.00 g, 2.91 mmol), 2-azabicyclo[2.1.1]hexane hydrochloride (37; 834 mg, 6.98 mmol, 2.4 eq), Pd2dba3 (266 mg, 0.291 mmol, 0.1 eq), XPhos (416 mg, 0.872 mmol, 0.3 eq), and Cs2CO3(4.55 g, 13.95 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x). Dioxane (14 mL) was added; after flushing the reaction again with nitrogen (3x), it was stirred at 100 °C for 18 h. It was then cooled to room temperature, filtered through Celite with CH2CI2, and evaporated. The residue was purified by silica gel chromatography (0-30% EtOAc / hexanes, linear gradient) to afford bis(3-(2-azabicyclo[2.1.1]hexan-2-yl)phenyl)sulfane (52; 403 mg, 40%)as an off-white solid. ’H NMR (CDCI3, 400 MHz) 87.11 (t, J= 7.9 Hz, 2H), 6.76 (dd, J= 2.4, 1.8 Hz, 2H), 6.69 (ddd, J= 7.7, 1.7, 0.9 Hz, 2H), 6.59 (ddd, J= 8.2, 2.4, 0.9 Hz, 2H), 4.26 (dt, J = 6.7, 1.8 Hz, 2H), 3.27 (bs, 4H), 2.94 - 2.86 (m, 2H), 1.94 - 1.84 (m, 4H), 1.45 - 1.36 (m, 4H);13C NMR (CDCI3, 101 MHz) 8 152.3 (C), 136.5 (C), 129.8 (CH), 119.9 (CH), 116.3 (CH), 112.8 (CH), 60.4 (CH), 55.0 (CH2), 40.0 (CH), 39.4 (CH2); HRMS (ESI) calcd for C22H25N2S [M+H]+349.1733, found 349.1733.

[0191] Step 2: The title compound 2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)thioxanthylium-9-yl)benzoate (45%, purple solid) was prepared from the product of Step 1 (52) and phthalaldehydic acid (54) according to the procedure described for Example 11, Step 2. ^NMR CDCh, 400 MHz) 87.95 (dt, 7.5, 1.1 Hz, 1H), 7.64 (ddd, J= 7.9, 6.9, 1.2 Hz, 1H), 7.59 - 7.52 (m, 2H), 6.80 (d, J= 8.8 Hz, 2H), 6.75 (d, J= 2.5 Hz, 2H), 6.43 (dd, J= 8.8, 2.5 Hz, 2H), 4.30 (dt, J= 6.8, 1.8 Hz, 2H), 3.32 - 3.25 (m, 4H), 2.96 - 2.89 (m, 2H), 1.97 - 1.87 (m, 4H), 1.44 - 1.36 (m, 4H);13C NMR (CDCb, 101 MHz) 8 170.3 (C), 153.4 (C), 151.3 (C), 134.3 (CH), 133.7 (C), 129.4 (CH), 128.3 (CH), 126.6 (C), 125.7 (CH), 124.3 (CH), 120.6 (C), 112.2 (CH), 110.0 (CH), 89.2 (C), 60.6 (CH), 54.4 (CH2), 39.8 (CH), 39.67 (CH2), 39.65 (CH2);Analytical HPLC: tR = 12.4 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 590 nm); HRMS (ESI) calcd for C3oH27N202S [M+H]‘ 479.1788, found 479.1787.

[0192] Example 24: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)thioxanthylium- 9-yl)-3,4,5,6-tetrafluorobenzoate (JFS592, 59)

[0193] The title compound (30%, dark purple solid) was prepared from bis(3-(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)phenyl)sulfane (Example 11, Step 1; 53) and tetrafluorophthalaldehydic acid (56; Grimm, I. B. et al. J. Am. Chem. Soc. 2023, 145, 23000-23013) according to the procedure described for Example 11, Step 2. 'll NMR (CDCI3, 400MHz) 56.75 (dd, J= 8.8, 0.9 Hz, 2H), 6.68 (d, J= 2.5 Hz, 2H), 6.44 (dd, J= 8.8, 2.5 Hz, 2H), 4.19 (dt, JHF = 21.2 Hz, J = 2.6 Hz, 2H), 3.39 - 3.31 (m, 4H), 2.19 - 2.10 (m, 4H), 2.09 - 1.98 (m, 4H);19F NMR (CDC13, 376 MHz) 5 -138.00 (td, J= 20.6, 4.1 Hz, IF), -138.35 (td, J= 19.9, 8.6 Hz, IF), -143.02 - -143.37 (m, IF), -150.42 (ddd, J= 21.6, 18.5, 4.1 Hz, IF), -164.79 (bd, JFH = 21.0 Hz, 2F); Analytical HPLC: tR = 12.3 min, >99% purity (10-95% MeCN / H₂O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 595 nm); HRMS (ESI) calcd for C30H21F6N2O2S [M+H]+587.1223, found 587.1223.

[0194] Example 25: 2-(3,6-Di(2-azabicyclo[2.1. l]hexan-2-yl)thioxanthylium-9-yl)-3,4,5,6-tetrafluorobenzoate (JFS606, 57)

[0195] The title compound (68%, dark purple solid) was prepared from bis(3-(2-azabicyclo[2.1.1]hexan-2-yl)phenyl)sulfane (Example 12, Step 1; 52) and tetrafluorophthalaldehydic acid (56) according to the procedure described for Example 11, Step 2. 'H NMR (CDCI3, 400 MHz) 87.07 (dd, J= 9.0, 0.9 Hz, 2H), 6.75 (d, J= 2.4 Hz, 2H), 6.60 (dd, J= 9.1, 2.5 Hz, 2H), 4.48 (dt, J= 6.7, 1.8 Hz, 2H), 3.43 - 3.33 (m, 4H), 3.05 - 2.97 (m, 2H), 2.10 -2.00 (m, 4H), 1.51 - 1.43 (m, 4H);19F NMR (CDCI3, 376 MHz) 8 -139.03 (ddd, J= 22.3, 16.6, 6.0 Hz, IF), -139.41 (ddd, J= 20.7, 16.7, 3.4 Hz, IF), -150.65 - -150.85 (m, IF), -152.07 (ddd, J= 21.9, 18.9, 3.5 Hz, IF); Analytical HPLC: tR= 12.7 min, >99% purity (10-95% MeCN / H₂O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 610 nm); HRMS (ESI) calcd for C30H23F4N2O2S [M+H]+551.1411, found 551.1411.

[0196] Example 26: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)benzoate (JFS607, 62)

[0197] The title compound (82%, light blue solid) was prepared from carbofluorescein ditriflate (60; Grimm, J. B. et al. ACS Chem. Biol. 2013, 5, 1303-1310) and 4-fluoro-2-azabicyclo[2.1.1]hexane hydrochloride (39) according to the procedure described for Example 1. 'HNMR (CDCI3, 400 MHz) 88.02 - 7.97 (m, 1H), 7.60 (td, J= 7.4, 1.5 Hz, 1H), 7.55 (td, J = 7.4, 1.2 Hz, 1H), 7.10 - 7.03 (m, 1H), 6.84 (d, J= 2.5 Hz, 2H), 6.58 (d, J= 8.6 Hz, 2H), 6.45 (dd, J= 8.6, 2.5 Hz, 2H), 4.19 (dt, JHF = 21.4 Hz, J= 2.5 Hz, 2H), 3.41 - 3.35 (m, 4H), 2.14 -2.02 (m, 8H), 1.84 (s, 3H), 1.74 (s, 3H);19F NMR (CDCI3, 376 MHz) 8 -164.39 (bd,. / in = 21.2 Hz);13C NMR (CDC13, 101 MHz) 8 170.8 (C), 155.3 (C), 150.5 (C), 147.1 (C), 134.6 (CH), 129.2 (CH), 129.1 (CH), 127.3 (C), 125.1 (CH), 124.0 (CH), 121.7 (C), 112.6 (CH), 110.7 (CH), 91.24 (d, CF = 265.0 Hz, CF), 87.8 (C), 53.6 (d,2JCF = 25.7 Hz, CH2), 50.4 (d, CF = 25.1 Hz, CH), 44.58 (d,2JCF = 19.2 Hz, CH2), 44.57 (d,2JCF = 19.3 Hz, CH2), 38.6 (C), 35.7 (CH3), 32.4 (CH3); Analytical HPLC: tR = 12.8 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 600 nm); HRMS (ESI) calcd for C33H3iF2N2O2[M+H]+525.2349, found 525.2348.

[0198] Example 27: 2-(3,6-Di(2-azabicyclo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9(10 7)-yl)benzoate (JFS619, 61)

[0199] The title compound (27%, blue solid) was prepared from carbofluorescein ditriflate (60) and 2-azabicyclo[2.1.1]hexane hydrochloride (37) according to the procedure described for Example 1. ‘HNMR ^DCh, 400 MHz) 88.00 - 7.96 (m, 1H), 7.59 (td, J= 7.4, 1.4 Hz, 1H), 7.54 (td, J= 7.4, 1.2 Hz, 1H), 7.09 (dt, J= 7.6, 1.0 Hz, 1H), 6.90 (d, J= 2.4 Hz, 2H), 6.55 (d, J= 8.7 Hz, 2H), 6.48 (dd, J= 8.7, 2.4 Hz, 2H), 4.34 (dt, J= 6.7, 1.8 Hz, 2H), 3.37- 3.31 (tn, 4H), 2.98 - 2.90 (m, 2H), 1.96 - 1.89 (tn, 4H), 1.84 (s, 3H), 1.74 (s, 3H), 1.48 - 1.39 (m, 4H);13C NMR (CDC13, 101 MHz) 8 171.0 (C), 155.5 (C), 151.9 (C), 147.2 (C), 134.4 (CH), 129.1 (CH), 128.9 (CH), 127.5 (C), 124.9 (CH), 124.2 (CH), 120.6 (C), 112.8 (CH), 110.8 (CH), 88.6 (C), 60.4 (CH), 54.8 (CH2), 40.0 (CH), 39.55 (CH2), 39.53 (CH2), 38.6 (C), 35.8 (CH3), 32.3 (CH3); Analytical HPLC: tR = 13.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 625 nm); HRMS (ESI) calcd for C33H33N2O2[M+H]+489.2537, found 489.2538.

[0200] Example 28: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-3,4,5,6-tetrafluorobenzoate (JFS632, 68)

[0201] Step 1: A vial was charged with 9,9-dimethyl-10-oxo-9,10-dihydroanthracene-2,7-diyl bi s(trifluorom ethanesulfonate) (63; Grimm, J. B. et al. J. Am. Chem. Soc. 2023, 145, 23000-23013; 1.00 g, 1.93 mmol), 4-fluoro-2-azabicyclo[2.1.1]hexane hydrochloride (39; 637 mg, 4.63 mmol, 2.4 eq), Pd2dba3(177 mg, 0.193 mmol, 0.1 eq), XPhos (276 mg, 0.579 mmol, 0.3 eq), and Cs2CO3(3.02 g, 9.26 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x). Dioxane (12 mL) was added; after flushing the reaction again with nitrogen (3x), it was stirred at 100 °C for 4 h. It was then cooled to room temperature, filtered through Celite with CH2Cl2, and evaporated. The residue was purified by silica gel chromatography (0-40% EtOAc / hexanes, linear gradient) to afford 3,6-bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9(10 / / )-one (65; 733 mg, 90%) as an off-white solid. *HNMR (CDCI3, 400 MHz) 88.25 (d, J= 8.7 Hz, 2H), 6.71 (d, J= 2.3 Hz, 2H), 6.70 (dd, J= 8.7, 2.4 Hz, 2H), 4.36 (dt, JHF = 21.3 HZ, J= 2.6 HZ, 2H), 3.49 - 3.44 (m, 4H), 2.24 - 2.15 (m, 4H), 2.14 -2.06 (m, 4H), 1.69 (s, 6H);19F NMR (CDCI3, 376 MHz) 8 -164.75 - -164.87 (m);13C NMR (CDC13, 101 MHz) 8 181.2 (C), 152.7 (C), 152.6 (C), 129.6 (CH), 121.5 (C), 111.7 (CH), 109.0 (CH), 91.0 (d, CF = 264.9 Hz, CF), 52.7 (d,2JCF= 26.5 Hz, CH2), 50.5 (d,3JCF = 24.4 Hz, CH), 45.1(d,2JCF = 19.0 Hz, CH2), 38.2 (C), 33.6 (CH3); HRMS (ESI) calcd for C26H27F2N2O [M+H]+421.2086, found 421.2086.

[0202] Step 2: A solution of 2,3,4,5-tetrafluorobenzoic acid (66; 554 mg, 2.85 mmol, 6 eq) in THF (10 mL) was cooled to -78 °C under nitrogen. A-Butyllithium (2.5 M in hexanes, 2.28 mL, 5.71 mmol, 12 eq) was added, and the reaction was stirred at -78 °C for 3 h. A solution of the product from Step 1 (65; 200 mg, 0.476 mmol) in THF (10 mL) was added; the reaction was warmed to room temperature and stirred for 18 h. It was subsequently quenched with 1 M HC1 (10 mL), vigorously stirred for 10 min, further diluted with water, and extracted with EtOAc (2x). The combined organic extracts were washed with saturated NaHCCL, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. Purification by silica gel chromatography (0-40% EtOAc / toluene, linear gradient) yielded 237 mg (83%) of the title compound 2-(3,6-bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)-l 0,10-dimethylanthracen-9-ylium-9(10 / 7)-yl)-3, 4,5,6-tetrafluorobenzoate as a blue solid. 'HNMR (CDCI3, 400 MHz) 56.81 (d, J = 2.5 Hz, 2H), 6.69 (d, J= 8.6 Hz, 2H), 6.52 (dd, J= 8.7, 2.5 Hz, 2H), 4.22 (dt, JHF = 21.3 Hz, J= 2.6 Hz, 2H), 3.43 - 3.36 (m, 4H), 2.18 -2.11 (m, 4H), 2.11 - 2.03 (m, 4H), 1.77 (s, 3H), 1.72 (s, 3H);,9FNMR (CDCI3, 376 MHz) 8 -139.43 (td, J= 20.0, 8.6 Hz, IF), -141.83 (td, J= 20.1, 3.9 Hz, IF), -143.32 (ddd, = 20.6, 18.3, 8.6 Hz, IF), -151.77 (ddd, J= 21.4, 18.7, 4.0 Hz, IF), -164.44 - -164.55 (m, 2F); Analytical HPLC: tR = 12.8 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 635 nm); HRMS (ESI) calcd for C33H27F6N2O2 [M+H]+597.1972, found 597.1972.

[0203] Example 29: 2-(3,6-Di(2-azabicyclo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9(10H)-yl)-3,4,5,6-tetrafluorobenzoate (JFS643, 67)

[0204] Step I: A vial was charged with 9,9-dimethyl-10-oxo-9,10-dihydroanthracene-2,7-diyl bi s(trifluorom ethanesulfonate) (63; 1.00 g, 1.93 mmol), 2-azabicyclo[2.1.1]hexane hydrochloride (37; 554 mg, 4.63 mmol, 2.4 eq), Pd2dba3(177 mg, 0.193 mmol, 0.1 eq), XPhos (276 mg, 0.579 mmol, 0.3 eq), and Cs2CO3(3.02 g, 9.26 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x). Dioxane (12 mL) was added; after flushing the reaction again with nitrogen (3x), it was stirred at 100 °C for 6 h. It was then cooled to room temperature, filtered through Celite with CH2C12, and evaporated. The residue was purified by silica gel chromatography (0-50% EtOAc / hexanes, linear gradient, with constant 40% v / v CH2C12additive) to afford 3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9(10H)-one (64; 672 mg, 91%) as a yellow solid. 'HNMR (CDCI3, 400 MHz) 88.23 (d, J= 8.6 Hz, 2H), 6.75 (d, J= 2.4 Hz, 2H), 6.72 (dd, J= 8.7, 2.3 Hz, 2H), 4.52 (dt, J= 6.8, 1.8 Hz, 2H), 3.42 (bs, 4H), 3.01 (dt, J= 6.7, 3.0 Hz, 2H), 2.06 - 1.96 (m, 4H), 1.70 (s, 6H), 1.53 - 1.45 (m, 4H);13C NMR (CDC13, 101 MHz) 8 181.1 (C), 153.8 (C), 152.7 (C), 129.5 (CH), 120.6 (C), 111.9 (CH), 109.0 (CH), 60.5 (CH), 53.6 (CH2), 40.1 (CH2), 39.6 (CH), 38.1 (C), 33.7 (CH3); HRMS (ESI) calcd for C26H29N2O [M+H]+385.2275, found 385.2275.[00205J Step 2: A solution of 2,3,4,5-tetrafluorobenzoic acid (66; 757 mg, 3.90 mmol, 6 eq) in THF (13 mL) was cooled to -78 °C under nitrogen. A-Butyllithium (2.5 M in hexanes, 3.12 mL, 7.80 mmol, 12 eq) was added, and the reaction was stirred at -78 °C for 3 h. A solution of the product from Step 1 (64; 250 mg, 0.650 mmol) in THF (25 mL) was added; the reaction was warmed to room temperature and stirred for 18 h. It was subsequently quenched with 1 M HC1 (20 mL), vigorously stirred for 10 min, further diluted with water, and extracted with EtOAc (2x). The combined organic extracts were washed with saturated NaHCCL, dried over anhydrous MgSCU, filtered, and concentrated in vacuo. Purification by silica gel chromatography (0-10% MeOH (2 M NH3) / CH2C12, linear gradient) yielded 201 mg (55%) of the title compound 2-(3,6-di(2-azabicyclo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9(10H)-yl)-3,4,5,6-tetrafluorobenzoate as a dark blue solid. 'H NMR (CD3OD, 400 MHz) 57.15 (dd, J =9.3, 1.0 Hz, 2H), 7.10 (d, J= 2.4 Hz, 2H), 6.73 (dd, J= 9.3, 2.3 Hz, 2H), 4.99 (dt, J= 6.8, 1.7 Hz, 2H), 3.66 (bs, 4H), 3.09 (dt, J= 6.6, 3.1 Hz, 2H), 2.31 - 2.21 (m, 4H), 1.78 (s, 3H), 1.72 (s, 3H), 1.60 - 1.52 (m, 4H);19F NMR (CD3OD, 376 MHz) 8 -139.75 (ddd, J= 22.0, 12.9, 3.2 Hz, IF), - 141.60 (ddd, J= 22.5, 12.5, 3.2 Hz, IF), -155.15 (ddd, J= 22.7, 19.3, 3.4 Hz, IF), -158.59 (ddd, J= 22.1, 19.3, 3.4 Hz, IF); Analytical HPLC: tR= 12.7 min, >99% purity (10-95% MeCN / H2O,linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C33H29F4N2O2[M+H]+561.2160, found 561.2158.

[0206] Example 30: 2-(3,7-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate (JFS645, 71)

[0207] The title compound (87%, off-white solid) was prepared from Si-fluorescein ditriflate (69; Grimm, J. B. et al. Nat. Methods 2015, 12, 244-250) and 4-fluoro-2-azabicyclo[2.1. l]hexane hydrochloride (39) according to the procedure described for Example 1. ^NMR CDCh, 400 MHz) 87.97 (dt, J= 7.6, 1.1 Hz, 1H), 7.66 (td, J= 7.5, 1.2 Hz, 1H), 7.56 (td, J= 7.5, 1.0 Hz, 1H), 7.33 (dt, J= 7.7, 0.9 Hz, 1H), 6.92 (d, J= 2.8 Hz, 2H), 6.78 (d, J= 8.8 Hz, 2H), 6.49 (dd, J= 8.8, 2.7 Hz, 2H), 4.18 (dt,. / HF = 21.4 Hz, J= 2.5 Hz, 2H), 3.38 - 3.32 (m, 4H), 2.13 - 2.01 (m, 8H), 0.63 (s, 3H), 0.60 (s, 3H);19F NMR (CDC13, 376 MHz) 8 -164.47 (bd, JFH = 21.3 HZ);13C NMR (CDCI3, 101 MHz) 8 170.6 (C), 153.9 (C), 149.1 (C), 137.6 (C), 133.9 (C), 133.8 (CH), 129.0 (CH), 128.3 (CH), 127.2 (C), 125.9 (CH), 124.8 (CH), 118.3 (CH), 114.4 (CH), 91.6 (C), 91.2 (d, CF = 264.9 Hz, CF), 53.5 (d, CF = 25.7 Hz, CH2), 50.3 (d,3. / cr = 25.1 Hz, CH), 44.5 (d, CF = 18.9 Hz, CH2), 0.5 (CH3), -1.6 (CH3); Analytical HPLC: tR= 15.4 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C32H31F2N2O2Si [M+H]+541.2118, found 541.2119.

[0208] Example 31: 2-(3,7-Di(2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate (JFS657, 70)

[0209] The title compound (47%, light blue solid) was prepared from Si-fluorescein ditriflate (69) and 2-azabicyclo[2.1.1]hexane hydrochloride (37) according to the procedure described for Example 1. ^NMR CDCh, 400 MHz) 57.96 (dt, J = 7.6, 1.0 Hz, 1H), 7.65 (td, J = 7.5, 1.2 Hz, 1H), 7.54 (td, J= 7.5, 1.0 Hz, 1H), 7.34 (dt, J= 7.7, 0.9 Hz, 1H), 6.97 (d, J= 2.7 Hz, 2H), 6.73 (d, J= 8.8 Hz, 2H), 6.52 (dd, J= 8.8, 2.7 Hz, 2H), 4.34 (dt, J= 6.7, 1.8 Hz, 2H), 3.31 (bs, 4H), 2.96 - 2.89 (m, 2H), 1.97 - 1.86 (m, 4H), 1.46 - 1.37 (m, 4H), 0.62 (s, 3H), 0.60 (s, 3H);13C NMR (CDC13, 101 MHz) 8 170.8 (C), 154.2 (C), 150.6 (C), 137.5 (C), 133.6 (CH), 132.8 (C), 128.8 (CH), 128.3 (CH), 127.4 (C), 125.8 (CH), 125.0 (CH), 118.6 (CH), 114.5 (CH), 92.3 (C), 60.3 (CH), 54.6 (CH2), 40.0 (CH), 39.55 (CH2), 39.53 (CH2), 0.6 (CH3), -1.6 (CH3); Analytical HPLC: tR = 12.7 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C32H33N2O2Si [M+H]+505.2306, found 505.2306.

[0210] Example 32: 2-(3,7-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-3,4,5,6-tetrafluorobenzoate (JFS670, 76)

[0211] Step 1: A vial was charged with bis(3-bromophenyl)dimethylsilane (72; 500 mg, 1.35 mmol), 4-fhioro-2-azabicyclo[2.1.1]hexane hydrochloride (39; 446 mg, 3.24 mmol, 2.4 eq), Pd2dba3(124 mg, 0.135 mmol, 0.1 eq), XPhos (193 mg, 0.405 mmol, 0.3 eq), and Cs2CO3(2.11 g, 6.48 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x). Dioxane (7 mL) was added; after flushing the reaction again with nitrogen (3*), it was stirred at 100 °C for 18 h. It was then cooled to room temperature, filtered through Celite with CH2Q2, and evaporated. The residue was purified by silica gel chromatography (0-30% Et2O / hexanes, linear gradient) to afford bis(3-(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)phenyl)dimethylsilane (74; 342 mg, 62%) as an off-white solid. 'H NMR (CDCI3, 400 MHz) 57.22 (dd, J= 8.2, 7.2 Hz, 2H), 6.94 (dt, J= 7.2, 1.0 Hz, 2H), 6.83 (dd, J= 2.6, 1.1 Hz, 2H), 6.70 (ddd, J= 8.2, 2.6, 1.0 Hz, 2H), 4.15 (dt, JHF = 21.7 Hz, J= 2.5 Hz, 2H), 3.36 - 3.30 (m, 4H), 2.10 -2.02 (m, 8H), 0.51 (s,6H);19F NMR (CDCh, 376 MHz) 5 -164.20 (bd, JFH = 21.2 Hz);13C NMR (CDCh, 101 MHz) 6 149.6 (C), 139.4 (C), 128.8 (CH), 124.5 (CH), 119.6 (CH), 114.6 (CH), 91.4 (d, CF = 264.8 Hz, CF), 54.0 (d, CF = 25.3 Hz, CH2), 50.2 (d,3JCF = 25.4 Hz, CH), 44.3 (d, CF = 19.0 Hz, CH2), -2.1 (CH3); HRMS (ESI) calcd for C24H29F2N2Si [M+H]+411.2063, found 411.2064.

[0212] Step 2: The product from Step 1 (74; 295 mg, 0.718 mmol) and tetrafluorophthalaldehydic acid (56; 239 mg, 1.08 mmol, 1.5 eq) were combined in 2, 2, 3, 3, 4,4,4-heptafluoro-1 -butanol (2.4 mL) in a crimp-top vial. The reaction mixture was sparged with O2from a balloon for 10 min, then stirred at 95 °C under the O2balloon for 72 h. The reaction was cooled to room temperature, diluted into EtOAc, washed with saturated NaHCCh and brine, dried over anhydrous MgSC, filtered, and concentrated in vacuo. Silica gel chromatography (0-35% EtOAc / hexanes, linear gradient) yielded 167 mg (38%) of the title compound 2-(3,7-bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[6,e]silin-l 0-ylium- 10(577)-yl)-3,4,5,6-tetrafluorobenzoate as a pale green solid. 'H NMR (CDCh, 400 MHz) 56.90 (d, J= 2.7 Hz, 2H), 6.77 (dd, J= 8.7, 1.0 Hz, 2H), 6.56 (dd, J= 8.8, 2.8 Hz, 2H), 4.22 (dt, JHF = 21.3 Hz, J = 2.6 Hz, 2H), 3.38 (bs, 4H), 2.17 - 2.09 (m, 4H), 2.09 - 2.02 (m, 4H), 0.58 (s, 3H), 0.56 (s, 3H);19F NMR (CDCh, 376 MHz) 5 -138.71 - -138.94 (m, 2F), -143.62 - -143.84 (m, IF), -151.39 - -151.58 (m, IF), -164.59 (bd, JFH = 21.3 Hz, 2F); Analytical HPLC: tR= 15.7 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 675 nm); HRMS (ESI) calcd for C32H27F6N2O2Si [M+H]+613.1741, found 613.1742.

[0213] Example 33: 2-(3,7-Di(2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-3,4,5,6-tetrafluorobenzoate (JFS678, 75)

[0214] Step 1: A vial was charged with bis(3-bromophenyl)dimethylsilane (72; 500 mg, 1.35 mmol), 2-azabicyclo[2.1.1]hexane hydrochloride (37; 388 mg, 3.24 mmol, 2.4 eq), Pd2dba3(124 mg, 0.135 mmol, 0.1 eq), XPhos (193 mg, 0.405 mmol, 0.3 eq), and Cs₂CO₃ (2.11 g, 6.48 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3×). Dioxane (7 mL) was added; after flushing the reaction again with nitrogen (3×), it was stirred at 100 °C for 18 h. It was then cooled to room temperature, filtered through Celite with CH2CI2, and evaporated. The residue was purified by silica gel chromatography (0-30% Et2O / hexanes, linear gradient) to afford bis(3-(2-azabicyclo[2.1.1]hexan-2-yl)phenyl)dimethylsilane (73; 378 mg, 75%) as a paleyellow gum. 'H NMR (CDCh, 400 MHz) 87.19 (ddd, J= 8.2, 7.1, 0.6 Hz, 2H), 6.92 - 6.90 (m, 2H), 6.89 (dt, J= 7.0, 1.0 Hz, 2H), 6.74 (ddd, J= 8.2, 2.6, 1.1 Hz, 2H), 4.31 (dt, J= 6.7, 1.8 Hz, 2H), 3.30 (bs, 4H), 2.94 - 2.86 (m, 2H), 1.94 - 1.84 (m, 4H), 1.47 - 1.39 (m, 4H), 0.51 (s, 6H);13C NMR (CDCh, 101 MHz) 8 151.1 (C), 139.3 (C), 128.6 (CH), 123.6 (CH), 120.0 (CH), 114.9 (CH), 60.2 (CH), 55.3 (CH2), 40.1 (CH), 39.3 (CH2), -2.0 (CH3); HRMS (ESI) calcd for C24H3iN2Si [M+H]+375.2252, found 375.2253.

[0215] Step 2: The product from Step 1 (73; 325 mg, 0.868 mmol) and tetrafluorophthalaldehydic acid (56; 289 mg, 1.30 mmol, 1.5 eq) were combined in 2, 2, 3, 3, 4,4,4-heptafluoro-1 -butanol (3 mL) in a crimp-top vial. The reaction mixture was sparged with O2from a balloon for 10 min, then stirred at 95 °C under the O2balloon for 48 h. The reaction was cooled to room temperature, diluted into EtOAc, washed with saturated NaHCCh and brine, dried over anhydrous MgSCh, filtered, and concentrated in vacuo. Silica gel chromatography (10-100% EtOAc / toluene, linear gradient) yielded 343 mg (69%) of the title compound 2-(3,7-di(2-azabicyclo[2.1.1 ]hexan-2-yl)-5,5-dimethyldibenzo[6,e]silin-l 0-ylium- 10(5 / / )-yl)-3, 4,5,6-tetrafluorobenzoate as a blue solid. 'H NMR (CDCh, 400 MHz) 86.94 (d, J= 2.7 Hz, 2H), 6.73 (dd, J= 8.8, 1.3 Hz, 2H), 6.58 (dd, J= 8.8, 2.7 Hz, 2H), 4.37 (dt, J= 6.8, 1.8 Hz, 2H), 3.33 (bs, 4H), 2.98 -2.91 (m, 2H), 1.99 - 1.89 (m, 4H), 1.47- 1.39 (m, 4H), 0.57 (s, 3H), 0.55 (s, 3H);19F NMR (CDCh, 376 MHz) 8 -138.82 (td, J= 19.9, 4.0 Hz, IF), -139.38 (td, J= 20.0, 8.4 Hz, IF), -144.22 (ddd, J= 20.8, 18.1, 8.4 Hz, IF), -152.06 (ddd, J= 21.4, 18.3, 3.8 Hz, IF);Analytical HPLC: tR = 13.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 685 nm); HRMS (ESI) calcd for C32H29F4N2O2Si [M+H]+577.1929, found 577.1929.

[0216] Example 34: 2,2'-(5,5-Dimethyl-3'H,5H-spiro[dibenzo[b,e]siline-10,1'-isobenzofuran]-3,7-diyl)bis(4-fluoro-2-azabicyclo[2.1.1]hexane) (JFS645b, 131):

[0217] A vial was charged with hydroxymethyl-Si-fluorescein ditriflate (130; Holland, K. L. et al. bioRxiv 2024, doi: 10.1101 / 2024.02.23.581625; 150 mg, 0.240 mmol), 4-fluoro-2-azabicyclo[2.1.1]hexane hydrochloride (39; 79.3 mg, 0.580 mmol, 2.4 eq), RuPhos-G3-palladacycle (20.1 mg, 24.0 μmol, 0.1 eq), RuPhos (11.2 mg, 24.0 μmol, 0.1 eq), and Cs₂CO₃ (376 mg, 1.15 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x). Dioxane (2 mL) was added, and the reaction was flushed again with nitrogen (3x). The reaction was then stirred at 100 °C for 3 h. It was subsequently cooled to room temperature, filtered through Celite with CH2CI2, and concentrated to dryness. Purification by silica gel chromatography (0-30% EtOAc / hexanes, linear gradient) afforded the title compound (93 mg, 74%) as a white solid. 'H NMR (CDCI3, 400 MHz) 57.33 - 7.28 (m, 2H), 7.28 - 7.23 (m, 1H), 7.07 (dt, J= 7.4, 1.1 Hz, 1H), 6.97 (d, J= 8.7 Hz, 2H), 6.91 (d, J= 2.7 Hz, 2H), 6.54 (dd, J = 8.7, 2.8 Hz, 2H), 5.21 (s, 2H), 4.17 (dt, JHF = 21.6 Hz, J= 2.4 Hz, 2H), 3.39 - 3.32 (m, 4H), 2.11 - 1.99 (m, 8H), 0.60 (s, 3H), 0.54 (s, 3H);19F NMR (CDCI3, 376 MHz) 5 -164.23 - -164.34 (m);13C NMR (CDCI3, 101 MHz) 8 148.4 (C), 145.7 (C), 140.1 (C), 140.0 (C), 136.1 (C), 128.7 (CH), 127.4 (CH), 127.3 (CH), 124.8 (CH), 121.3 (CH), 118.4 (CH), 114.7 (CH), 92.6 (C), 91.4 (d, VCF = 264.8 Hz, CF), 72.4 (CH2), 53.7 (d, VCF = 25.3 Hz, CH2), 50.3 (d, VCF = 25.4 Hz, CH), 44.41 (d, VCF = 19.0 Hz, CH2), 44.38 (d, CF = 18.9 Hz, CH2), 0.7 (CH3), -1.2 (CH3); Analytical HPLC: tR = 12.7 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 655 nm); MS (ESI) calcd for C32H33F2N2OSi [M+H]+527.2, found 527.3.

[0218] Example 35: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)-4-( / e / -butoxy carbonyl )benzoate (6- / crz-butoxycarbonyl-JFS548, 89)...

[0219] The title compound (81%, red-pink solid) was prepared from 6-tert-butoxycarbonylfluorescein ditriflate (82; Grimm, J. B. et al. Nat. Methods 2015, 12, 244-250) and 4-fluoro-2-azabicyclo[2.1.1]hexane hydrochloride (39) according to the procedure described for Example 1. ^NMR ^DCh, 400 MHz) 88.19 (dd,. / = 8.0, 1.3 Hz, 1H), 8.03 (dd,. / = 8.0, 0.7 Hz, 1H), 7.78 - 7.73 (m, 1H), 6.57 (d, J= 8.7 Hz, 2H), 6.47 (d, J= 2.4 Hz, 2H), 6.34 (dd, J= 8.7, 2.5 Hz, 2H), 4.18 (dt,. / IIF = 21.3 Hz, J= 2.6 Hz, 2H), 3.41 - 3.31 (m, 4H), 2.18 - 2.10 (m, 4H), 2.10 - 2.02 (m, 4H), 1.55 (s, 9H);19F NMR (CDCI3, 376 MHz) 8 -164.64 (bd, JFH = 21.4 Hz);13C NMR (CDCI3, 101 MHz) 8 169.0 (C), 164.5 (C), 153.1 (C), 152.9 (C), 151.9 (C), 138.2 (C), 130.7 (CH), 130.6 (C), 129.2 (CH), 125.3 (CH), 124.9 (CH), 110.1 (CH), 108.2 (C), 100.0 (CH), 91.0 (d,1JCF= 265.0 Hz, CF), 85.7 (C), 82.6 (C), 53.2 (d,2JCF= 26.2 Hz, CH2), 50.7 (d,3JCF= 24.8 Hz, CH), 44.8 (d, J= 18.9 Hz, CH2), 28.2 (CH3); Analytical HPLC: tR= 13.5 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C35H33F2N2O5[M+H]+599.2353, found 599.2353.

[0220] Example 36: 4-(ter / -Butoxycarbonyl)-2-(3,6-di(2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (6- / t?rt-butoxycarbonyl-JFS56i, 83)

[0221] The title compound (82%, dark purple solid) was prepared from 6-tert-butoxy carbonylfluorescein ditriflate (82) and 2-azabicyclo[2.1.1]hexane hydrochloride (37) according to the procedure described for Example 1.NMR (CD3OD, 400 MHz) 88.19 (dd, J = 8.1, 1.7 Hz, 1H), 8.10 (d, J= 8.1 Hz, 1H), 7.77 (d, J= 1.6 Hz, 1H), 7.15 (d, J = 9.3 Hz, 2H), 6.89 (bd, J= 9.4 Hz, 2H), 6.80 (bs, 2H), 4.87 (dt, J= 6.8, 1.7 Hz, 2H), 3.58 (s, 4H), 3.09 (dt, J=6.7, 3.2 Hz, 2H), 2.28 - 2.20 (m, 4H), 1.59 (s, 9H), 1.57 - 1.51 (m, 4H);13C NMR (CD3OD, 101 MHz) 8 172.5 (C), 166.1 (C), 160.4 (C), 159.2 (C), 156.4 (C), 145.7 (C), 133.8 (C), 133.6 (C), 132.9 (CH), 131.4 (CH), 131.3 (CH), 130.9 (CH), 115.2 (CH), 114.8 (C), 96.7 (CH), 83.1 (C), 64.0 (CH), 53.1 (CH2), 41.8 (CH2), 39.8 (CH), 28.4 (CH3); Analytical HPLC: tR= 13.4 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 565 nm); MS (ESI) calcd for C35H35N2O5[M+H]+563.3, found 563.4.

[0222] Example 37: 2-(3, 6-Bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)-l 0,10-dimethylanthracen-9-ylium-9(10 / / )-yl)-4-(to -butoxycarbonyl)benzoate (6-terf-butoxycarbonyl-JFS607, 103)

[0223] The title compound (85%, pale blue solid) was prepared from 6- / C / 7-butoxycarbonylcarbofluorescein ditriflate (96; Grimm, J. B. et al. Nat. Methods 2017, 14, 987-994) and 4-fluoro-2-azabicyclo[2.1.1 ]hexane hydrochloride (39) according to the procedure described for Example 1. ^NMR^DCh, 400 MHz) 88.15 (dd, J = 8.0, 1.4 Hz, 1H), 8.01 (dd, J= 8.0, 0.8 Hz, 1H), 7.63 (dd, J= 1.3, 0.8 Hz, 1H), 6.84 (d, J= 2.4 Hz, 2H), 6.57 (d, J= 8.6 Hz, 2H), 6.46 (dd, J= 8.7, 2.5 Hz, 2H), 4.20 (dt,. / = 21.3 Hz, J= 2.5 Hz, 2H), 3.42 - 3.36 (m, 4H), 2.16 -2.03 (m, 8H), 1.84 (s, 3H), 1.75 (s, 3H), 1.53 (s, 9H);19F NMR (CDCI3, 376 MHz) 8 - 164.41 (bd, JFH = 21.4 Hz);13C NMR (CDCI3, 101 MHz) 8 170.0 (C), 164.6 (C), 155.5 (C), 150.5 (C), 147.0 (C), 137.9 (C), 130.2 (CH), 130.1 (C), 129.2 (CH), 125.1 (CH), 124.9 (CH), 120.9 (C), 112.7 (CH), 110.8 (CH), 91.2 (d, CF = 265.0 Hz, CF), 88.1 (C), 82.5 (C), 53.6 (d,2JCF = 25.8 Hz, CH2), 50.4 (d,3, / CF = 25.1 Hz, CH), 44.6 (d,2JCF = 19.1 Hz, CH2), 38.5 (C), 35.5 (CH3), 32.9 (CH3), 28.2 (CH3); Analytical HPLC: tR= 11.2 min, >99% purity (30-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 615 nm); HRMS (ESI) calcd for C38H39F2N2O4[M+H]+625.2873, found 625.2873.

[0224] Example 38: 4-(terz-Butoxycarbonyl)-2-(3,6-di(2-azabicyclo[2.1.1 ]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)benzoate (6- / e / 7-butoxycarbonyl-JFS6i9, 97)

[0225] The title compound (86%, blue solid) was prepared from 6- / e / 7-butoxycarbonylcarbofluorescein ditriflate (96) and 2-azabicyclo[2.1.1]hexane hydrochloride (37) according to the procedure described for Example 1.rH NMR (CDCh, 400 MHz) 58.14 (dd, J = 8.0, 1.3 Hz, 1H), 8.00 (dd, J= 8.0, 0.8 Hz, 1H), 7.65 (dd, J= 1.3, 0.7 Hz, 1H), 6.90 (d, J= 2.4 Hz, 2H), 6.54 (d, J= 8.7 Hz, 2H), 6.49 (dd, J= 8.7, 2.4 Hz, 2H), 4.35 (dt, J= 6.7, 1.8 Hz, 2H), 3.38 - 3.3 l (m, 4H), 2.97 -2.92 (m, 2H), 1.97 - 1.90 (m, 4H), 1.85 (s, 3H), 1.75 (s, 3H), 1.53 (s, 9H), 1.48 - 1.41 (m, 4H);13C NMR (CDCI3, 101 MHz) 5 170.2 (C), 164.6 (C), 155.5 (C), 152.0 (C), 147.1 (C), 137.7 (C), 130.4 (C), 130.0 (CH), 129.1 (CH), 125.2 (CH), 124.8 (CH), 119.8 (C), 112.9 (CH), 110.9 (CH), 89.3 (C), 82.3 (C), 60.4 (CH), 54.7 (CH2), 40.0 (CH), 39.61 (CH2), 39.59 (CH2), 38.5 (C), 35.5 (CH3), 32.8 (CH3), 28.2 (CH3); Analytical HPLC: tR= 11.9 min, >99% purity (30-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 625 nm); HRMS (ESI) calcd for C38H41N2O4[M+H]+589.3061, found 589.3062.

[0226] Example 39: 2-(3,7-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin- 10-ylium- 10(57 )-yl)-4-(te / 7-butoxycarbonyl)benzoate (6-tert-butoxycarbonyl-JFS645, 118)UXJ*

[0227] The title compound (76%, off-white solid) was prepared from 6-tert-butoxycarbonyl-Si-fluorescein ditriflate (113; Grimm, J. B. et al. Nat. Methods 2015, 72, 244-250) and 4-fluoro-2-azabicyclo[2.1.1 ]hexane hydrochloride (39) according to the procedure described for Example 1.rH NMR (CDCh, 400 MHz) 58.13 (dd, J= 8.0, 1.3 Hz, 1H), 7.97 (dd, J= 8.0, 0.8 Hz, 1H), 7.85 (dd, J= 1.3, 0.7 Hz, 1H), 6.91 (d, J= 2.7 Hz, 2H), 6.84 (d, J= 8.8 Hz, 2H), 6.54 (dd, J= 8.8, 2.7 Hz, 2H), 4.19 (dt,. / m = 21.4 Hz, J= 2.6 Hz, 2H), 3.39 - 3.34 (m, 4H), 2.14 - 2.07 (m, 4H), 2.07 - 2.00 (m, 4H), 1.56 (s, 9H), 0.66 (s, 3H), 0.60 (s, 3H);19F NMR (CDCh, 376 MHz) 5 -164.49 (bd, JFH = 21.3 Hz);13C NMR (CDCh, 101 MHz) 5 170.2 (C), 164.5 (C), 154.9 (C), 149.1 (C), 137.3 (C), 136.8 (C), 133.4 (C), 130.1 (CH), 129.3 (C), 128.0 (CH), 125.8 (CH), 125.3 (CH), 118.3 (CH), 114.7 (CH), 91.5 (C), 91.2 (d, CF = 264.9 Hz, CF), 82.5 (C), 53.4 (d,2JCF = 25.8 Hz, CH2), 50.3 (d,3. / CF = 25.1 Hz, CH), 44.61 (d, CF = 19.0 Hz, CH2), 44.59 (d, CF = 18.9 Hz, CH2), 28.2 (CH3), 0.2 (CH3), -0.8 (CH3); Analytical HPLC: tR= 16.7 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C37H39F2N2O4Si [M+H]+641.2642, found 641.2643.

[0228] Example 40: 4-(teH-Butoxycarbonyl)-2-(3,7-di(2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin-10-ylium-10(5 / / )-yl)benzoate (6-ter / -butoxycarbonyl-JFS657, 114)

[0229] The title compound (44%, yellow solid) was prepared from 6- / C / 7-butoxycarbonyl-Si-fluorescein ditriflate (113) and 2-azabicyclo[2.1.1]hexane hydrochloride (37) according to the procedure described for Example 1.rH NMR (CDCh, 400 MHz) 58.12 (dd, J = 8.0, 1.3 Hz, 1H), 7.96 (dd, J= 8.0, 0.8 Hz, 1H), 7.86 (dd, J= 1.3, 0.8 Hz, 1H), 6.96 (d, J = 2.7 Hz, 2H), 6.79 (d, J= 8.8 Hz, 2H), 6.56 (dd, J= 8.8, 2.7 Hz, 2H), 4.35 (dt, J= 6.8, 1.8 Hz, 2H), 3.35 - 3.29 (m, 4H), 2.96 -2.91 (m, 2H), 1.96 - 1.88 (m, 4H), 1.55 (s, 9H), 1.46 - 1.39 (m, 4H), 0.65 (s, 3H), 0.59 (s, 3H);13C NMR (CDCh, 101 MHz) 5 170.4 (C), 164.6 (C), 155.3 (C), 150.5 (C), 137.1 (C), 136.7 (C), 132.2 (C), 129.9 (CH), 129.5 (C), 128.0 (CH), 125.6 (CH), 125.4 (CH), 118.5 (CH), 114.9 (CH), 92.0 (C), 82.3 (C), 60.3 (CH), 54.5 (CH2), 40.0 (CH), 39.61 (CH2), 39.58 (CH2), 28.2 (CH3), 0.3 (CH3), -0.8 (CH3); Analytical HPLC: tR= 14.3 min, >99%purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); MS (ESI) calcd for C37H41N2O4Si [M+H]+605.3, found 605.5.

[0230] Example 41: 2-(3,7-Bis(4-(methoxycarbonyl)-2-azabicyclo[2.1. l]hexan-2-yl)-5,5-dimethyldibenzo[Z»,e]silin-10-ylium-10(57 / )-yl)-4-(ter / -butoxycarbonyl)benzoate (126)I 1 J jJ< < -If '

[0231] The title compound (88%, pale yellow solid) was prepared from 6- / c' / 7-butoxycarbonyl-Si-fluorescein ditriflate (113) and methyl 2-azabicyclo[2.1.1]hexane-4-carboxylate hydrochloride (48) according to the procedure described for Example 1.JH NMR (CDC13, 400 MHz) 5 8.12 (dd, J= 8.0, 1.3 Hz, 1H), 7.97 (dd, J= 8.0, 0.8 Hz, 1H), 7.84 (dd, J = 1.3, 0.7 Hz, 1H), 6.96 (d, J= 2.7 Hz, 2H), 6.83 (d, J= 8.8 Hz, 2H), 6.57 (dd, J= 8.8, 2.7 Hz, 2H), 4.32 (t, J = 1.9 Hz, 2H), 3.75 (s, 6H), 3.58 - 3.51 (m, 4H), 2.28 - 2.19 (m, 4H), 1.84- 1.76 (m, 4H), 1.58 (s, 9H), 0.67 (s, 3H), 0.60 (s, 3H);13C NMR (CDCI3, 101 MHz) 8 171.0 (C), 170.3 (C), 164.5 (C), 155.1 (C), 149.7 (C), 137.3 (C), 136.7 (C), 133.1 (C), 130.0 (CH), 129.2 (C), 128.0 (CH), 125.7 (CH), 125.2 (CH), 118.6 (CH), 114.9 (CH), 91.6 (C), 82.4 (C), 58.1 (CH), 55.3 (CH2), 52.4 (C), 52.1 (CH3), 42.7 (CH2), 28.2 (CH3), 0.2 (CH3), -0.7 (CH3); Analytical HPLC: tR = 14.1 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 660 nm); HRMS (ESI) calcd for C4iH45N2O8Si [M+H]+721.2940, found 721.2939.

[0232] Example 42: / c V-Butyl 3,7-bis(4-fluoro-2-azabicyclo[2.1 l]hexan-2-yl)-5,5-dimethyl-37 / ,5Z / -spiro[dibenzo[Z>,e]siline-10,r-isobenzofuran]-6'-carboxylate (6-tert-butoxycarbonyl-IFS64sb, 133)

[0233] A vial was charged with 6- / c / 7-butoxycarbonyl-hydroxymethyl-Si-fluorescein ditriflate (132; Holland, K. L. et al. bioRxiv 2024, doi: 10.1101 / 2024.02.23.581625; 250 mg, 0.340 mmol), 4-fluoro-2-azabicyclo[2.1.1]hexane hydrochloride (39; 114 mg, 0.830 mmol, 2.4 eq), RuPhos-G3-palladacycle (28.9 mg, 34.0 μmol, 0.1 eq), RuPhos (16.1 mg, 34.0 μmol, 0.1 eq), and Cs₂CO₃ (540 mg, 1.66 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x). Dioxane (3 mL) was added, and the reaction was flushed again with nitrogen (3x). The reaction was then stirred at 100 °C for 3 h. It was subsequently cooled to room temperature, filtered through Celite with CH2C12, and concentrated to dryness. Purification by silica gel chromatography (0-30% EtOAc / hexanes, linear gradient) afforded the title compound (158 mg, 73%) as a white solid. 'H NMR (CDCh, 400 MHz) 87.92 (dd, J= 7.9, 1.4 Hz, 1H), 7.62 (dd, J = 1.5, 0.6 Hz, 1H), 7.32 (dd, J= 7.9, 0.8 Hz, 1H), 7.05 (d, J= 8.7 Hz, 2H), 6.90 (d, J= 2.7 Hz, 2H), 6.59 (dd, J= 8.8, 2.8 Hz, 2H), 5.36 (s, 2H), 4.18 (dt, J= 21.4, 2.4 Hz, 2H), 3.39 - 3.33 (m, 4H), 2.12 -2.00 (m, 8H), 1.52 (s, 9H), 0.65 (s, 3H), 0.54 (s, 3H);19F NMR (CDCh, 376 MHz) 8 -164.26 - -164.37 (m);13C NMR (CDCh, 101 MHz) 8 165.6 (C), 148.5 (C), 147.0 (C), 143.5 (C), 140.3 (C), 135.3 (C), 131.9 (C), 129.0 (CH), 128.5 (CH), 125.4 (CH), 121.2 (CH), 118.2 (CH), 115.0 (CH), 92.5 (C), 91.4 (d, CF = 264.8 Hz, CF), 81.3 (C), 73.0 (CH2), 53.7 (d,2. / CF = 25.3 Hz, CH2), 50.3 (d, CF = 25.4 Hz, CH), 44.5 (d, CF = 18.9 Hz, CH2), 44.4 (d,2JCF = 18.9 Hz, CH2), 28.3 (CH3), 0.4 (CH3), -0.4 (CH3); Analytical HPLC: tR= 14.2 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 655 nm); MS (ESI) calcd for C37H41F2N2O3Si [M+H]+627.3, found 627.3.

[0234] Example 43: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)xanthylium-9-yl)-4-carboxybenzoate (6-carboxy-JFS548, 90)

[0235] 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1. I ]hexan-2-yl)xanthylium-9-yl)-4-( / c / 7-butoxycarbonyl)benzoate (Example 24; 89; 153 mg, 0.256 mmol) was taken up in CH2CI2 (5 mL), and trifluoroacetic acid (1 mL) was added. The reaction was stirred at room temperature for 6 h. Toluene (6 mL) was added; the reaction mixture was concentrated to dryness and then azeotroped with MeOH three times to provide the title compound as a dark red-purple solid (159 mg, 95%, TFA salt). Analytical HPLC and NMR indicated that the material was >95% pure and did not require further purification prior to amide coupling.JH NMR (CD3OD, 400 MHz, 325 K) 88.41 (dd, J= 8.2, 0.8 Hz, 1H), 8.38 (dd, J = 8.2, 1.5 Hz, 1H), 7.96 (dd, J= 1.5, 0.7 Hz, 1H), 7.12 (d, J= 9.4 Hz, 2H), 6.94 (dd, J= 9.3, 2.1 Hz, 2H), 6.90 (d, J= 2.2 Hz, 2H), 4.84 (dt,, / i n = 20.6 Hz, J= 2.5 Hz, 2H), 3.68 (bs, 4H), 2.46 - 2.37 (m, 4H), 2.21 - 2.13 (m, 4H);19F NMR (CD3OD, 376 MHz, 325 K) 8 -75.61 (s, 3F), -166.38 (bd,. / in = 20.4 Hz, 2F);13C NMR (CD3OD, 101 MHz, 325 K) 8 167.7 (C), 167.4 (C), 160.0 (C), 159.4 (C), 156.3 (C), 136.2 (C), 136.0 (C), 135.3 (C), 132.8 (CH), 132.5 (CH), 132.34 (CH), 132.26 (CH), 115.4 (C), 115.2 (CH), 96.8 (CH), 91.0 (d, VCF = 263.7 Hz, CF), 53.8 (d,3. / CT = 22.4 Hz, CH), 51.8 (d,2JCF = 29.4 Hz, CH2), 46.72 (d,2JCF = 19.3 Hz, CH2); Analytical HPLC: tR= 11.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C31H25F2N2O5[M+H]+543.1727, found 543.1728.

[0236] Example 44: 4-Carboxy-2-(3,6-di(2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (6-carboxy-JFS56i, 84)

[0237] The title compound (-100%, dark purple solid, TFA salt) was prepared from 4-(tert-butoxycarbonyl)-2-(3,6-di(2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (Example 25; 83) according to the procedure described for Example 32.1H NMR (CD3OD, 400 MHz, 325 K) 88.39 (dd, J = 8.3, 0.8 Hz, 1H), 8.36 (dd, J= 8.2, 1.5 Hz, 1H), 7.96 (dd, J= 1.5, 0.8 Hz, 1H), 7.04 (d, J= 9.3 Hz, 2H), 6.91 (bd, J= 9.5 Hz, 2H), 6.84 (bs, 2H), 4.88 (dt, J= 6.8, 1.7 Hz, 2H), 3.60 (s, 4H), 3.10 (dt, J= 6.5, 3.1 Hz, 2H), 2.29 - 2.21 (m, 4H), 1.60 - 1.53 (m, 4H);13C NMR (CD3OD, 101 MHz, 300 K) 8 167.7 (C), 167.4 (C), 159.1 (C), 158.2 (C), 156.4 (C), 136.1 (C), 135.9 (C), 135.6 (C), 132.8 (CH), 132.4 (CH), 132.19 (CH), 132.16 (CH), 115.5 (CH), 114.6 (C), 96.8 (CH), 64.2 (CH), 53.1 (CH2), 41.9 (CH2), 39.7 (CH); Analytical HPLC: tR= 10.6 min, 98.2% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 565 nm); MS (ESI) calcd for C31H27N2O5[M+H]+507.2, found 507.2.

[0238] Example 45: 4-Carboxy-2-(3,6-di(2-azabicyclo[2.1. l]hexan-2-yl)thioxanthylium-9-yl)benzoate (6-carboxy-JFSs82, 94)

[0239] The title compound (53%, dark red solid, acetate salt) was prepared from bis(3-(2-azabicyclo[2.1.1]hexan-2-yl)phenyl)sulfane (Example 12, Step 1; 52) and 2-formylterephthalic acid (93) according to the procedure described for Example 11, Step 2. 'H NMR (CD3OD, 400 MHz, 325 K) 38.39 (dd, J = 8.2, 0.6 Hz, 1H), 8.35 (dd, J= 8.1, 1.6 Hz, 1H), 7.90 (dd, J= 1.6, 0.7 Hz, 1H), 7.16 (bs, 2H), 7.11 (d, J= 9.5 Hz, 2H), 6.91 (bd, J= 9.5 Hz, 2H), 4.87 (dt, J= 6.9, 1.7 Hz, 2H), 3.58 (bs, 4H), 3.08 (dt, J= 6.7, 3.2 Hz, 2H), 2.28 – 2.18 (m, 4H), 1.58 - 1.49 (m, 4H);13C NMR (CD3OD, 101 MHz, 325 K) 8 167.9 (C), 167.5 (C), 160.0 (C), 152.9 (C), 145.1 (C), 139.1 (C), 136.6 (CH), 136.2 (C), 135.9 (C), 132.7 (CH), 132.6 (CH), 131.7 (CH), 119.9 (C), 116.7 (CH), 106.2 (CH), 64.0 (CH), 53.0 (CH2), 41.9 (CH2), 39.7 (CH); Analytical HPLC: tR= 10.8 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFAadditive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 590 nm); HRMS (ESI) calcd for C31H27N2O4S [M+H]+523.1687, found 523.1686.

[0240] Example 46: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)-l 0,10-dimethylanthracen-9-ylium-9(10 / 7)-yl)-4-carboxybenzoate (6-carboxy-JFS607, 104)

[0241] The title compound (99%, blue solid, TFA salt) was prepared from 2-(3,6-bis(4-fluoro-2-azabicy clo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9( 10 / / )-yl )-4-(Ze / 7-butoxycarbonyl)benzoate (Example 26; 103) according to the procedure described for Example 32. ’H NMR (CD3OD, 400 MHZ, 325 K) 58.35 - 8.30 (m, 2H), 7.85 (t, J= 1.1 Hz, 1H), 7.13 (d, J= 2.4 Hz, 2H), 6.96 (d, J= 9.2 Hz, 2H), 6.69 (dd, J= 9.2, 2.3 Hz, 2H), 4.88 (dt, JHF = 20.6 Hz, J = 2.5 Hz, 2H), 3.68 (bs, 4H), 2.43 - 2.34 (m, 4H), 2.19 -2.11 (m, 4H), 1.87 (s, 3H), 1.76 (s, 3H);19F NMR (CD3OD, 376 MHz, 325 K) 5 -75.56 (s, 3F), -166.29 (bd, JFH = 20.7 Hz, 2F); Analytical HPLC: tR = 11.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 615 nm); HRMS (ESI) calcd for C34H31F2N2O4[M+H]+569.2247, found 569.2247.

[0242] Example 47: 4-Carboxy-2-(3, 6-di(2-azabicyclo[2.1.1 ]hexan-2-yl)-l 0,10-dimethylanthracen-9-ylium-9(107 )-yl)benzoate (6-carboxy-JFS6i9, 98)\ / XX"stXXT'''HO.. C -

[0243] The title compound (-100%, blue solid, TFA salt) was prepared from 4-( / er / -butoxycarbonyl)-2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10 / )-yl)benzoate (Example 27; 97) according to the procedure described for Example 32. 'H NMR (CD3OD, 400 MHz, 325 K) 58.33 (d, J= 8.2 Hz, 1H), 8.30 (dd, J= 8.3, 1.4 Hz, 1H), 7.87(d, J= 1.5 Hz, 1H), 7.13 (d, J =2.3 Hz, 2H), 6.93 (d, J = 9.3 Hz, 2H), 6.68 (dd, J= 9.3, 2.3 Hz, 2H), 4.95 (d, J= 6.7 Hz, 2H), 3.64 (bs, 4H), 3.09 (dt, J= 6.6, 3.1 Hz, 2H), 2.30 - 2.21 (m, 4H), 1.86 (s, 3H), 1.75 (s, 3H), 1.60 - 1.52 (m, 4H);13C NMR (CD3OD, 101 MHz, 325 K) 5 168.0 (C), 167.7 (C), 163.5 (C), 158.2 (C), 155.3 (C), 139.2 (C), 137.8 (CH), 136.5 (C), 135.4 (C), 132.6 (CH), 132.4 (CH), 131.3 (CH), 121.6 (C), 113.9 (CH), 111.7 (CH), 64.1 (CH), 53.1 (CH2), 42.9 (C), 41.9 (CH2), 39.6 (CH), 35.5 (CH3), 32.4 (CH3); Analytical HPLC: tR= 11.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 625 nm); HRMS (ESI) calcd for C34H33N2O4[M+H]+533.2435, found 533.2435.

[0244] Example 48: 2-(3,7-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin- 10-ylium- 10(5 / / )-yl)-4-carboxybenzoate (6-carboxy-IF S645, 119)ZJ

[0245] The title compound (99%, dark blue solid, TFA salt) was prepared from 2-(3,7-bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin-10-ylium-10(5 / / )-yl)-4-( / c77-butoxycarbony I (benzoate (Example 28; 118) according to the procedure described for Example 32. ‘HNMR (CD3OD, 400 MHz, 325 K) 58.26 (dd, J= 8.1, 1.5 Hz, 1H), 8.19 (d, J= 8.1 Hz, 1H), 7.83 (d, J= 1.4 Hz, 1H), 7.16 (d, J= 2.8 Hz, 2H), 6.88 (d, J= 9.2 Hz, 2H), 6.62 (dd, J= 9.2, 2.7 Hz, 2H), 4.74 -4.64 (m, 2H), 3.55 (bs, 4H), 2.33 -2.23 (m, 4H), 2.13 -2.03 (m, 4H), 0.65 (s, 3H), 0.57 (s, 3H);19F NMR (CD3OD, 376 MHz, 325 K) 8 -75.88 (s, 3F), -164.80 --166.52 (m, 2F); Analytical HPLC: tR= 13.5 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C33H31F2N2O4Si [M+H]+585.2016, found 585.2018.

[0246] Example 49: 4-Carboxy-2-(3,7-di(2-azabicyclo[2.1. l]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin- 10-ylium- 10(57 )-yl)benzoate (6-carboxy-JF S657, 115)

[0247] The title compound (-100%, dark blue solid, TFA salt) was prepared from 4-( / e / 7-butoxycarbonyl)-2-(3,7-di(2-azabicyclo[2.1.1 ]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin- 10-ylium- 10(57 )-yl)benzoate (Example 29; 114) according to the procedure described for Example 32. 'H NMR (CD3OD, 400 MHz, 325 K) 88.30 (dd, J= 8.2, 0.7 Hz, 1H), 8.27 (dd, J= 8.2, 1.5 Hz, 1H), 7.82 (dd, J= 1.6, 0.8 Hz, 1H), 7.22 (d, J= 2.7 Hz, 2H), 6.88 (d, J= 9.5 Hz, 2H), 6.63 (dd, J= 9.5, 2.7 Hz, 2H), 4.95 (bd, J= 6.7 Hz, 2H), 3.63 (s, 4H), 3.08 (dt, J= 6.6, 3.2 Hz, 2H), 2.28 -2.20 (m, 4H), 1.58 - 1.51 (m, 4H), 0.63 (s, 3H), 0.57 (s, 3H);13C NMR (CD3OD, 101 MHz, 300 K) 8 168.0 (C), 167.7 (C), 152.6 (C), 148.73 (C), 142.84 (C), 141.2 (CH), 136.0 (C), 135.3 (C), 132.5 (CH), 132.3 (CH), 130.9 (CH), 128.8 (C), 121.8 (CH), 118.8 (C), 114.8 (CH), 64.2 (CH), 53.0 (CH2), 41.9 (CH2), 39.5 (CH), -0.8 (CH3), -1.8 (CH3); Analytical HPLC: tR= 10.7 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); MS (ESI) calcd for C33H33N2O4Si [M+H]+549.2, found 549.3.

[0248] Example 50: 2-(3,7-Bis(4-(methoxycarbonyl)-2-azabicyclo[2.1,l]hexan-2-yl)-5,5-dimethyldibenzo[ / >,t?]silin-10-ylium-10(5 / / )-yl)-4-carboxybenzoate (127)

[0249] The title compound (-100%, dark blue solid, TFA salt) was prepared from 2-(3,7-bis(4-(methoxycarbonyl)-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin-10-ylium-10(5 / / )-yl)-4-( / crt-butoxycarbonyl)benzoate (Example 30; 126) according to the procedure described for Example 32. 'H NMR (CD3OD, 400 MHz, 325 K) 88.27 (dd,.7= 8.1, 1.5 Hz, 1H), 8.24 (d, J= 8.2 Hz, 1H), 7.86 - 7.81 (m, 1H), 7.21 (d, J= 2.6 Hz, 2H), 6.89 (d, J= 9.3 Hz, 2H), 6.66 (dd, J= 9.3, 2.7 Hz, 2H), 4.84 (s, 2H), 3.80 - 3.74 (m, 10H), 2.48 - 2.38 (m,4H), 1.91 - 1.81 (m, 4H), 0.65 (s, 3H), 0.58 (s, 3H); Analytical HPLC: tR= 11.3 min, 98.8% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 660 nm); HRMS (ESI) calcd for C37H37N2O8Si [M+H]+665.2314, found 665.2314.

[0250] Example 51: 3,7-Bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)-5,5-dimethyl-37 / ,57 / -spiro[dibenzo[Z>,e]siline-10, l'-isobenzofuran]-6'-carboxylic acid (6-carboxy-JFS645b, 134)

[0251] / c / 7-Butyl 3,7-bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)-5,5-dimethyl-3' / / ,5 / / -spiro[dibenzo[Z»,e]siline-10,^-isobenzofuran]-6'-carboxylate (Example 31; 133; 135 mg, 0.215 mmol) was taken up in CH2CI2 (4 mL), and trifluoroacetic acid (0.8 mL) was added. The reaction was stirred at room temperature for 3 h. Toluene (3 mL) was added; the reaction mixture was concentrated to dryness and then azeotroped with MeOH (3x). The residue was redissolved in 1: 1 THF / MeOH (3 mL), and 1 M NaOH (600 pL, 0.600 mmol, 3 eq) was added. After stirring the reaction at room temperature for 1 h, it was acidified with 1 M HC1 (600 pL), diluted with water, and extracted with CH2CI2 (2x). The combined organic extracts were washed with brine, dried over anhydrous MgSCU, filtered, and concentrated in vacuo to afford 108 mg (88%) of the title compound as a blue solid. Analytical HPLC and NMR indicated that the material was >99% pure and did not require further purification prior to amide coupling.1H NMR (CDCI3, 400 MHz) 68.02 (dd, J= 7.9, 1.5 Hz, 1H), 7.76 (dd, J= 1.5, 0.6 Hz, 1H), 7.38 (dd, J= 8.0, 0.8 Hz, 1H), 6.99 (d, J= 8.7 Hz, 2H), 6.91 (d, J= 2.7 Hz, 2H), 6.57 (dd, J= 8.8, 2.8 Hz, 2H), 5.33 (s, 2H), 4.17 (dt,. / in = 21.6 Hz, J= 2.5 Hz, 2H), 3.40 - 3.32 (m, 4H), 2.11 -2.01 (m, 8H), 0.65 (s, 3H), 0.53 (s, 3H);19F NMR (CDCI3, 376 MHz) 8 -164.36 (bd, JFII = 21.3 Hz);13C NMR (CDCI3, 101 MHz) 6 171.1 (C), 148.5 (C), 147.1 (C), 145.4 (C), 139.9 (C), 135.7 (C), 129.7 (CH), 129.1 (C), 128.4 (CH), 126.5 (CH), 121.5 (CH), 118.6 (CH), 115.0 (CH), 92.5 (C), 91.4 (d, '. / ci = 264.9 Hz, CF), 72.7 (CH2), 53.7 (d, CF = 25.3 Hz, CH2), 50.3 (d, CF = 25.4 Hz, CH), 44.39 (d,2JCF = 19.1 Hz, CH2), 44.38 (d,2JCF = 18.9 Hz, CH2), 0.6 (CH3), -0.9 (CH3); Analytical HPLC: tR= 11.5 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 655 nm); MS (ESI) calcd for C33H33F2N2O3Si [M+H]+571.2, found 571.2.

[0252] Example 52: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1,l]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(1077)-yl)-4-(dicyano(methoxymethoxy)methyl)-3,5,6-trifluorobenzoate (6-(MOM-MAC)-JFS632, 110)

[0253] 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)-3,4,5,6-tetrafluorobenzoate (Example 17; 68; 250 mg, 0.419 mmol) and 2-(methoxymethoxy)malononitrile (108; 52.9 mg, 0.419 mmol, 1 eq) were combined in DMF (5 mL), and DIEA (146 pL, 0.838 mmol, 2 eq) was added. After stirring the reaction at room temperature for 2 h, it was concentrated in vacuo and purified by silica gel chromatography (10-100% EtOAc / toluene, linear gradient) to yield 141 mg (48%) of the title compound as a bluegreen foam. ’H NMR (CDC13, 400 MHz) 56.82 (d, J= 2.5 Hz, 2H), 6.66 (d, J= 8.6 Hz, 2H), 6.53 (dd, J= 8.7, 2.5 Hz, 2H), 5.12 (s, 2H), 4.24 (dt, J= 21.3, 2.5 Hz, 2H), 3.50 (s, 3H), 3.44 -3.38 (m, 4H), 2.19-2.12 (m, 4H), 2.12 - 2.07 (m, 4H), 1.77 (s, 3H), 1.72 (s, 3H);19F NMR (CDC13, 376 MHz) 8 -116.37 (d, J= 22.9 Hz, IF), -127.19 (d, J= 20.3 Hz, IF), -140.02 (dd, J = 22.9, 20.2 Hz, IF), -164.49 - -164.61 (m, 2F); Analytical HPLC: tR= 12.8 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 640 nm); HRMS (ESI) calcd for C38H32F5N4O4[M+H]+703.2339, found 703.2338.

[0254] Example 53: 2-(3,6-Di(2-azabicyclo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)-4-(dicyano(methoxymethoxy)methyl)-3,5,6-trifluorobenzoate (6-(M0M-MAC)-JFS643, 109)X r!-fciC f

[0255] The title compound (36%, blue solid) was prepared from 2-(3,6-di(2-azabicy clo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9(l 0 / / )-yl)-3,4,5,6-tetrafluorobenzoate (Example 18; 67) according to the procedure described for Example 41. ’H NMR (CDCh, 400 MHz) 86.86 (d, J= 2.5 Hz, 2H), 6.73 (d, J= 8.7 Hz, 2H), 6.56 (dd, J= 8.8, 2.4 Hz, 2H), 5.12 (s, 2H), 4.44 (dt, J= 6.8, 1.8 Hz, 2H), 3.51 (s, 3H), 3.40 (bs, 4H), 2.99 (dt, J= 6.5, 3.1 Hz, 2H), 2.05 - 1.95 (m, 4H), 1.77 (s, 3H), 1.73 (s, 3H), 1.52 - 1.44 (m, 4H);19FNMR (CDCh, 376 MHz) 8 -115.94 (d, J= 21.9 Hz, IF), -128.07 (d, J= 20.7 Hz, IF), -140.36 (t, J = 21.2 Hz, IF); Analytical HPLC: tR = 12.8 min, >99% purity (10-95% MeCN / HzO, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C38H34F3N4O4 [M+H]+667.2527, found 667.2526.

[0256] Example 54: 2-(3,7-Bis(4-fluoro-2-azabicyclo[2.1,l]hexan-2-yl)-5,5-dimethyldibenzo[ / >,e]silin- 10-ylium- 10(51 / )-yl)-4-(dicyano(methoxymethoxy)methyl)-3, 5,6-trifluorobenzoate (6-(MOM-MAC)-JFSe7o, 123)

[0257] The title compound (50%, light green solid) was prepared from 2-(3,7-bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)-5, 5-dimethyldibenzo[6,e]silin-l 0-ylium- 10(5 / / )-yl)-3,4,5,6-tetrafluorobenzoate (Example 21; 76) according to the procedure described for Example 41. ^NMRXCDCh, 400 MHz) 86.90 (d, J= 2.7 Hz, 2H), 6.73 (dd, J= 8.8, 1.2 Hz, 2H), 6.58 (dd, J= 8.8, 2.7 Hz, 2H), 5.18 (s, 2H), 4.23 (dt, JHF = 21.3 Hz, J= 2.5 Hz, 2H), 3.53 (s, 3H), 3.39 (bs, 4H), 2.19 - 2.10 (m, 4H), 2.09 -2.02 (m, 4H), 0.58 (s, 3H), 0.56 (s, 3H);19F NMR (CDCh,376 MHz) 8 -113.19 (d, J = 22.8 Hz, IF), -127.23 (d, J= 19.9 Hz, IF), -139.38 (dd, J= 22.8, 20.1 Hz, IF), -164.63 (bd,. in = 21.0 Hz, 2F); Analytical HPLC: tR= 14.2 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 675 nm); HRMS (ESI) calcd for C37H32F5N4O4Si [M+H]+719.2108, found 719.2108.

[0258] Example 55: 2-(3,7-Di(2-azabicyclo[2.1,l]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin- 10-ylium- 10(5 / / )-yl)-4-(dicyano(methoxymethoxy)methyl)-3, 5, 6-trifluorobenzoate (6-(MOM-MAC)-JFSe78, 122)

[0259] The title compound (48%, green solid) was prepared from 2-(3,7-di(2-azabicyclo[2.1.1 ]hexan-2-yl)-5, 5-dimethyldibenzo[ / >, e]silin-l 0-ylium- 10(5 7)-yl)-3, 4,5,6-tetrafluorobenzoate (Example 22; 75) according to the procedure described for Example 41. 'H NMR (CDCh, 400 MHz) 56.94 (d, J= 2.7 Hz, 2H), 6.70 (dd, J= 8.8, 1.2 Hz, 2H), 6.60 (dd, J = 8.8, 2.7 Hz, 2H), 5.17 (s, 2H), 4.38 (dt, J= 6.7, 1.8 Hz, 2H), 3.53 (s, 3H), 3.34 (bs, 4H), 2.98 -2.93 (m, 2H), 2.00 - 1.90 (m, 4H), 1.48 - 1.40 (m, 4H), 0.57 (s, 3H), 0.55 (s, 3H);19F NMR (CDCh, 376 MHz) 8 -113.15 (d, J= 22.5 Hz, IF), -127.88 (d, J= 20.2 Hz, IF), -139.94 (dd, J = 22.7, 20.2 Hz, IF); Analytical HPLC: tR= 13.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 685 nm); HRMS (ESI) calcd for C37H34F3N4O4Si [M+H]+683.2296, found 683.2297.

[0260] Example 56: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (JFS548-HaloTag® ligand, 91)

[0261] 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)-4-carboxybenzoate (Example 32; 90; TFA salt; 50 mg, 76.2 ymol) was combined with DSC (49.1 mg, 0.192 mmol, 2.5 eq) in DMF (4 m ). After adding EtsN (66.7 pL, 0.479 mmol, 6.3 eq) and DMAP (1.0 mg, 7.98 pmol, 0.1 eq), the reaction was stirred at room temperature for 30 min. 2-(2-((6-Chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85; 58.1 mg, 0.223 mmol, 2.9 eq) was then added. The reaction was stirred an additional 2 h at room temperature. Purification of the crude reaction mixture by reverse phase HPLC (20-70% MeCN / FEO, linear gradient, with constant 0.1% v / v TFA additive) afforded 50.5 mg (77%, TFA salt) of the title compound as a dark red-purple solid. 'H NMR (CD3OD, 400 MHz, 325 K) 58.39 (d, J = 8.2 Hz, 1H), 8.20 (dd, J = 8.2, 1.8 Hz, 1H), 7.81 (d, J= 1.7 Hz, 1H), 7.13 (d, J = 9.3 Hz, 2H), 6.94 (dd, J = 9.3, 2.3 Hz, 2H), 6.90 (d, J= 2.3 Hz, 2H), 4.85 (dt, JHF = 20.6 Hz, J= 2.5 Hz, 2H), 3.71 - 3.55 (m, 12H), 3.52 (t, J= 6.6 Hz, 2H), 3.44 (t, J= 6.5 Hz, 2H), 2.46 -2.36 (m, 4H), 2.21 -2.12 (m, 4H), 1.76 - 1.68 (m, 2H), 1.56 - 1.48 (m, 2H), 1.46 - 1.29 (m, 4H);19F NMR (CD3OD, 376 MHz, 325 K) 6 -75.51 (s, 3F), -166.36 (bd, JFH = 20.7 Hz, 2F); Analytical HPLC: tR= 13.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C41H45CIF2N3O6 [M+H]+748.2960, found 748.2958.

[0262] Example 57: 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)benzoate (JFSsei-HaloTag® ligand, 86)

[0263] The title compound (74%, red-pink solid, TFA salt) was prepared from 4-carboxy-2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)benzoate (Example 33; 84) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 45. 'HNMR (CD3OD, 400 MHz, 325 K) 88.37 (d, J= 8.2 Hz, 1H), 8.18 (dd, J= 8.2, 1.8 Hz, 1H), 7.81 (d, J= 1.8 Hz, 1H), 7.05 (d, J= 9.3 Hz, 2H), 6.91 (bd, J= 9.5 Hz, 2H), 6.84 (bs, 2H), 4.88 (dt, J= 6.8, 1.7 Hz, 2H), 3.69 - 3.56 (m, 12H), 3.51 (t, J= 6.6 Hz, 2H), 3.43 (t, J= 6.5 Hz, 2H), 3.10 (dt, J= 6.7, 3.1 Hz, 2H), 2.29 - 2.21 (m, 4H), 1.77- 1.68 (m, 2H), 1.60 - 1.53 (m, 4H), 1.54 - 1.47 (m, 2H), 1.44 - 1.29 (m, 4H); Analytical HPLC: tR= 10.4 min, 98.8% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 550 nm); MS (ESI) cal cd for C41H47CIN3O6 [M+H]+712.3, found 712.4.

[0264] Example 58: 2-(3-(2-Azabicyclo[2.1. l]hexan-2-yl)-6-(4-(methoxycarbonyl)-2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (139)

[0265] Step 1: A vial was charged with 6- / er / -butoxycarbonylfluorescein ditriflate (800 mg, 1.15 mmol), 2-azabicyclo[2.1.1]hexane hydrochloride (37; 172 mg, 1.44 mmol, 1.25 eq), Pd2dba3 (52.6 mg, 57.0 pmol, 0.05 eq), XPhos (82.1 mg, 0.172 mmol, 0.15 eq), and CS2CO3 (1.12 g, 3.45 mmol, 3 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x).Dioxane (8 mL) was added, and the reaction was flushed again with nitrogen (3x). The reaction was then stirred at 80 °C for 3 h. It was subsequently cooled to room temperature, diluted with MeOH, deposited onto Celite, and concentrated to dryness. The crude was purified twice by silica gel chromatography (0-10% MeOH (2 M NH3) / CH2C12, linear gradient, dry load with Celite; then, 0-30% EtOAc / hexanes, linear gradient) to afford to7-butyl 3'-(2-azabicyclo[2.1.l]hexan-2-yl)-3-oxo-6'-(((trifluoromethyl)sulfonyl)oxy)-3 / 7-spiro[isobenzofuran-l,9'-xanthene]-6-carboxylate (136; 68.9 mg, 10%) as an orange solid. 'H NMR (CDCh, 400 MHz) 88.23 (dd, J= 8.0, 1.3 Hz, 1H), 8.06 (dd, J = 8.0, 0.7 Hz, 1H), 7.76 (dd, J= 1.3, 0.8 Hz, 1H), 7.23 (d, J= 2.5 Hz, 1H), 6.93 (dd, J= 8.8, 2.5 Hz, 1H), 6.84 (d, J= 8.8 Hz, 1H), 6.56 (d, J = 8.7 Hz, 1H), 6.52 (d, J= 2.4 Hz, 1H), 6.43 (dd, J= 8.8, 2.4 Hz, 1H), 4.35 (dt, J= 6.8, 1.8 Hz, 1H), 3.35 - 3.30 (m, 2H), 2.97 (dt, J= 6.6, 3.0 Hz, 1H), 2.02 - 1.94 (m, 2H), 1.56 (s, 9H), 1.47 -1.41 (m, 2H);13C NMR (CDCh, 101 MHz) 8 168.5 (C), 164.2 (C), 153.4 (C), 152.8 (C), 152.6 (C), 152.3 (C), 150.2 (C), 138.7 (C), 131.2 (CH), 130.2 (CH), 129.9 (C), 129.0 (CH), 125.19 (CH), 125.18 (CH), 119.7 (C), 118.8 (q, CF = 321.0, CF3), 116.5 (CH), 111.2 (CH), 110.6 (CH), 105.3 (C), 99.6 (CH), 83.2 (C), 82.8 (C), 60.8 (CH), 54.0 (CH2), 39.9 (CH2), 39.8 (CH), 28.2 (CH3); Analytical HPLC: tR = 14.7 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 280 nm); MS (ESI) calcd for C31H27F3NO8S [M+H]+630.1, found 630.2.

[0266] Step 2: A vial was charged with the product from Step 1 (136; 192 mg, 0.305 mmol), methyl 2-azabicyclo[2.1.1]hexane-4-carboxylate hydrochloride (48; 130 mg, 0.732 mmol, 2.4 eq), Pd2dba3 (27.9 mg, 30.5 pmol, 0.1 eq), XPhos (43.6 mg, 91.5 pmol, 0.3 eq), and Cs2CCh (477 mg, 1.46 mmol, 4.8 eq). The vial was sealed and evacuated / backfilled with nitrogen (3x). Dioxane (3 mL) was added, and the reaction was flushed again with nitrogen (3x). The reaction was then stirred at 100 °C for 3 h. It was subsequently cooled to room temperature, diluted with MeOH, deposited onto Celite, and concentrated to dryness. Purification by silica gel chromatography (0-10% MeOH (2 M NH3) / CH2C12, linear gradient; dry load with Celite) afforded 2-(3-(2-azabicyclo[2.1,l]hexan-2-yl)-6-(4-(methoxycarbonyl)-2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)-4-( / ert-butoxycarbonyl)benzoate (137; 98.7 mg, 52%) as a pink solid. Analytical HPLC: tR = 13.1 min, 99.0% purity (10-95% MeCN / H2O, lineargradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 565 nm); MS (ESI) calcd for C37H37N2O7 [M+H]+621.3, found 621.3.

[0267] Step 3: The product from Step 2 (137) was subjected to the conditions described for Example 32 to afford 2-(3-(2-azabicyclo[2.1.1]hexan-2-yl)-6-(4-(methoxycarbonyl)-2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)-4-carboxybenzoate (138; -100%, dark purple solid, TFA salt). Analytical HPLC: tR = 10.4 min, 97.8% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 565 nm); MS (ESI) calcd for C33H29N2O7 [M+H]+565.2, found 565.3.

[0268] Step 4: The product of Step 3 (138) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) were subjected to the conditions described for Example 45 to provide the title compound 2-(3-(2-azabicyclo[2.1.1]hexan-2-yl)-6-(4-(methoxycarbonyl)-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (25%, pink solid, TFA salt).NMR (CD3OD, 400 MHz, 325 K) 88.58 (bs, 1H), 8.37 (d, J= 8.2 Hz, 1H), 8.19 (dd, J= 8.2, 1.8 Hz, 1H), 7.81 (d, J = 1.7 Hz, 1H), 7.11 - 7.04 (m, 2H), 6.98 - 6.82 (m, 4H), 4.91 (dt, J= 6.8, 1.7 Hz, 1H), 4.87 (t, J= 1.8 Hz, 1H), 3.80 (s, 2H), 3.79 (s, 3H), 3.69 - 3.55 (m, 10H), 3.51 (t, J= 6.6 Hz, 2H), 3.43 (t, J= 6.5 Hz, 2H), 3.11 (dt, J= 6.5, 3.1 Hz, 1H), 2.53 - 2.45 (m, 2H), 2.31 - 2.23 (m, 2H), 1.94 - 1.87 (m, 2H), 1.76 - 1.67 (m, 2H), 1.61 - 1.55 (m, 2H), 1.55 - 1.47 (m, 2H), 1.45 -1.29 (m, 4H); Analytical HPLC: tR = 12.3 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 565 nm); MS (ESI) calcd for C43H49CIN3O8 [M+H]+770.3, found 770.3.

[0269] Example 59: 2-(3-(2-Azabicyclo[2.1. l]hexan-2-yl)-6-(4-carboxy-2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (140)

[0270] 2-(3-(2-Azabicyclo[2.1.1]hexan-2-yl)-6-(4-(methoxycarbonyl)-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (Example 47; 139; 27 mg, 30.5 pmol) was dissolved in MeOH (3 mL), and 1 M NaOH (415 μL, 0.415 mmol, 4 eq) was added. After stirring the reaction at room temperature for 1 h, it was acidified with 1 M HCl (400 μL) and concentrated under vacuum to provide 51.7 mg (-100%) of the title compound as a purple solid that was used without further purification.JH NMR (CD3OD, 400 MHz, 325 K) 58.38 (d, J = 8.2 Hz, 1H), 8.19 (dd,, / = 8.2, 1.7 Hz, 1H), 7.82 (d,.7= 1.8 Hz, 1H), 7.07 (d,.7= 9.3 Hz, 2H), 6.99 - 6.82 (m, 4H), 4.94 - 4.89 (m, 1H), 4.89 - 4.85 (m, 1H), 3.79 (s, 2H), 3.70 - 3.54 (m, 10H), 3.51 (t, J= 6.6 Hz, 2H), 3.43 (t, J= 6.5 Hz, 2H), 3.14 - 3.08 (m, 1H), 2.52 - 2.43 (m, 2H), 2.31 -2.23 (m, 2H), 1.94 - 1.85 (m, 2H), 1.76 - 1.68 (m, 2H), 1.60 - 1.55 (m, 2H), 1.55 - 1.47 (m, 2H), 1.44 - 1.29 (m, 4H); Analytical HPLC: tR= 11.6 min, 98.2% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 565 nm); MS (ESI) calcd for C42H47CIN3O8 [M+H]+756.3, found 756.4.

[0271] Example 60: 2-(3-(2-Azabicyclo[2.1. I hexan^-y -b-^-^-P^XS-^aSA^ba ^-oxohexahydro-l / Z-thieno^A- imidazoM-yl)pentanamido)ethoxy)ethoxy)ethyl)carbamoyl)-2-azabicyclo[2.1.1 ]hexan-2-yl)xanthylium-9-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (biotin- JFSsei-HaloTag® ligand, 142)

[0272] 2-(3-(2-Azabicyclo[2.1. l]hexan-2-yl)-6-(4-carboxy-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (Example 48; 140; 15 mg, 20 μmol) and HATU (11.3 mg, 30 μmol, 1.5 eq) were combined in DMF (1 mL); N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (141; 11.1 mg, 30 μmol, 1.5 eq) and DIEA (10.4 μL, 60 μmol, 3 eq) were added, and the reaction was stirred at room temperature for 2 h. The solvent was removed by rotary evaporation, and the crude material was purified by reverse phase HPLC (30-50% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive) to yield 0.9 mg (4.1%) of the title compound as a pink solid. MS (ESI) calcd for C58H75CIN7O11S [M+H]+1112.5, found 1112.5.

[0273] Example 61: 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,6-di(2-azabicyclo[2.1.l]hexan-2-yl)thioxanthylium-9-yl)benzoate (JFSs82-HaloTag® ligand, 95)

[0274] The title compound (77%, dark red-purple solid, TFA salt) was prepared from 4-carboxy-2-(3,6-di(2-azabicyclo[2.1. l]hexan-2-yl)thioxanthylium-9-yl)benzoate (Example 34; 94) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 45.JH NMR (CD3OD, 400 MHz, 325 K) 88.38 (d, J= 8.2 Hz, 1H), 8.18 (dd,.7= 8.2, 1.8 Hz, 1H), 7.76 (d, J= 1.8 Hz, 1H), 7.16 (bs, 2H), 7.12 (d, J= 9.5 Hz,2H), 6.90 (bd, J= 9.6 Hz, 2H), 4.87 (dt, J= 6.8, 1.7 Hz, 2H), 3.69- 3.64 (m, 2H), 3.64 - 3.54 (m, 10H), 3.51 (t, J= 6.7 Hz, 2H), 3.42 (t, J= 6.5 Hz, 2H), 3.08 (dt, J= 6.7, 3.2 Hz, 2H), 2.28 – 2.18 (m, 4H), 1.76- 1.68 (m, 2H), 1.57 - 1.46 (m, 6H), 1.45 - 1.30 (m, 4H); Analytical HPLC: tR= 12.8 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 590 nm); HRMS (ESI) calcd for C41H47CIN3O5S [M+H]+728.2920, found 728.2919.

[0275] Example 62: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (JFS607–HaloTag® ligand, 105)

[0276] The title compound (77%, blue solid) was prepared from 2-(3,6-bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-carboxybenzoate (Example 35; 104) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 45.1H NMR (CDCh, 400 MHz) 88.03 (dd, J = 7.9, 0.7 Hz, 1H), 7.93 (dd, J= 8.0, 1.4 Hz, 1H), 7.47 (dd, J = 1.5, 0.7 Hz, 1H), 6.84 (d, J = 2.5 Hz, 2H), 6.77 (t, J= 5.4 Hz, 1H), 6.55 (d, J= 8.7 Hz, 2H), 6.45 (dd, J= 8.7, 2.5 Hz, 2H), 4.20 (dt, JHF = 21.3 Hz, J= 2.5 Hz, 2H), 3.65 - 3.57 (m, 6H), 3.56 - 3.52 (m, 2H), 3.51 (t, J= 6.6 Hz, 2H), 3.40 (t, J = 6.6 Hz, 2H), 3.39 - 3.35 (m, 4H), 2.16 - 2.02 (m, 8H), 1.85 (s, 3 H), 1.79 - 1.70 (m, 2H), 1.74 (s, 3H), 1.57 - 1.49 (m, 2H), 1.46 - 1.38 (m, 2H), 1.37 - 1.29 (m, 2H);19F NMR (CDCI3, 376 MHz) δ -164.41 (bd, JFH = 21.5 Hz); Analytical HPLC: tR= 13.0 min, 98.6% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 615 nm); HRMS (ESI) calcd for C44H51CIF2N3O5 [M+H]+774.3480, found 774.3479.

[0277] Example 63: 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)benzoate (JFSei9-HaloTag® ligand, 99)

[0278] The title compound (79%, blue solid, TFA salt) was prepared from 4-carboxy-2-(3,6-di(2 -azabi cy clo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9( 10 / / )-yl)benzoate (Example 36; 98) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 45.JH NMR (CD3OD, 400 MHz, 325 K) δ 8.56 (t, J = 5.4 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.14 (dd, J= 8.2, 1.8 Hz, 1H), 7.75 (d, J= 1.8 Hz, 1H), 7.13 (d, J= 2.3 Hz, 2H), 6.94 (d, J= 9.3 Hz, 2H), 6.68 (dd, J= 9.3, 2.3 Hz, 2H), 4.95 (d, J= 6.7 Hz, 2H), 3.69 - 3.55 (m, 12H), 3.51 (t, J= 6.6 Hz, 2H), 3.44 (t, J= 6.5 Hz, 2H), 3.10 (dt, J = 6.4, 3.0 Hz, 2H), 2.30-2.21 (m, 4H), 1.85 (s, 3H), 1.76 (s, 3H), 1.75 - 1.67 (m, 2H), 1.59 - 1.54 (m, 4H), 1.54 - 1.47 (m, 2H), 1.44 - 1.30 (m, 4H); Analytical HPLC: tR= 13.1 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 625 nm); HRMS (ESI) calcd for C44H53CIN3O5 [M+H]+738.3669, found 738.3665.

[0279] Example 64: 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3,5,6-trifluorobenzoate (JFS632–HaloTag® ligand, 112)

[0280] 2-(3,6-Bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)-l 0, 10-dimethylanthracen-9-ylium-9(1077)-yl)-4-(dicyano(methoxymethoxy)methyl)-3,5,6-trifluorobenzoate (Example 41; 110; 50 mg, 71.2 pmol) was taken up in CH2CI2 (3 mL); triethylsilane (300 pL) was added, followed by trifluoroacetic acid (600 pL). The reaction was stirred at room temperature for 8 h. Toluene (4 mL) was added, and the reaction mixture was concentrated to dryness. The residue was combined with a premixed solution of 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85; 37.0 mg, 0.142 mmol, 2 eq) and DIEA (124 pL, 0.712 mmol, 10 eq) in DMF (3 mL), and the reaction was stirred at room temperature for 18 h. The solvent was removed by rotary evaporation, and the crude material was purified by reverse phase HPLC (20–70% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive) to afford the title compound as a dark blue solid (49.8 mg, 74%, TFA salt). ’H NMR (CD3OD, 400 MHz, 325 K) 87.17 (dd, J = 9.3, 0.8 Hz, 2H), 7.12 (d, J= 2.3 Hz, 2H), 6.76 (dd, J = 9.2, 2.3 Hz, 2H), 4.91 (dt, JHF = 20.6 Hz, J= 2.5 Hz, 2H), 3.71 (bs, 4H), 3.68 -3.54 (m, 8H), 3.51 (t,.7= 6.6 Hz, 2H), 3.44 (t, J= 6.5 Hz, 2H), 2.46 - 2.36 (m, 4H), 2.21 - 2.12 (m, 4H), 1.82 (s, 3H), 1.76 - 1.67 (m, 2H), 1.71 (s, 3H), 1.57 - 1.49 (m, 2H), 1.46 - 1.29 (m, 4H);19F NMR (CD3OD, 376 MHz, 325 K) 6 -75.56 (s, 3F), -117.55 (d, J= 15.9 Hz, IF), -133.69 (dd, J= 21.7, 2.3 Hz, IF), -140.73 (dd, J = 22.0, 15.9 Hz, 1F), -166.39 (bd,. / in = 20.7 Hz, 2F); Analytical HPLC: tR= 13.1 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 635 nm); HRMS (ESI) calcd for C44H48CIF5N3O5 [M+H]+828.3198, found 828.3194.

[0281] Example 65: 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,6-di(2-azabicyclo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)-3, 5,6-trifluorobenzoate (JFS643–HaloTag® ligand, 111)

[0282] The title compound (71%, dark blue solid, TFA salt) was prepared from 2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)-4-(dicyano(methoxymethoxy)methyl)-3,5,6-trifluorobenzoate (Example 42; 109) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 53. 'H NMR (CD3OD, 400 MHz, 325 K) δ 7.132 (dd, J = 9.3, 1.0 Hz, 2H), 7.127 (d, J = 2.1 Hz, 2H), 6.75 (dd, J= 9.3, 2.3 Hz, 2H), 4.99 (dt, J= 6.8, 1.7 Hz, 2H), 3.67 (bs, 4H), 3.67 -3.55 (m, 8H), 3.50 (t, J= 6.6 Hz, 2H), 3.44 (t, J= 6.5 Hz, 2H), 3.11 (dt, J= 6.5, 3.2 Hz, 2H), 2.32 -2.23 (m, 4H), 1.81 (s, 3H), 1.75 - 1.68 (m, 2H), 1.70 (s, 3H), 1.61 - 1.55 (m, 4H), 1.55 -1.47 (m, 2H), 1.45 - 1.30 (m, 4H);19F NMR (CD3OD, 376 MHz, 325 K) 6 -75.53 (s, 3F), -117.13 (d, J = 15.7 Hz, 1F), -133.98 (dd, J = 22.2, 2.3 Hz, 1F), -140.85 (dd, J = 22.0, 15.3 Hz, 1F); Analytical HPLC: tR = 13.1 min, >99% purity (10–95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 645 nm); HRMS (ESI) calcd for C44H50CIF3N3O5 [M+H]+792.3386, found 792.3380.

[0283] Example 66: 2-(3,7-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (JFS645–HaloTag® ligand, 120)

[0284] The title compound (82%, blue solid, TFA salt) was prepared from 2-(3,7-bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)-5, 5-dimethyldibenzo[Z»,e]silin-l 0-ylium- 10(5 / / )-yl)-4-carboxybenzoate (Example 37; 119) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 45. 'H NMR (CD3OD, 400 MHz, 325 K) δ 8.18 (d, J = 8.2 Hz, 1H), 8.08 (dd, J= 8.1, 1.7 Hz, 1H), 7.69 (dd, J= 1.6, 0.6 Hz, 1H), 7.15 (d, J = 2.7 Hz, 2H), 6.87 (d, J= 9.2 Hz, 2H), 6.62 (dd, J= 9.2, 2.7 Hz, 2H), 4.76 - 4.62 (m, 2H), 3.68 - 3.52 (m, 12H), 3.50 (t, J= 6.6 Hz, 2H), 3.42 (t, J= 6.5 Hz, 2H), 2.33 - 2.24 (m, 4H), 2.12 -2.03 (m, 4H), 1.75 - 1.66 (m, 2H), 1.54- 1.46 (m, 2H), 1.44 - 1.28 (m, 4H), 0.64 (s,3H), 0.57 (s, 3H);19F NMR (CD3OD, 376 MHz, 325 K) 5 -75.84 (s, 3F), -165.14 - -166.10 (m, 2F); Analytical HPLC: tR = 15.4 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C43H51ClF2N3O5Si [M+H]+790.3250, found 790.3245.

[0285] Example 67: 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,7-di(2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[ ’,e]silin-l 0-ylium- 10(5 / / )-yl)benzoate (JFSe57-HaloTag® ligand, 116)

[0286] The title compound (32%, pale blue solid) was prepared from 4-carboxy-2-(3,7-di(2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate (Example 38; 115) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 45.1H NMR (CDCE, 400 MHz) δ 7.99 (dd, J = 7.9, 0.7 Hz, 1H), 7.91 (dd, J= 8.0, 1.4 Hz, 1H), 7.71 (dd, J= 1.4, 0.6 Hz, 1H), 6.96 (d, J=2.7 Hz, 2H), 6.79 (s, 1H), 6.73 (d, J= 8.8 Hz, 2H), 6.53 (dd, J= 8.8, 2.7 Hz, 2H), 4.34 (dt, J= 6.7, 1.8 Hz, 2H), 3.67 - 3.61 (m, 6H), 3.56 - 3.53 (m, 2H), 3.50 (t, J= 6.7 Hz, 2H), 3.39 (t, J= 6.7 Hz, 2H), 3.32 (bs, 4H), 2.93 (dt, J= 6.2, 2.9 Hz, 2H), 1.96 - 1.87 (m, 4H), 1.77 - 1.69 (m, 2H), 1.54 - 1.48 (m, 2H), 1.46 - 1.36 (m, 6H), 1.33 - 1.28 (m, 2H), 0.65 (s, 3H), 0.58 (s, 3H);Analytical HPLC: tR = 13.0 min, >99% purity (10-95% MeCN / HzO, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); MS (ESI) calcd for C43H53ClN3O5Si [M+H]+754.3, found 754.4.

[0287] Example 68: 2-(3,7-Bis(4-(methoxycarbonyl)-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (128)

[0288] The title compound (96%, pale blue-green solid) was prepared from 2-(3,7-bis(4-(methoxycarbonyl)-2-azabicyclo[2.1. l]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin-10-ylium-10(57 / )-yl)-4-carboxybenzoate (Example 39; 127) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 45.XH NMR (CDC13, 400 MHz) δ 8.00 (dd, J = 7.9, 0.7 Hz, 1H), 7.91 (dd, J = 8.0, 1.4 Hz, 1H), 7.72 (dd, J = 1.5, 0.7 Hz, 1H), 6.95 (d, J= 2.7 Hz, 2H), 6.83 (s, 1H), 6.77 (d, J= 8.8 Hz, 2H), 6.54 (dd, J= 8.8, 2.7 Hz, 2H), 4.32 (t, J= 1.9 Hz, 2H), 3.75 (s, 6H), 3.68 - 3.60 (m, 6H), 3.58 - 3.52 (m, 6H), 3.50 (t,,7= 6.6 Hz, 2H), 3.40 (t, J= 6.7 Hz, 2H), 2.28 - 2.19 (m, 4H), 1.85 - 1.77 (m, 4H), 1.77 - 1.69 (m, 2H), 1.56 - 1.48 (m, 2H), 1.45 - 1.27 (m, 4H), 0.66 (s, 3H), 0.59 (s, 3H); Analytical HPLC: tR = 13.3 min, 98.0% purity (10–95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 660 nm); HRMS (ESI) calcd for C47H57ClN3O9Si [M+H]+870.3548, found 870.3546.

[0289] Example 69: 2-(3,7-Bis(4-carboxy-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (JFS657i–HaloTag® ligand, 129)

[0290] 2-(3,7-Bis(4-(methoxycarbonyl)-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (Example 57; 128; 50 mg, 57.4 pmol) was dissolved in MeOH (5 mL), and 1 MNaOH (230 pL, 0.230 mmol, 4 eq) was added. After stirring the reaction at room temperature for 18 h, it was acidified with 1 M HC1 (300 pL) and directly purified by reverse phase HPLC (35-45% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive) to provide 50.5 mg (92%, TFA salt) of the title compound as a blue solid. 1H NMR (CD3OD, 400 MHz, 325 K) δ 8.24 (d, J= 8.2 Hz, 1H), 8.10 (dd, J = 8.2, 1.7 Hz, 1H), 7.71 (d, J = 1.6 Hz, 1H), 7.22 (d, J = 2.6 Hz, 2H), 6.90 (d, J = 9.3 Hz, 2H), 6.65 (dd, J = 9.4, 2.6 Hz, 2H), 4.84 (s, 2H), 3.76 (bs, 4H), 3.67 - 3.54 (m, 8H), 3.50 (t, J = 6.6 Hz, 2H), 3.43 (t, J = 6.5 Hz, 2H), 2.47 -2.38 (m, 4H), 1.90 - 1.82 (m, 4H), 1.75 - 1.67 (m, 2H), 1.55 - 1.47 (m, 2H), 1.45 - 1.30 (m, 4H), 0.64 (s, 3H), 0.58 (s, 3H); Analytical HPLC: tR= 11.4 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 660 nm); HRMS (ESI) calcd for C45H53ClN3O9Si [M+H]+842.3235, found 842.3228.

[0291] Example 70: 2-(3,7-Bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3,5,6-trifluorobenzoate (JFS670–HaloTag® ligand, 125)

[0292] The title compound (73%, dark blue solid, TFA salt) was prepared from 2-(3,7-bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin- 10-ylium- 10(5Z7)-yl)-4-(dicyano(methoxymethoxy)methyl)-3,5,6-trifIuorobenzoate (Example 43; 123) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 53. 'H NMR (CD3OD, 400 MHz) δ 9.14 (t, J = 5.4 Hz, 1H), 7.15 (d, 2.7 Hz, 2H), 7.00 (dd, J= 9.0, 0.8 Hz, 2H), 6.71 (dd, J = 9.1, 2.7 Hz, 2H), 4.64 (dt, JHF = 21.0 Hz, J = 2.8 Hz, 2H), 3.68 - 3.53 (m, 12H), 3.51 (t, J= 6.7 Hz, 2H), 3.45 (t, J= 6.5 Hz, 2H), 2.33 - 2.24 (m, 4H),2.13 - 2.04 (m, 4H), 1.76 - 1.68 (m, 2H), 1.57 - 1.49 (m, 2H), 1.46 - 1.30 (m, 4H), 0.62 (s, 3H), 0.55 (s, 3H);19F NMR (CD3OD, 376 MHz) 8 -75.47 (s, 3F), -118.30 (d, J = 19.3 Hz, 1F), -134.56 (d, J = 21.1 Hz, 1F), -141.99 – -142.63 (m, 1F), -164.78 – -165.12 (m, 2F); Analytical HPLC: tR = 15.2 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 675 nm); HRMS (ESI) calcd for C43H48ClF5N3O5Si [M+H]+844.2967, found 844.2967.

[0293] Example 71: 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,7-di(2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-3,5,6-trifluorobenzoate (JFS678–HaloTag® ligand, 124)

[0294] The title compound (68%, dark blue solid, TFA salt) was prepared from 2-(3,7-di(2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[ / >,e]silin-10-ylium-10(5 / / )-yl)-4-(dicyano(methoxymethoxy)methyl)-3,5,6-trifluorobenzoate (Example 44; 122) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85) according to the procedure described for Example 53.JH NMR (CD3OD, 400 MHz) δ 9.10 (t, J = 5.4 Hz, 1H), 7.23 (d, J= 2.6 Hz, 2H), 7.10 (dd, J= 9.5, 0.6 Hz, 2H), 6.70 (dd, J= 9.5, 2.7 Hz, 2H), 5.00 (dt, J= 6.8, 1.7 Hz, 2H), 3.69 - 3.55 (m, 12H), 3.50 (t, J= 6.6 Hz, 2H), 3.44 (t, J= 6.5 Hz, 2H), 3.09 (dt, J= 6.4, 3.1 Hz, 2H), 2.31 – 2.22 (m, 4H), 1.75 – 1.67 (m, 2H), 1.61 – 1.54 (m, 4H), 1.54 – 1.48 (m, 2H), 1.43 - 1.29 (m, 4H), 0.60 (s, 3H), 0.53 (s, 3H);19F NMR (CD3OD, 376 MHz) 8 -75.30 (s, 3F), -117.88 (d, J = 15.7 Hz, IF), -134.89 (d, J= 22.3 Hz, IF), -141.68 (dd, J= 22.0, 16.0 Hz, IF); Analytical HPLC: tR = 13.4 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 685 nm); HRMS (ESI) calcd for C43H50ClF3N3O5Si [M+H]+808.3155, found 808.3147.

[0295] Example 72:? / -(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3,7-bis(4-fluoro-2-azabicyclo[2.1.1 ]hexan-2-yl)-5,5-dimethyl-37 / ,5 / / -spiro[dibenzo[Z>,e]siline- 10,1'-isobenzofuran]-6'-carboxamide (JFS64sb-HaloTag® ligand, 135)O s

[0296] 3,7-Bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)-5,5-dimethyl-3'Ff,5Ff-spiro[dibenzo[Z>,e]siline-10,l'-isobenzofuran]-6'-carboxylic acid (Example 40; 134; 30 mg, 52.0 pmol) and HATU (29.9 mg, 79.0 pmol, 1.5 eq) were combined in DMF (2 mL); 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine hydrochloride (85; 20.5 mg, 79.0 pmol, 1.5 eq) and DIEA (27.4 pL, 0.158 mmol, 3 eq) were added, and the reaction was stirred at room temperature for 2 h. The solvent was removed by rotary evaporation, and the crude material was purified by reverse phase HPLC (40-70% MeCN / H₂O, linear gradient, with constant 0.1% v / v TFA additive). The pooled HPLC product fractions were partially concentrated to remove MeCN, diluted with saturated NaHCCh, and extracted with CH2CI2 (2x). The organic extracts were dried over anhydrous MgSO₄, filtered, and evaporated to yield 24.9 mg (61%) of the title compound as a light blue solid. ’HNMR (CD3OD, 400 MHz) 87.75 (dd, J= 7.9, 1.6 Hz, 1H), 7.46 (dd, J= 8.0, 0.8 Hz, 1H), 7.35 (dd, J= 1.4, 0.7 Hz, 1H), 7.04 (d, J= 8.8 Hz, 2H), 6.98 (d, J= 2.7 Hz, 2H), 6.68 (dd, J= 8.8, 2.7 Hz, 2H), 5.41 (s, 2H), 4.30 (dt,. / in = 21.4 Hz, J= 2.6 Hz, 2H), 3.61 - 3.55 (m, 4H), 3.54- 3.45 (m, 6H), 3.38 (t, J= 6.5 Hz, 2H), 3.33 (s, 4H), 2.14 - 2.07 (m, 4H), 2.01 - 1.93 (m, 4H), 1.72 - 1.63 (m, 2H), 1.50 - 1.42 (m, 2H), 1.37 - 1.24 (m, 4H), 0.64 (s, 3H), 0.50 (s, 3H);19F NMR (CD3OD, 376 MHz) 8 -163.48 - -163.59 (m); Analytical HPLC: tR= 13.2 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 655 nm); MS (ESI) calcd for C43H53ClF2N3O4Si [M+H]+776.3, found 776.3.

[0297] Example 73: 4-((4-(((2-Amino-6-chloropyrimidin-4-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,6-bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (JFSs48-cp-SNAP-tag ligand, 92)

[0298] The title compound (80%, pink solid, TFA salt) was prepared from 2-(3,6-bis(4-fluoro-2-azabicyclo[2.1,l]hexan-2-yl)xanthylium-9-yl)-4-carboxybenzoate (Example 32; 90) and 4-((4-(aminomethyl)benzyl)oxy)-6-chloropyrimidin-2-amine (87) according to the procedure described for Example 45. 'HNMR (CD3OD, 400 MHz, 325 K) 58.39 (d, J= 8.2 Hz, 1H), 8.21 (dd, J= 8.2, 1.8 Hz, 1H), 7.82 (d, J= 1.8 Hz, 1H), 7.41 - 7.34 (m, 4H), 7.12 (d, J= 9.3 Hz, 2H), 6.92 (bd, J= 9.3 Hz, 2H), 6.89 (bs, 2H), 6.07 (s, 1H), 5.34 (s, 2H), 4.84 (dt, JHF = 20.6 Hz, J = 2.5 Hz, 2H), 4.59 - 4.57 (m, 2H), 3.67 (s, 4H), 2.46 - 2.37 (m, 4H), 2.20 - 2.12 (m, 4H);19F NMR (CD3OD, 376 MHz, 325 K) 5 -75.72 (s, 3F), -166.37 (bd, JFH= 20.6 Hz, 2F); Analytical HPLC: tR = 12.3 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 555 nm); MS (ESI) cal cd for C43H36ClF2N6O5 [M+H]+789.2, found 789.4.

[0299] Example 74: 4-((4-(((2-Amino-6-chloropyrimidin-4-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,6-di(2-azabicyclo[2.1. l]hexan-2-yl)xanthylium-9-yl)benzoate (JFSsei-cp-SNAP-tag ligand, 88)

[0300] The title compound (32%, pink solid, TFA salt) was prepared from 4-carboxy-2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)xanthylium-9-yl)benzoate (Example 33; 84) and 4-((4-(aminomethyl)benzyl)oxy)-6-chloropyrimidin-2-amine (87) according to the procedure described for Example 45. 'H NMR (CD3OD, 400 MHz, 325 K) 58.37 (d, J= 8.2 Hz, 1H), 8.20 (dd, J = 8.2, 1.8 Hz, 1H), 7.82 (d, J= 1.8 Hz, 1H), 7.41 - 7.34 (m, 4H), 7.04 (d, J= 9.3 Hz, 2H), 6.89 (bd, J= 9.5 Hz, 2H), 6.83 (bs, 2H), 6.08 (s, 1H), 5.33 (s, 2H), 4.87 (dt, J= 6.8, 1.7 Hz, 2H), 4.61 - 4.58 (m, 2H), 3.60 (s, 4H), 3.13 - 3.07 (m, 2H), 2.30 -2.20 (m, 4H), 1.60 - 1.52 (m, 4H); Analytical HPLC: tR = 12.6 min, >99% purity (10—95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 570 nm); MS (ESI) calcd for C43H38CIN6O5 [M+H]+753.3, found 753.4.

[0301] Example 75: 4-((4-(((2-Amino-6-chloropyrimidin-4-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,6-bis(4-fluoro-2-azabicyclo[2.1. l]hexan-2-yl)-10, 10-dimethylanthracen-9-ylium-9(1077)-yl)benzoate (IFS607-cp-SNAP-tag ligand, 106)L o'A A. a

[0302] The title compound (81%, pale blue solid) was prepared from 2-(3,6-bis(4-fluoro- 2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10 / 7)-yl)-4-carboxybenzoate (Example 35; 104) and 4-((4-(aminomethyl)benzyl)oxy)-6-chloropyrimidin-2-amine (87)according to the procedure described for Example 45. 'H NMR (CD3OD, 400 MHz, 325 K) 8 8.06 (dd, J= 8.1, 1.4 Hz, 1H), 8.03 (dd, J= 8.0, 0.8 Hz, 1H), 7.48 (dd, J= 1.5, 0.8 Hz, 1H), 7.30 (AB quartet, vA= 2929.8 Hz, vB= 2908.8 Hz, JAB = 8.2 Hz, 4H), 6.99 - 6.95 (m, 2H), 6.59 -6.52 (m, 4H), 6.06 (s, 1H), 5.30 (s, 2H), 4.47 (s, 2H), 4.34 (dt, JHF = 21.3 Hz, J= 2.6 Hz, 2H), 3.36 (bs, 4H), 2.16 - 2.09 (m, 4H), 2.02 - 1.95 (m, 4H), 1.85 (s, 3H), 1.74 (s, 3H);19F NMR (CD3OD, 376 MHz, 325 K) 8 -163.94 (bd,. / in = 21.1 Hz); Analytical HPLC: tR= 12.7 min, >99% purity (10-95% MeCN / H20, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 615 nm); MS (ESI) calcd for C46H42CIF2N6O4 [M+H]+815.3, found 815.5.

[0303] Example 76: 4-((4-(((2-Amino-917-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,6-bis(4-fhjoro-2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)benzoate (JFSeo?- SNAP -tag ligand, 107)

[0304] The title compound (65%, pale blue solid) was prepared from 2-(3,6-bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)-4-carboxybenzoate (Example 35; 104) and 6-((4-(aminomethyl)benzyl)oxy)-9 / / -purin-2-amine (101) according to the procedure described for Example 45. 'H NMR (CD3OD, 400 MHz, 325 K) 88.07 (dd, J = 8.0, 1.4 Hz, 1H), 8.03 (dd, J= 8.0, 0.8 Hz, 2H), 7.79 (s, 1H), 7.51 - 7.46 (m, 1H), 7.36 (AB quartet, vA= 2975.6 Hz, vB= 2917.6 Hz, JAB= 8.1 Hz, 4H), 6.99 - 6.95 (m, 2H), 6.59 - 6.54 (m, 4H), 5.51 (s, 2H), 4.49 (s, 2H), 4.35 (dt, JHF = 21.4 Hz, J= 2.6 Hz, 2H), 3.36 (bs, 4H), 2.16-2.09 (m, 4H), 2.03 - 1.96 (m, 4H), 1.85 (s, 3H), 1.74 (s, 3H);19F NMR (CD3OD, 376 MHz, 325 K) 8 -163.94 (bd, JFH = 21.3 Hz); Analytical HPLC: tR= 10.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow;ESI; positive ion mode; detection at 615 nm); MS (ESI) calcd for C47H43F2N8O4 [M+H]+821.3, found 821.5.

[0305] Example 77: 4-((4-(((2-Amino-6-chloropyrimidin-4-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,6-di(2-azabicyclo[2.1.1 ]hexan-2-yl)- 10,10-dimethylanthracen-9-ylium-9(10 / y)-yl)benzoate (JFSei9-cp- SNAP -tag ligand, 100)

[0306] The title compound (62%, blue solid) was prepared from 4-carboxy-2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(107 / )-yl)benzoate (Example 36; 98) and 4-((4-(aminomethyl)benzyl)oxy)-6-chloropyrimidin-2-amine (87) according to the procedure described for Example 45.JH NMR (CD3OD, 400 MHz, 325 K) 88.07 - 8.01 (m, 2H), 7.60 - 7.56 (m, 1H), 7.34 (AB quartet, vA= 2944.8 Hz, vB= 2931.7 Hz, JAB = 8.3 Hz, 4H), 7.04 (d, J= 2.4 Hz, 2H), 6.89 (d, J= 9.1 Hz, 2H), 6.62 (dd, J = 9.1, 2.4 Hz, 2H), 6.08 (s, 1H), 5.32 (s, 2H), 4.73 (dt, J= 6.8, 1.8 Hz, 2H), 4.54 (s, 2H), 3.51 (s, 4H), 3.03 (dt, J= 6.6, 3.1 Hz, 2H), 2.17 -2.10 (m, 4H), 1.81 (s, 3H), 1.76 (s, 3H), 1.52- 1.44 (m, 4H); Analytical HPLC: tR= 12.9 min, >99% purity (10-95% MeCN / H₂O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 625 nm); MS (ESI) calcd for C46H44CIN6O4 [M+H]+779.3, found 779.5.

[0307] Example 78: 4-((4-(((2-Amino-9 / f-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)benzoate (JFS619-SNAP-tag ligand, 102)

[0308] The title compound (67%, blue solid) was prepared from 4-carboxy-2-(3,6-di(2-azabicyclo[2.1.1]hexan-2-yl)-10,10-dimethylanthracen-9-ylium-9(10 / / )-yl)benzoate (Example 36; 98) and 6-((4-(aminomethyl)benzyl)oxy)-9 / / -purin-2-amine (101) according to the procedure described for Example 45. ’H NMR (CD3OD, 400 MHz, 325 K) 88.08 - 8.01 (m, 2H), 7.79 (s, 1H), 7.60 - 7.57 (m, 1H), 7.40 (AB quartet, vA= 2986.2 Hz, vB= 2936.6 Hz, B = 8.1 Hz, 4H), 7.04 (d, J= 2.4 Hz, 2H), 6.89 (d, J= 9.1 Hz, 2H), 6.62 (dd, J= 9.1, 2.4 Hz, 2H), 5.53 (s, 2H), 4.73 (dt, J= 7.0, 1.7 Hz, 2H), 4.54 (s, 2H), 3.51 (s, 4H), 3.02 (dt, J= 6.3, 2.8 Hz, 2H), 2.16 -2.09 (m, 4H), 1.81 (s, 3H), 1.76 (s, 3H), 1.52 - 1.44 (m, 4H); Analytical HPLC: tR= 10.0 min, >99% purity (10-95% MeCN / H₂O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 625 nm); MS (ESI) calcd for C47H45N8O4 [M+H]+785.4, found 785.5.

[0309] Example 79: 4-((4-(((2-Amino-9-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2- (3,7-bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin-10-ylium-10(5 / / )-yl)benzoate (JFS645-SNAP-tag ligand, 121)

[0310] The title compound (78%, pale blue solid) was prepared from 2-(3,7-bis(4-fluoro-2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[Z>,e]silin-10-ylium-10(5Z / )-yl)-4-carboxybenzoate (Example 37; 119) and 6-((4-(aminomethyl)benzyl)oxy)-9J / -purin-2-amine (101) according to the procedure described for Example 45. 'H NMR (CD3OD, 400 MHz, 325 K) 88.03 (dd, J= 8.0, 1.4 Hz, 1H), 7.99 (dd, J= 8.0, 0.8 Hz, 1H), 7.79 (s, 1H), 7.71 - 7.66 (m, 1H), 7.39 (AB quartet, vA= 2983.4 Hz, vB= 2930.2 Hz, JAB= 8.1 Hz, 4H), 7.01 (d, J= 2.7 Hz, 2H), 6.75 (d, J= 8.8 Hz, 2H), 6.62 (dd, J= 8.9, 2.7 Hz, 2H), 5.52 (s, 2H), 4.52 (s, 2H), 4.31 (dt, JHF = 21.3 Hz, J= 2.6 Hz, 2H), 3.32 (bs, 4H), 2.14 - 2.07 (m, 4H), 2.01 - 1.93 (m, 4H), 0.62 (s, 3H), 0.55 (s, 3H);19F NMR (CD3OD, 376 MHz, 325 K) 8 -163.94 (bd,. / HI = 21.2 Hz);Analytical HPLC: tR = 11.3 min, 98.4% purity (10-95% MeCN / H₂O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 650 nm); MS (ESI) calcd for C46H43F2N8O4Si [M+H]+837.3, found 837.5.

[0311] Example 80: 4-((4-(((2-Amino-9Ff-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,7-di(2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[Z»,e]silin-10-ylium-10(5Z / )-yl)benzoate (IFS657- SNAP -tag ligand, 117)

[0312] The title compound (67%, pale blue solid) was prepared from 4-carboxy-2-(3,7-di(2-azabicyclo[2.1.1]hexan-2-yl)-5,5-dimethyldibenzo[ / »,e]silin-10-ylium-10(5J / )-yl)benzoate (Example 38; 115) and 6-((4-(aminomethyl)benzyl)oxy)-9 / / -purin-2-amine (101) according to the procedure described for Example 45.JH NMR (CD3OD, 400 MHz, 325 K) 88.03 (dd, J = 8.1, 1.4 Hz, 1H), 7.99 (dd, J= 8.0, 0.8 Hz, 1H), 7.80 (s, 1H), 7.70 - 7.65 (m, 1H), 7.39 (AB quartet, VA = 2983.2 Hz, vB= 2930.1 Hz, JAB = 8.1 Hz, 4H), 7.05 (d, J= 2.7 Hz, 2H), 6.72 (d, J= 8.9 Hz, 2H), 6.63 (dd, J= 8.9, 2.8 Hz, 2H), 5.52 (s, 2H), 4.52 (s, 2H), 4.42 (dt, J= 6.7, 1.8 Hz, 2H), 3.32 (bs, 4H), 2.93 (dt, J= 6.3, 3.0 Hz, 2H), 2.01 - 1.93 (m, 4H), 1.42 - 1.35 (m, 4H), 0.60(s, 3H), 0.54 (s, 3H); Analytical HPLC: tR= 10.0 min, >99% purity (10-95% MeCN / H2O, linear gradient, with constant 0.1% v / v TFA additive; 20 min run; 1 mL / min flow; ESI; positive ion mode; detection at 665 nm); MS (ESI) calcd for C46H45NsO4Si [M+H]+801.3, found 801.5.

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[0314] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein.Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMSWhat is claimed is1. A compound of the following structure:whereinQ is selected from the group consisting of C(alkyl), C(alkyl)2, NH, N(alkyl), O, S, SO2, Si(alkyl)2, P(O)(aryl), P(O)(alkyl), PO2H, PO2(alkyl), Se, and replaced with two H atoms;Ri, R2, R3, and R4 are independently selected from the group consisting of H, D, halogen, alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CHO, C(O)(alkyl), C(O)(aryl), CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NH2, NH(alkyl), N(alkyl)2, NH(aryl), N(aryl)2, NHC(O)alkyl, NO2, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), SO3H, and PO3H2;X1is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl substituted alkenyl, alkynyl substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), N3, NH2, NH(alkyl), N(alkyl)2, OH, and O(alkyl);X2is independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, substituted alkyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, and C(O)NH(aryl);when Y is not present in the structure, Z is selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl, and substituted alkenyl, CH2OH, CO2, CO2H, CO2(alkyl), CO2(aryl), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, OH,O(alkyl), O(aryl), SO3, SO3H, SO2NH(alkyl), SO2N(alkyl)2, SO2NH(aryl), SO2N(aryl)2, PO32, PO3H, and PO3H2;when Y is not present in the structure and when Z is a monoanionic substituent then the compound is optionally prepared as a net-neutral inner salt form;when Y is not present in the structure and when Z is not a monoanionic substituent then the compound is optionally prepared in a salt form with an appropriate counterion;when Y is present in the structure, Z is selected from the group consisting of C(O), SO2, PO2H, and CR2where each R is independently selected from the group consisting of H, alkyl, and substituted alkyl; and Y is selected from the group consisting of O, S, C(O), C(N2), NH, N(alkyl), N(aryl), N(SO2R) where R can be alkyl, substituted alkyl, and CN; andR5, R6, R7, and R8are independently selected from the group consisting of H, D, halogen, alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NH2, NHC(O)(alkyl), NH(alkyl), N(alkyl)2, NH(aryl), N(aryl)2, NO2, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), SO3H, SO2NH2, SO2NH(alkyl), SO2N(alkyl)2, SO2NH(aryl), SO2N(aryl)2, and PO3H2.

2. The compound of claim 1, wherein R6is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NHC(O)(alkyl), NH( alkyl), and N(alkyl)2.

3. The compound of claim 1, wherein R6is selected from the group consisting of4. The compound of claim 1, wherein Q is selected from the group consisting of \ / \ / O. Of-Bu O OH OvPh X°X, X5X, X Z, XsX, XSX, X' X, X'^X, XPX, and replaced with two H atoms.

5. The compound of claim 1, wherein Q is selected from the group consisting of6. The compound of claim 1, wherein each X2is H.

7. The compound of claim 1, wherein each X1is independently selected from the group consisting of H, F, OCH3, N3, NHBoc, NH2, CO2CH3, CO2H, andOo o8. The compound of claim 1, wherein each R1, R2, R3, R4, R5, R7, and R8is independently selected from the group consisting of H and F.

9. The compound of claim 1, having the following structure:whereino sQ is selected from the group consisting of X 'Z " X, X, and X X;X1is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl substituted alkenyl, alkynyl substituted alkynyl, CN, CO2H, CCh alkyl), CO2(aryl),CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), N3, NH2, NH(alkyl), N(alkyl)2, OH, and O(alkyl);R5, R7, and R8are selected from the group consisting of H and F; andR6is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NHC(O)(alkyl), NH(alkyl), and N(alkyl)2.

10. The compound of claim 1, having the following structure:whereinQ is selected from the group consisting ofV0' / -, X and X'S''X;X1is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl substituted alkenyl, alkynyl substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), N3, NH2, NH(alkyl), N(alkyl)2, OH, and O(alkyl);R5, R7, and R8are selected from the group consisting of H and F; andR6is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NHC(O)(alkyl), NH(alkyl), and N(alkyl)2.

11. The compound of claim 1, having the following structure:whereinQ is selected from the group consisting of " oX s X ^4 Z, and X ';X1is selected from the group consisting of H, halogen, alkyl, substituted alkyl, alkenyl substituted alkenyl, alkynyl substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), N3, NH2, NH(alkyl), N(alkyl)2, OH, and O(alkyl);R5, R7, and R8are selected from the group consisting of H and F; andR6is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, CN, CO2H, CO2(alkyl), CO2(aryl), CO2(imide), C(O)NH(alkyl), C(O)N(alkyl)2, C(O)NH(aryl), C(O)N(aryl)2, N3, NHC(O)(alkyl), NH(alkyl), and N(alkyl)2.R9is selected from the group consisting of OH, O(alkyl), O(aryl), NH(alkyl), N(alkyl)2, NH(aryl) N(aryl)2.

12. The compound of claim 1, having a structure chosen from:O'13. The compound of claim 1, having a structure chosen from:

14. A method for detecting a target substance, comprising:contacting a sample with the compound of claim 1, anddetecting an emission light from the compound, the emission light indicating the presence of the target substance.

15. The method of claim 14, wherein the target substance is selected from a protein, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a substrate, a metabolite, an inhibitor, a drug, a nutrient, a growth factor, a lipoprotein, and a combination thereof.

16. The method of claim 14, wherein the detecting step is performed with a microscope.

17. The method of claim 14, wherein the contact step and the detecting step are performed in a live cell.

18. The method of claim 14, wherein: the compound includes a first compound and a second compound; the first compound being selective for a first target substance and capable of emitting a first emission light; the second compound being selective for a second target substance and capable of emitting a second emission light, and the detecting step includes detecting the first emission light that indicates the presence of the first target substance and the second emission light that indicates the presence of the second target substance.

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