Combinatorial phototagging of cells

Abstract

The present disclosure provides compounds comprising photocaged chemical moieties (e.g., tetrazines) and a click chemistry handle or a halotag ligand. The compounds described herein are capable of serving as "photo-click" adapters between recognition groups on the surfaces of cells and diverse chemical payloads. The present disclosure also provides compositions, systems, and kits comprising any of the compounds provided herein. Methods for optical and chemical tagging of cells using the compounds described herein are also provided by the present disclosure. Such methods may be used, for example, to monitor the expression and / or subcellular location of the one or more proteins of interest.

Description

COMBINATORIAL PHOTOTAGGING OF CELLSRELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S.S.N. 63 / 746,516, filed January 17, 2025, which is incorporated herein by reference.BACKGROUND

[0002] Light can control chemical reactions with high resolution in space and time. This capability is particularly useful in biological systems, as a means to probe spatial and temporal aspects of biological signaling. Many methods are known in the art to photo-tag cells, for example, methods to select cells based on visually discernable traits by photocrosslinking fluorescent probes to optically targeted cells of interest have been developed [1,2, 3, 4], More recently, methods of photo-tagging cells with oligonucleotides have been developed, coupling information about their location to genomics data obtained in a later sequencing step [5,6,7]. The development of further light- dependent live cell targeting strategies would advance the art.SUMMARY

[0003] The present disclosure describes compositions, systems, compounds, kits, and methods for intersectional optical and chemical tagging of live cells. Photocaged tetrazines serve as “photo-click” adapters between recognition groups on the cell surface and diverse chemical payloads. Two functionalized photocaged tetrazine structures that add a light-gating step to three cell-targeting chemical methods (HaloTag / chloroalkane labeling, non-specific primary amine labeling, and antibody labeling) are described. Light-gated versions of these three techniques are demonstrated in live cultured cells. The use of the compounds and methods provided herein for monitoring tissue flows on the surface of developing zebrafish embryos, and for combinatorial multicolor labeling and sorting of optically defined groups of cells, is also described. The photo-click adapters described herein add optical control to cell tagging schemes, with modularity in both tag and cell attachment chemistry.

[0004] Thus, the methods provided herein comprise attaching one or more compounds comprising one or more photocaged moieties (e.g., tetrazines) to the surface of one or more cells, wherein the one or more compounds are attached to the cell surface using a cell-1 / 62#14793713v2targeting chemical method (such as a halotag-chloroalkane system, non-specific reaction with amines on cell surface proteins, and / or reaction with an antibody that binds a cell surface protein). The photocaged moieties are then uncaged (either sequentially at particular times of interest (e.g., over the course of seconds, minutes, or hours), or at the same time depending on what biological process is being monitored), for example, by contacting the cells with light of a particular wavelength (e.g., about 405 nm). Next, the uncaged moieties are contacted with one or more visualizable dyes capable of reacting with them, and the cells are then imaged to determine the presence and / or locations of the visualizable dyes. In some embodiments, the one or more visualizable dyes are fused to a click chemistry handle. For example, the visualizable dyes may be fused to a transcyclooctene (TCO), such that the TCO can react with a tetrazine moiety following the uncaging step of the method.

[0005] In some embodiments, at least one of the one or more compounds comprising a photocaged moiety is attached to the cell surface using a halotag-chloroalkane system (e.g., as described in Los, G. V., et al., HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3 (6), 373-382). In such an embodiment, the method may further comprise expressing in the cells a halotag fused to one or more proteins of interest. The method can thereby be used to monitor the expression and / or subcellular location of the one or more proteins of interest. In some embodiments, at least one of the one or more compounds comprising a photocaged moiety is attached to the cell surface by reaction with an antibody that binds a cell surface protein. The method can thereby be used to monitor the expression and / or subcellular location of the cell surface protein. In some embodiments, at least one of the one or more compounds comprising a photocaged moiety is attached to the cell surface using non-specific reaction with amines on cell surface proteins. The method can thereby be used to non-specifically label the cell surface with a visualizable dye. In some embodiments, at least one of the compounds comprising a photocaged moiety is attached to the cell surface using a halotag-chloroalkane system, at least one of the compounds comprising a photocaged moiety is attached to the cell surface by reaction with an antibody that binds a cell surface protein, and at least one of the compounds comprising a photocaged moiety is attached to the cell surface using non-specific reaction with amines on cell surface proteins.2 / 62#14793713v2

[0006] In some embodiments, at least one of the compounds comprising a photocaged moietycomprises the structure: . In some embodiments, at least one of the compounds comprising a photocaged moiety comprises the structure:compounds comprising a photocaged moiety comprises the structure:compounds comprising a photocaged moiety comprises the structure:3 / 62#14793713v2BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figures 1A-1E show cell-targeted photocaged tetrazines. Figure 1A shows that illumination of photocaged dihydrotetrazine (pcDTz) uncages it to form active tetrazine, which then reacts with TCO-conjugated dye. X denotes the functional group. Figure IB shows where the functional groups azide and chloroalkane “HaloTag” ligand are attached to pcDTz. Figure 1C is a schematic showing attachment of chloroalkane-conjugated pcDTz to a membrane-expressed HaloTag receptor. Figure ID is a schematic showing the two-step attachment of pcDTz to membrane proteins via reactive ester-amine coupling and azide-DBCO click chemistry. Figure IE is a schematic showing two-step attachment of pcDTz to antibody via reactive ester-amine coupling and azide-DBCO click chemistry.

[0008] Figures 2A-2D show opto-chemical tagging of cultured cells. Figure 2A shows genetically targeted surface labeling. Fluorescence images show subcellular multicolor lightgated labeling via HTL-pcDTz on an HTR-PDGFR-expressing MDCK cell. The cell was simultaneously labeled with low-concentration HTL-AF488 to indicate HTR expression. The cell was then illuminated with two horizontal bars of 405 nm uncaging light (rectangles; top left) and stained with TCO-Cy3 (top middle). The cell was then illuminated with vertical bars of 405 nm light (bottom left) and stained with TCO-Cy5 (bottom middle). Rightmost image merges the fluorescence channels and shows line profiles of Cy3 and Cy5 intensity across the patterned regions. Scale bar: 10 pm. Figure 2B shows: Top: Fluorescence image of a confluent layer of HEK cells expressing HTR-PDGFR and treated with HTL-pcDTz. The pcDTz was photouncaged in a 5 mm diameter circle and then labeled with TCO-Cy3. Scale bar: 1 mm. Bottom: histogram of cell average intensities inside and outside the patterned region. Figure 2C shows nonspecific surface labeling. Brightfield (left) and fluorescence (right) images of a wild-type MDCK cell after treating with sulfoNHS-DBCO and azide-pcDTz, illuminating with patterned 405 nm light (left), and perfusion with TCO-Cy5 dye. Dashed line shows cell outline. Scale bar: 10 pm. Figure 2D shows antibody-targeted surface labeling. HEK cells were transfected with CD44-HaloTag, treated with biotinylated antiCD44-pcDTz, and photopattemed. Left image shows fluorescence from streptavidin-AF488, indicating binding of the antibody. Right images show fluorescence before (top) and after (bottom) uncaging and perfusion with TCO-Cy5. Scale bar: 10 pm.

[0009] Figures 3A-3F show amine-targeted photo-click on live zebrafish embryos. Figure 3A is a schematic showing chemical treatment and iterative patterning of stripes on the zebrafish embryo tail. Figure 3B shows a zebrafish embryo, 14 hours post-fertilization (hpf), treated with sulfoNHS-DBCO and azide-pcDTz, patterned iteratively with 405 nm light,4 / 62#14793713v2TC0-Cy3 (dye #1), and TC0-Cy5 (dye #2). Figure 3C shows a zebrafish embryo that underwent the same patterning process described in Figure 3B, then left for 12 hours before fixation and imaging. Figure 3D shows a zebrafish embryo, with Cy3 stripes patterned across the tail at ~18 hpf, followed by immediate fixation and imaging. Figure 3E shows a zebrafish embryo, with Cy3 stripes patterned across the tail at ~18 hpf, then left until 24 hpf before fixation and imaging. Inset shows boxed region, magnified. Figure 3F shows TCO-Cy5 was patterned on the surface of a zebrafish embryo ubiquitously expressing cell-membrane-localized mNeonGreen. Three-dimensional imaging showed that the patterning was confined to a superficial layer. Scale bars: 100 pm.

[0010] Figures 4A-4C show multicolor combinatorial cell sorting. Figure 4A shows: Left: schematic showing expected dye retention in each iteratively patterned region. Right: merged fluorescence image and schematic showing regions of HEK cells photo-patterned in partially overlapping regions by sequentially targeted illumination and TCO-dye labeling (TCO-Cy3, TCO-Cy5, TCO-AZDye488). Scale bar 1 mm. Figure 4B shows a schematic showing FACS-sorted cell populations after patterning via sulfoNHS-DBCO + azide-pcDTz. Figure 4C shows that FACS data showed separable populations with all seven combinations of the three dyes.

[0011] Figure 5 shows sub-cellular intersectional genetic and optical targeting. Brightfield (left) and merged fluorescence (right) show images of MDCK cells expressing HTR-PDGFR and stained with a mixture of HTL-pcDTz and HTL-AF488, uncaged in a small region with 405 nm light, and perfused with TCO-Cy5 dye. AF488 fluorescence indicates HaloTag expression, Cy5 fluorescence shows TCO-dye binding, the dash line outlines all cells in brightfield image, and the solid line indicates the region targeted with uncaging light. The pattern of Cy5 fluorescence demonstrates that the photochemical targeting only occurred in cells expressing HTR-PDGFR. The blurry objects at the bottom of the transmitted light image are the channels of the BioPen micro-pipette used for local dye perfusion. Scale bars 10 pm.

[0012] Figures 6A-6B show photo-click reaction kinetics. Uncaging and binding kinetics on cells expressing HTR-PDGFR and treated with HTL-pcDTz and HTL-AF488 (10:1) are shown. Fluorescence intensity of the TCO-Cy5 signal is normalized by the HTL-AF488 signal on the same cell, to control for variations in expression level. Figure 6A shows normalized Cy5 fluorescence intensity vs. illumination time (50 W / cm2, 405 nm) after uncaging and 60 seconds of perfusion with 10 pM TCO-Cy5, fit to a two-parameter exponential model I = A(1 - e“t / r), with characteristic timescale T = 5.8 seconds [95% CI: 5.0, 6.6] and saturation value A = 0.73 [95% CI: 0.70, 0.76]. N = 5 cells, at room temperature.5 / 62#14793713v2Error bars show s.e.m. Figure 6B shows normalized Cy5 fluorescence intensity vs. dye perfusion time. Region of interest was first illuminated for 5 seconds at 125 W / cm2(625 J / cm2), then perfused with 10 pM TCO-Cy5 dye. Fit to two-parameter exponential model, with characteristic timescale T = 665 seconds [95% CI: 577, 752] and saturation value A = 2.85 [95% CI: 2.59, 3.11]. N = 4 cells, at room temperature. Error bars show s.e.m.

[0013] Figures 7A-7C show control experiments for antibody mediated photo-click. Figure 7A shows: (top) untransfected, (bottom) CD44-HaloTag-expressing HEK cells, stained with biotinylated anti-CD44 and streptavidin- AF488 and HTL-JF635. Left: brightfield images, middle: AF488 fluorescence, right: JF635 fluorescence. Scale bar 100 pm. Figure 7B shows merged brightfield and fluorescence images of CD44-HaloTag-expressing HEK cells. Left: stained with biotinylated antiCD44 and streptavidin-AF488; right: stained with pcDTz-modified biotinylated antiCD44 and streptavidin- AF488. Scale bar 100 pm. Figure 7C shows HEK cells transfected with CD44-HaloTag, treated with antiCD44-pcDTz and photopattemed. Left image shows fluorescence from HaloTag ligand- JF635, indicating expression of the construct, overlayed on brightfield images. Right images show fluorescence of TCO-dye. Top: first uncaging and perfusion with TCO-AZdye488. Bottom: second uncaging and perfusion with TCO-Cy3. Scale bar: 10 pm. Asterisk indicates untransfected cell.

[0014] Figure 8 shows that fish developed normally after photo-patterning. Representative images of zebrafish, taken at 60 hours post fertilization, after control (fish water rinses, left) or photo-click surface patterning (right) at 14 hpf.

[0015] Figures 9A-9B show combinatorial photo-labeling of HTR-PDGFR-expressing HEK cells, analyzed by image processing. Figure 9A shows images of stained cells in each color channel separately (left: TCO-Cy3; middle: TCO-Cy5; right: TCO-AZDye488). Scale bar: 1 mm. Figure 9B shows the frequency of cell intensities. Thick lines show cell intensities inside all regions illuminated at indicated stage and intensities for cells not illuminated at that stage. Thin lines denote all possible overlap regions, with solid lines indicating regions expected to retain dye, and dashed lines indicating those expected to be dark.

[0016] Figure 10 shows a spectrum of the HTL-pcDTz,1H NMR.

[0017] Figure 11 shows a spectrum of the HTL-pcDTz,13C NMR.

[0018] Figure 12 shows a spectrum of the compound S6,1H NMR.

[0019] Figure 13 shows a spectrum of the compound S6,13C NMR.

[0020] Figure 14 shows a spectrum of the compound S7,1H NMR.

[0021] Figure 15 shows a spectrum of the compound S7,13C NMR.6 / 62#14793713v2

[0022] Figure 16 shows a spectrum of the azide-pcDTz,!H NMR.

[0023] Figure 17 shows a spectrum of the azide-pcDTz,13C NMR.

[0024] Figure 18 shows a spectrum of the HTL-pcDTz, mass spectrum.

[0025] Figure 19 shows the compound S6, mass spectrum.

[0026] Figure 20 shows the compound S7, mass spectrum.

[0027] Figure 21 shows the azide-pcDTz, mass spectrum.

[0028] Figures 22A-22B show PDX-496, a compound uncaged by near infrared (635-800 nm) light. Figure 22A shows the structure of PDX-496. Figure 22B shows an activation spectrum for PDX-496.

[0029] Figures 23A-23B show targeting of PDX-496 to cells.

[0030] Figure 24 shows targeted fluorescent labeling of HEK293 cells

[0031] Figure 25 shows targeting of biological macromolecules such as antibodies to cells using pcDTz or PDX-496.

[0032] Figure 26 shows the fluorescence of the AF488 dye, demonstrating patterning of an antibody in a checkerboard patternDEFINITIONS

[0033] Definitions of specific functional groups and terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Michael B. Smith, March’s Advanced Organic Chemistry, 7thEdition, John Wiley & Sons, Inc., New York, 2013; Richard C. Larock, Comprehensive Organic Transformations, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987.

[0034] “Click chemistry” is a chemical strategy tailored to generate substances quickly and reliably by joining small units together. See, e.g., Kolb, Finn, and Sharpless, Angew Chem Int Ed 2001, 40, 2004; Evans, Australian Journal of Chemistry 2007, 60, 384. The term “click chemistry” does not refer to a specific reaction or set of reaction conditions, but instead refers to a class of reactions (e.g., coupling reactions). Exemplary coupling reactions (some of which may be classified as “click chemistry”) include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides;7 / 62#14793713v2nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-yne addition; imine formation; and Michael additions (e.g., maleimide addition). Examples of click chemistry reactions can be found in, e.g., Kolb, H. C.; Finn, M. G. and Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004; Kolb, H. C. and Sharpless, K. B. Drug Disc. Today 2003, 8, 112; Rostovtsev, V. V.; Green L. G.; Fokin, V. V. and Sharpless, K. B. Angew. Chem. Int. Ed. 2002, 41, 2596; Tomoe, C. W.; Christensen, C. and Meldal, M. J. Org. Chem. 2002, 67, 3057; Wang, Q. et al. J. Am. Chem. Soc. 2003, 125, 3192; Lee, L. V. et al. J. Am. Chem. Soc. 2003, 125, 9588; Lewis, W. G. et al. Angew. Chem. Int. Ed. 2002, 41, 1053; Manetsch, R. et al., J. Am. Chem. Soc. 2004, 126, 12809; Mocharla, V. P. et al. Angew. Chem. Int. Ed.2005, 44, 116; each of which is incorporated by reference herein. In some embodiments, the click chemistry reaction involves a reaction with an alkyne moiety comprising a carboncarbon triple bond (i.e., an alkyne handle). In some embodiments, the click chemistry reaction is a copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. A CuAAC reaction generates a l,4-disubstituted-l,2,3-triazole product i.e., a 5-membered heterocyclic ring). See, e.g., Hein J. E.; Fokin V. V. Chem Soc Rev, 2010, 39, 1302; which is incorporated herein by reference.

[0035] ‘ ‘Click chemistry handles” are chemical moieties that are capable of reacting in a click chemistry reaction as described above and may comprise, for example, an ester functional group, such as N-hydroxy succinimidyl (NHS) esters or a functional group in succinimidyl valeric acid (SVA), acrylate or methacrylate, n-acetylgalactosamine, maleimide, an azide group, an alkyne group (e.g., a cyclooctyne group), a tetrazine group, a trans-cyclooctene group, or a hexynyl group.

[0036] A “photocaged moiety” refers to a chemical moiety comprising a protecting group that can be removed with light. In some embodiments, any of the click chemistry handles described above, such as a tetrazine group, may be photocaged. Examples of photolabile protecting groups that can be used in photocaged moieties include, but are not limited to, nitrobenzyl-based protecting groups, carbonyl-based protecting groups (such as phenacyl protecting groups), and benzyl-based protecting groups. Methods of attaching photolabile protecting groups to compounds, including to click chemistry handles, are well known and will be readily apparent to those of skill in the art.

[0037] A “halotag ligand” refers to a synthetic compound containing a chloroalkane linker attached to a functional group or other small molecule. Halotag ligands form highly specific covalent bonds with the halotag protein. The use of the halotag ligand-protein system enables 8 / 62#14793713v2tagging proteins of interest with fluorescent dyes or other molecules to facilitate imaging, purification, or analysis of protein interactions. Halotag ligands are further described, for example, in Los, G. V., et al., HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3 (6), 373-382, which is incorporated herein by reference.

[0038] A “visualizable dye” refers to a small molecule compound that absorbs and emits specific wavelengths of light. This allows the visualizable dye to be observed, located, or tracked using various imaging techniques, such as microscopy (e.g., fluorescence microscopy, confocal microscopy, or electron microscopy).

[0039] The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“Ci-20 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“Ci-12 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“Ci-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“Ci-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”).Examples of Ci-6 alkyl groups include methyl (Ci), ethyl (C2), propyl (C3) (e.g., zz-propyl, isopropyl), butyl (C4) (e.g., zz-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tert-amyl), and hexyl (Ce) (e.g., n-hexyl). Additional examples of alkyl groups include zz-heptyl (C7), n-octyl (Cs), zz-dodecyl (C12), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as F). In certain embodiments, the alkyl group is an unsubstituted Ci-12 alkyl (such as unsubstituted Ci-6 alkyl, e.g., -CH3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (z-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted zz-butyl (zz-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu or s-Bu), unsubstituted isobutyl (z-Bu)). In certain embodiments, the alkyl group is a substituted Ci-12 alkyl (such as9 / 62#14793713v2substituted Ci6alkyl, e.g., -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2, -CH2CF3, or benzyl (Bn)).

[0040] The term “aryl” refers to a radical of a monocyclic or polycyclic e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 n electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“Ce-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“Ce aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“Cio aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“Ci4 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted Ce-14 aryl. In certain embodiments, the aryl group is a substituted Ce-14 aryl. In some embodiments, an aryl group is a substituted or unsubstituted benzyl group.

[0041] A group is optionally substituted unless expressly provided otherwise. The term “optionally substituted” refers to being substituted or unsubstituted. In certain embodiments, alkyl, aryl, and acyl groups are optionally substituted. “Optionally substituted” refers to a group which is substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl or “substituted” or “unsubstituted” aryl). In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds and includes any of the substituents described herein that results in the formation of a stable compound. The present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation 10 / 62#14793713v2of a stable moiety. The disclosure is not limited in any manner by the exemplary substituents described herein.

[0042] Exemplary carbon atom substituents include halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -ORaa, -ON(Rbb)2, -N(Rbb)2, -N(Rbb)3+X“, -N(ORcc)Rbb, -SH, -SRaa, -SSRCC, -C(=O)Raa, -CO2H, -CHO, -C(ORCC)2, -CO2Raa, -OC(=O)Raa, -OCO2Raa, -C(=O)N(Rbb)2, -OC(=O)N(Rbb)2, -NRbbC(=O)Raa, -NRbbCO2Raa, -NRbbC(=O)N(Rbb)2, -C(=NRbb)Raa, -C(=NRbb)ORaa, -OC(=NRbb)Raa, -OC(=NRbb)ORaa, -C(=NRbb)N(Rbb)2, -OC(=NRbb)N(Rbb)2, -NRbbC(=NRbb)N(Rbb)2, -C(=O)NRbbSO2Raa, -NRbbSO2Raa, -SO2N(Rbb)2, -SO2Raa, -SO2ORaa, -OSO2Raa, -S(=O)Raa, -OS(=O)Raa, -Si(Raa)3, -OSi(Raa)3-C(=S)N(Rbb)2, -C(=O)SRaa, -C(=S)SRaa, -SC(=S)SRaa, -SC(=O)SRaa, -OC(=O)SRaa, -SC(=O)ORaa, -SC(=O)Raa, -P(=O)(Raa)2, -P(=O)(ORCC)2, -OP(=O)(Raa)2, -OP(=O)(ORCC)2, -P(=O)(N(Rbb)2)2, -OP(=O)(N(Rbb)2)2, -NRbbP(=O)(Raa)2, -NRbbP(=O)(ORcc)2, -NRbbP(=O)(N(Rbb)2)2, -P(RCC)2, -P(ORCC)2, -P(RCC)3+X“, -P(ORCC)3+X“, -P(RCC)4, -P(ORCC)4, -OP(RCC)2, -OP(RCC)3+X-, -OP(ORCC)2, -OP(ORCC)3+X-, -OP(RCC)4, -OP(ORCC)4, -B(Raa)2, -B(ORCC)2, -BRaa(ORcc), C1 20 alkyl, C 1 20 perhaloalkyl, C1-20 alkenyl, Ci-20 alkynyl, heteroCi-20 alkyl, heteroCi-20 alkenyl, heteroCi-20 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups;or two geminal hydrogens on a carbon atom are replaced with the group =0, =S, =NN(Rbb)2, =NNRbbC(=O)Raa, =NNRbbC(=O)ORaa, =NNRbbS(=O)2Raa, =NRbb, or =NORCC;wherein:each instance of Raais, independently, selected from Ci-20 alkyl, Ci-20 perhaloalkyl, Ci-20 alkenyl, Ci-20 alkynyl, heteroCi-20 alkyl, heteroCi-2oalkenyl, heteroCi-2oalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5- 14 membered heteroaryl, or two Raagroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each of the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups;each instance of Rbbis, independently, selected from hydrogen, -OH, -ORaa, -N(RCC)2, -CN, -C(=O)Raa, -C(=O)N(RCC)2, -CO2Raa, -SO2Raa, -C(=NRcc)ORaa, -C(=NRCC)N(RCC)2, -SO2N(RCC)2, -SO2RCC, -SO2ORCC, -SORaa, -C(=S)N(RCC)2, -C(=O)SRCC, -C(=S)SRCC, -P(=O)(Raa)2, -P(=O)(ORCC)2, -P(=O)(N(RCC)2)2, Ci-2o 11 / 62#14793713v2alkyl, Ci-20 perhaloalkyl, Ci-20 alkenyl, Ci-20 alkynyl, heteroCi-2oalkyl, heteroCi- 2oalkenyl, heteroCi-2oalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two Rbbgroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups; each instance of Rccis, independently, selected from hydrogen, Ci-20 alkyl, Ci-20 perhaloalkyl, Ci-20 alkenyl, Ci-20 alkynyl, heteroCi-20 alkyl, heteroCi-20 alkenyl, heteroCi-20 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two Rccgroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups;each instance of Rddis, independently, selected from halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -ORee, -ON(Rff)2, -N(Rff)2, -N(Rff)3+X“, -N(ORee)Rff, -SH, -SRee, -SSRee, -C(=O)Ree, -CO2H, -CO2Ree, -OC(=O)Ree, -OCO2Ree, -C(=O)N(Rff)2, -OC(=O)N(Rff)2, -NRffC(=O)Ree, -NRffCO2Ree, -NRffC(=O)N(Rff)2, -C(=NRff)ORee, -OC(=NRff)Ree, -OC(=NRff)ORee, -C(=NRff)N(Rff)2, -OC(=NRff)N(Rff)2, -NRffC(=NRff)N(Rff)2, -NRffSO2Ree, -SO2N(Rff)2, -SO2Ree, -SO2ORee, -OSO2Ree, -S(=O)Ree, -Si(Ree)3, -OSi(Ree)3, -C(=S)N(Rff)2, -C(=O)SRee, -C(=S)SRee, -SC(=S)SRee, -P(=O)(ORee)2, -P(=O)(Ree)2, -OP(=O)(Ree)2, -OP(=O)(ORee)2, Ci-io alkyl, Ci-io perhaloalkyl, Ci-io alkenyl, Ci-io alkynyl, heteroCi-ioalkyl, heteroCi-ioalkenyl, heteroCi-ioalkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, Ce-io aryl, and 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgggroups, or two geminal Rddsubstituents are joined to form =0 or =S;each instance of Reeis, independently, selected from Ci-10 alkyl, Ci-10 perhaloalkyl, Ci-10 alkenyl, Ci-10 alkynyl, heteroCi-10 alkyl, heteroCi-10 alkenyl, heteroCi-10 alkynyl, C3-10 carbocyclyl, Ce-io aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgggroups;12 / 62#14793713v2each instance of Rffis, independently, selected from hydrogen, Ci-io alkyl, Ci- 10 perhaloalkyl, Ci-io alkenyl, Ci-io alkynyl, heteroCi-io alkyl, heteroCi-io alkenyl, heteroCi-io alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, Ce-io aryl, and 5- 10 membered heteroaryl, or two Rffgroups are joined to form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgggroups;each instance of Rggis, independently, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OCi-6 alkyl, -ON(Ci-6alkyl)2, -N(Ci^ alkyl)2, -N(Ci^ alkyl^X’, -NH(CI-6 alkyl)2+X“, -NH2(CI6alkyl)+X“, -NH3+X“, -N(OCi6alkyl)(Ci-6 alkyl), -N(OH)(CI-6 alkyl), -NH(OH), -SH, -SCi-6alkyl, -SS(Ci-6alkyl), -C(=O)(Ci-6 alkyl), -CO2H, -CO2(CI6alkyl), -OC(=O)(Ci-6 alkyl), -OCO2(Ci6alkyl), -C(=O)NH2, -C(=O)N(CI-6 alkyl)2, -OC(=O)NH(Ci6alkyl), -NHC(=O)( Ci-6alkyl), -N(CI-6 alkyl)C(=O)( Ci-6alkyl), -NHCO2(CI6alkyl), -NHC(=O)N(Ci-6alkyl)2, -NHC(=O)NH(CI-6alkyl), -NHC(=O)NH2, -C(=NH)O(Ci6alkyl), -OC(=NH)(CI-6alkyl), -OC(=NH)OCi6alkyl, -C(=NH)N(Ci6alkyl)2, -C(=NH)NH(CI-6alkyl), -C(=NH)NH2, -OC(=NH)N(Ci-6alkyl)2, -OC(NH)NH(Ci 6 alkyl), -OC(NH)NH2, -NHC(NH)N(CI6alkyl)2, -NHC(=NH)NH2, -NHSO2(CI6alkyl), -SO2N(CI-6alkyl)2, -SO2NH(CI6alkyl), -SO2NH2, -SO2Ci6alkyl, -SO2OCi-6alkyl, -OSO2Ci6alkyl, -SOCi6alkyl, -Si(Ci-6alkyl)3, -OSi(Ci-6alkyl)3-C(=S)N(CI-6 alkyl)2, C(=S)NH(Ci-6alkyl), C(=S)NH2, -C(=O)S(Ci-6 alkyl), -C(=S)SC1^ alkyl, -SC(=S)SCi-6alkyl, -P(=O)(OCi-6 alkyl)2, -P(=O)(Ci-6 alkyl)2, -OP(=O)(Ci-6 alkyl)2, -OP(=O)(OCi-6 alkyl)2, Ci-10 alkyl, Ci-10 perhaloalkyl, Ci-10 alkenyl, Ci-10 alkynyl, heteroCi-io alkyl, heteroCi-io alkenyl, heteroCi-io alkynyl, C3- 10 carbocyclyl, Ce-io aryl, 3-10 membered heterocyclyl, or 5-10 membered heteroaryl; or two geminal Rggsubstituents are joined to form =0 or =S; andeach X“ is a counterion.

[0043] In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci-6 alkyl, -ORaa, -SRaa, -N(Rbb)2, -CN, -SCN, -NO2, -C(=O)Raa, -CO2Raa, -C(=O)N(Rbb)2, -OC(=O)Raa, -OCO2Raa, -OC(=O)N(Rbb)2, -NRbbC(=O)Raa, -NRbbCO2Raa, or -NRbbC(=O)N(Rbb)2. In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci-10 alkyl, -ORaa, -SRaa, -N(Rbb)2,13 / 62#14793713v2-CN, -SCN, -N02, -C(=O)Raa, -CCER32, -C(=O)N(Rbb)2, -OC(=O)Raa, -OCO2Raa, -OC(=O)N(Rbb)2, -NRbbC(=O)Raa, -NRbbCO2Raa, or -NRbbC(=O)N(Rbb)2, wherein Raais hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci-10 alkyl, an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl) when attached to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl) when attached to a sulfur atom; and each Rbbis independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci-10 alkyl, or a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted C1-6 alkyl, -ORaa, -SRaa, -N(Rbb)2, -CN, -SCN, or -NO2. In certain embodiments, each carbon atom substituent is independently halogen, substituted (e.g., substituted with one or more halogen moieties) or unsubstituted Ci-10 alkyl, -ORaa, -SR33, -N(Rbb)2, -CN, -SCN, or-N02, wherein Raais hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci-10 alkyl, an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl) when attached to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl) when attached to a sulfur atom; and each Rbbis independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci-10 alkyl, or a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts).

[0044] In certain embodiments, the molecular weight of a carbon atom substituent is lower than 250, lower than 200, lower than 150, lower than 100, or lower than 50 g / mol. In certain embodiments, a carbon atom substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, nitrogen, and / or silicon atoms. In certain embodiments, a carbon atom substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, and / or nitrogen atoms. In certain embodiments, a carbon atom substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, and / or iodine atoms. In certain embodiments, a carbon atom substituent consists of carbon, hydrogen, fluorine, and / or chlorine atoms.

[0045] The term “acyl” refers to a group having the general formula -C(=O)RX1, -C(=O)ORX1, -C(=O)-O-C(=O)RX1, -C(=O)SRX1, -C(=O)N(RX1)2, -C(=S)RX1, -C(=S)N(RX1)2, and -C(=S)S(RX1), -C(=NRX1)RX1, -C(=NRX1)ORX1, -C(=NRX1)SRX1, and 14 / 62#14793713v2-C(=NRX1)N(RX1)2, wherein RX1is hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di- aliphaticamino, mono- or di- heteroaliphaticamino, mono- or di- alkylamino, mono- or di- heteroalkylamino, mono- or di-arylamino, or mono- or diheteroarylamino; or two RX1groups taken together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-CO2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, hetero aliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0046] The term “small molecule” refers to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, a small molecule is an organic compound (e.g., it contains carbon). The small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.). In certain embodiments, the molecular weight of a small molecule is not more than about 1,000 g / mol, not more than about 900 g / mol, not more than about 800 g / mol, not more than about 700 g / mol, not more than about 600 g / mol, not more than about 500 g / mol, not more than about 400 g / mol, not more than about 300 g / mol, not more than about 200 g / mol, or not more than about 100 g / mol. In certain embodiments, the molecular weight of a small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 80015 / 62#14793713v2g / mol, or at least about 900 g / mol, or at least about 1,000 g / mol. Combinations of the above ranges (e.g., at least about 200 g / mol and not more than about 500 g / mol) are also possible. In certain embodiments, the small molecule is a visualizable dye.

[0047] A “biological macromolecule” refers to an organic compound that is not a small molecule. In certain embodiments, the molecular weight of a biological macromolecule is greater than about 2,000 g / mol, greater than about 3,000 g / mol, greater than about 4,000 g / mol, or greater than about 5,000 g / mol. In certain embodiments, the molecular weight of a biological macromolecule is at most about 100,000 g / mol, at most about 30,000 g / mol, at most about 10,000 g / mol, at most about 5,000 g / mol, or at most about 2,000 g / mol.Combinations of the above ranges (e.g., greater than about 2,000 g / mol and at most about 10,000 g / mol) are also possible. In certain embodiments, the biological macromolecule is a therapeutically active agent such as a drug (e.g., a molecule approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (C.F.R.)). In some embodiments, a biological macromolecule is a nucleic acid (e.g., DNA or RNA). In some embodiments, a biological macromolecule is a protein (e.g., an antibody).DETAILED DESCRIPTION

[0048] The present disclosure provides compounds comprising photocaged moieties (e.g., tetrazines) capable of serving as “photo-click” adapters between recognition groups on the surface of cells (including, e.g., primary amines) and diverse chemical payloads. Two functionalized photocaged tetrazine structures that add a light-gating step to cell-targeting chemical methods (such as HaloTag / chloroalkane labeling, non-specific primary amine labeling, and antibody labeling) are described. Light-gated versions of these three techniques are demonstrated in live cultured cells. The present disclosure also provides compositions, systems, kits, and methods for optical and chemical tagging of cells.Compounds

[0049] In some aspects, the present disclosure provides compounds comprising photocaged moieties and click chemistry handles. In some embodiments, the compounds can be used as adapters to attach small molecules, such as visualizable dyes, or biological macromolecules, such as antibodies, antibody fragments, or antibody variants, to the surface of a cell.

[0050] In some aspect, the present disclosure provides compounds comprising the structure:[photocaged moiety] -[click chemistry handle]. In some embodiments, the compound16 / 62#14793713v2comprises the structure: [photocaged tetrazine] -[click chemistry handle]. In some embodiments, the compound comprises the structure: [photocaged tetrazine] -[azide].

[0051] In some embodiments, the compound comprises the structure:wherein:R1is a photocaging moiety;R2is an alkyl group substituted with a click chemistry handle, wherein the alkyl group may optionally be substituted with one or more further substituents; andR3is an optionally substituted aryl group.

[0052] In some embodiments, the photocaging moiety comprises a substituted ester. In some embodiments, the click chemistry handle comprises an ester, acrylate, methacrylate, maleimide, an azide, an alkyne (e.g., a cyclooctyne), or a trans-cyclooctene. In certain embodiments, the click chemistry handle comprises an azide. In some embodiments, R2is Ci-Ce alkyl substituted with the click chemistry handle. In certain embodiments, R2is Ci-Ce alkyl substituted with an azide. In some embodiments, R3is an unsubstituted aryl group. In some embodiments, R3is an optionally substituted benzyl group. In certain embodiments, R3is an unsubstituted benzyl group.

[0053] In some embodiments, the compound comprises the structure:

[0054] In some embodiments, the compound comprises the structure:17 / 62#14793713v2

[0055] In some embodiments, the compound comprises the structure:

[0056] In some embodiments, the compound comprises the structure:wherein R4is an optionally substituted alkyl group.

[0057] In certain embodiments, the compound comprises the structure:

[0058] In certain embodiments, the compound comprises the structure:

[0059] In another aspect, the present disclosure provides compounds comprising the structure: [photocaged moiety]-[halotag ligand]. In some embodiments, the compound comprises the structure: [photocaged tetrazine]-[halotag ligand].18 / 62#14793713v2

[0060] In some embodiments, the compound comprises the structure:wherein:R1is a photocaging moiety;R2is an alkyl group substituted with a halotag ligand, wherein the alkyl group may optionally be substituted with one or more further substituents; andR3is an optionally substituted aryl group.

[0061] In some embodiments, the photocaging moiety comprises a substituted ester. In some embodiments, R2is Ci-Ce alkyl substituted with the halotag ligand. In certain embodiments, the halotag ligand comprises the structure:unsubstituted aryl group. In some embodiments, R3is an optionally substituted benzyl group. In certain embodiments, R3is an unsubstituted benzyl group.

[0062] In some embodiments, the compound comprises the structure:

[0063] In some embodiments, the compound comprises the structure:

[0064] In some embodiments, the compound comprises the structure:19 / 62#14793713v2

[0065] In some embodiments, the compound comprises the structure:wherein R4is an optionally substituted alkyl group.

[0066] In certain embodiments, the compound comprises the structure:

[0067] In certain embodiments, the compound comprises the structure:20 / 62#14793713v2Compositions

[0068] The present disclosure also provides compositions comprising any of the compounds disclosed herein, or salts thereof, and optionally an excipient. Such compositions may be useful for practicing the methods described herein, e.g., by contacting one or more cells with the composition. In certain embodiments, the compositions described herein comprises a compound disclosed herein, or a salt thereof, and an excipient. Compositions described herein can be prepared by any method known in the art. In general, such preparatory methods include bringing the compound described herein (z.e., the “active ingredient”) into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping, and / or packaging the product into a desired single- or multi-dose unit. Excipients used in the manufacture of the provided compositions include inert diluents or fillers, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, and / or buffering agents.

[0069] Exemplary diluents or fillers include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, starches (such as dry starch, cornstarch), sugars (such as powdered sugar), calcium trisulfate, carboxymethylcellulose calcium, dextrate, dextrin, dextrose, fructose, lactitol, lactose, magnesium carbonate, magnesium, maltitol, maltodextrin, maltose, sucrose, glucose, mannitol, silicic acid, xylitol, and mixtures thereof.

[0070] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0071] Exemplary surface active agents and / or emulsifiers include natural emulsifiers e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol,21 / 62#14793713v2oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.

[0072] Exemplary disintegrating agents or disintegrants include agar, algin, alginic acid, sodium alginate, silicates, sodium carbonate, calcium carbonate, carboxymethylcellulose, cellulose, clay, colloidal silicon dioxide, croscarmellose sodium, crospovidone, rubber, magnesium silicate, methylcellulose, potassium krillin, hydroxypropylcellulose (e.g., low substituted hydroxypropylcellulose), crosslinked polyvinylpyrrolidone, hydroxypropylcellulose, and starch (e.g., sodium glycolate starch, potato or tapioca starch).

[0073] Exemplary binding agents include starch (e.g., glycolate starch, cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and / or mixtures thereof.

[0074] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives,22 / 62#14793713v2acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0075] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0076] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.

[0077] Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0078] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0079] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0080] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0081] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, NeoIone®, Kathon®, and Euxyl®.

[0082] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic 23 / 62#14793713v2acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, and mixtures thereof.Kits

[0083] Also encompassed by the disclosure are kits. Such kits may be useful for practicing the methods described herein, e.g., by contacting one or more cells with the compounds provided in the kit. The kits provided may comprise one or more compounds or compositions described herein and a container e.g., a vial, ampule, bottle, syringe, and / or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising an excipient for dilution or suspension of one or more compounds or compositions described herein. In some embodiments, one or more compounds or compositions described herein provided in the first container and the second container are combined.

[0084] Thus, in one aspect, provided are kits including a first container comprising one or more compounds or compositions described herein. In certain embodiments, the kits are useful for tagging cells (e.g., with one or more small molecules, such as a visualizable dye, or with one or more biological macromolecules, such as an antibody). In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency. In certain embodiments, the kits and instructions provide for tagging cells (e.g., with one or more small molecules, such as a visualizable dye, or with one or more biological macromolecules, such as an antibody).Methods

[0085] In some aspects, the present disclosure provides methods for tagging cells (e.g., with small molecules, such as visualizable dyes, or with biological macromolecules, such as antibodies, antibody fragments, or antibody variants). The methods provided herein comprise attaching one or more compounds comprising one or more photocaged moieties (e.g., tetrazines) to the surface of one or more cells. This is accomplished using a cell-targeting chemical method (such as a halotag-chloroalkane system, non-specific reaction with amines 24 / 62#14793713v2on cell surface proteins, and / or reaction with an antibody that binds a cell surface protein). Photocaging is accomplished by adding a protecting group that is labile upon contact with light with a reactive chemical moiety, and it ensures that the reactive chemical moiety will not undergo a chemical reaction until a desired time, upon which it can be uncaged by light. The photocaged moieties may then be uncaged, for example, by contacting the cells with light of a particular wavelength as described herein. Next, the uncaged moieties are contacted with one or more small molecules (e.g., one or more visualizable dyes) and / or one or more biological macromolecules (e.g., one or more antibodies, antibody fragments, or antibody variants) capable of reacting with the uncaged moieties. The cells can then be imaged to determine the presence and / or locations of the small molecules and / or biological macromolecules. In some embodiments, the one or more visualizable dyes are fused to a click chemistry handle. For example, the small molecules and / or biological macromolecules may be fused to a transcyclooctene (TCO), such that the TCO can react with a tetrazine moiety following the uncaging step of the method.

[0086] Thus, in one aspect, the present disclosure provides methods comprising attaching one or more compounds (e.g., covalently) comprising one or more photocaged moieties to the surface of one or more cells, wherein the one or more compounds are attached to the cell surface using a cell-targeting chemical method; uncaging the one or more photocaged moieties; and contacting the uncaged moieties with one or more small molecules or biological macromolecules capable of reacting with the uncaged moieties. In some embodiments, the one or more small molecules or biological macromolecules comprise one or more visualizable dyes. In some embodiments, the one or more small molecules or biological macromolecules comprise one or more antibodies, antibody fragments, or antibody variants. In some embodiments, the method further comprises a step of imaging the one or more cells to determine the presence and / or locations of the one or more small molecules or biological macromolecules.

[0087] In another aspect, the present disclosure provides methods comprising attaching one or more compounds comprising one or more photocaged moieties to the surface of one or more cells, wherein the one or more compounds are attached to the cell surface using a celltargeting chemical method; uncaging the one or more photocaged moieties; contacting the uncaged moieties with one or more visualizable dyes capable of reacting with the uncaged moieties; and imaging the one or more cells to determine the presence and / or locations of the one or more visualizable dyes.25 / 62#14793713v2

[0088] Any of the compounds described herein may be used in the presently disclosed methods. In some embodiments, at least one of the photocaged moieties comprises a tetrazine. In some embodiments, the one or more photocaged moieties is uncaged by contacting the cell with visible light (e.g., light at a wavelength of about 380-750 nm). In some embodiments, the one or more photocaged moieties is uncaged by contacting the cell with visible light at a wavelength of about 380, about 385, about 390, about 395, about 400, about 405, about 410, about 415, or about 420 nm. In certain embodiments, the one or more photocaged moieties is uncaged by contacting the cell with visible light at a wavelength of about 405 nm. In some embodiments, the one or more photocaged moieties is uncaged by contacting the cell with infrared light or near infrared light (e.g., light at a wavelength of about 635-800 nm). In some embodiments, the one or more photocaged moieties is uncaged by contacting the cell with infrared or near infrared light at a wavelength of about 635, about 640, about 645, about 650, about 655, about 660, about 665, about 670, about 675, about 680, about 685, about 690, about 700, about 705, about 710, about 715, about 720, about 725, about 730, about 735, about 740, about 745, about 750, about 755, about 760, about 765, about 770, about 775, about 780, about 785, about 790, about 795, or about 800 nm. In some embodiments, the one or more photocaged moieties are uncaged at different times or at the same time.

[0089] In some embodiments, the one or more small molecules or biological macromolecules are fused to a click chemistry handle. In some embodiments, the click chemistry handle comprises an ester, acrylate, methacrylate, maleimide, an azide, an alkyne (e.g., a cyclooctyne), or a trans-cyclooctene. In certain embodiments, the click chemistry handle comprises a transcyclooctene (TCO). In some embodiments, at least one of the photocaged moieties comprises a tetrazine, and the TCO reacts with the tetrazine.

[0090] In some embodiments, the cell-targeting chemical method comprises a halotag-chloroalkane system, non-specific reaction with amines on cell surface proteins, and / or reaction with an antibody that binds a cell surface protein. In some embodiments, at least one of the one or more compounds comprising a photocaged moiety is attached to the cell surface using a halotag-chloroalkane system (e.g., as described in Los, G. V., et al., HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3 (6), 373-382). In such an embodiment, the method may further comprise expressing in the cells a halotag fused to one or more proteins of interest. The method can thereby be used to monitor the expression and / or subcellular location of the one or more proteins of interest.26 / 62#14793713v2

[0091] In some embodiments, at least one of the one or more compounds comprising a photocaged moiety is attached to the cell surface by reaction with an antibody that binds a cell surface protein. In such an embodiment, the method may be used to monitor the expression and / or subcellular location of the cell surface protein.

[0092] In some embodiments, at least one of the one or more compounds comprising a photocaged moiety is attached to the cell surface using non-specific reaction with amines on cell surface proteins and / or reaction with an antibody that binds a cell surface protein. In some embodiments, attaching the compound comprising a photocaged moiety to the cell surface using non-specific reaction with amines on cell surface proteins comprises contacting the amines with a second compound comprising an electrophile and an alkyne moiety. In some embodiments, the alkyne moiety reacts with an azide in the compound comprising a photocaged moiety via click chemistry. In some embodiments, attaching the compound comprising a photocaged moiety to the cell surface by reaction with an antibody that binds a cell surface protein comprises contacting the cell with the antibody. In some embodiments, the method further comprises contacting a primary amine group on the antibody with a second compound comprising an electrophile and an alkyne moiety. In some embodiments, the alkyne moiety reacts with an azide in the compound comprising a photocaged moiety via click chemistry.EXAMPLESExample 1: Tools for intersectional optical and chemical tagging on cell surfaces

[0093] A light-gating element was added into several non-light-dependent live cell targeting strategies, with modularity in the cell attachment chemistry and the chemical payload. To accomplish this, photocaged dihydrotetrazine (pcDTz) [8] was used as a “photo-click adaptor.” The caged dihydrotetrazine is uncaged by visible light (405 nm), whereafter it can react quickly via a bio-orthogonal reaction with strained alkenes, including transcyclooctene (TCO) [9] (Figure 1A). This strategy enables fast, bio-orthogonal, light-controlled chemistry on the surface of live cells. Reference [8] presented the first of these compounds and demonstrated light-gated fluorescent labeling of an individual cell in which a lipid-conjugated pcDTz was incorporated into the membrane.

[0094] pcDTz was leveraged as a photo-click adaptor in three cell targeting strategies. First, light-control was coupled to the Halotag-chloroalkane system

[0010] for intersectional genetic and optical targeting. Second, nonspecific surface protein targeting via reactive esters was used for wide-area optical targeting

[0011] , Third, the pcDTz was incorporated onto antibodies 27 / 62#14793713v2for intersectional immunohistochemical and optical labeling. By adding light control to these cell-targeting strategies, the motion of tagged components can be followed; and labeling specificity can be increased by optically targeting specific morphological or functional features of the sample. To add optical control to these three methods, two new functional pcDTz structures were used. The structures and cell-targeting strategies were tested in live cultured cells, demonstrating multicolor labeling at both large and subcellular scale. Two applications were explored: tracking tissue flows in a developing zebrafish embryo, and combinatorial labeling for live cell sorting.HaloTag mediated photo-click in cultured cells

[0095] HaloTag is a powerful genetically encoded tool for tagging proteins of interest. The HaloTag receptor (HTR) forms a covalent bond with exogenous labels conjugated to a chloroalkane “HaloTag ligand” (HTL). With pcDTz fused to an HTL (Figure IB), lightgating was added to the HaloTag toolbox.

[0096] As a demonstration, the HTR fused to the extracellular side of a transmembrane protein (platelet- derived growth factor receptor beta (PDGFR) transmembrane domain) was expressed

[0012] , The cells were incubated with a mixture of HTL-pcDTz and HTL-AF488, with the latter added at a small mole fraction to mark HTR-PDGFR-expressing cells. After rinsing out unbound HTL reagents, the bound HTL-pcDTz was uncaged with patterned 405 nm light, and a TCO-conjugated fluorescent dye was added (Figure 1C). Figure 2A shows iterative photo-uncaging and TCO-dye labeling steps on subcellular regions (~10 pm) of an HTR-PDGFR-expressing MDCK cell. A small amount of background fluorescence is visible; the cell-localized background in the Cy3 channel (Figure 2A, top middle) is primarily due to off-target excitation of the HTL-AF488 co-stain by the Cy3-excitation laser. The diffuse background in the Cy5 channel (Figure 2A, bottom middle) is due to incomplete rinsing of the TCO-dye. It was confirmed that TCO-dye only bound to cells (1) expressing HTR-PDGFR, as confirmed by the co-stain with HTL-AF488, and (2) within the illuminated region (Figure 5).

[0097] Figure 2B demonstrates the same technique on a larger scale. A 5 mm circular region was patterned on a confluent layer of HTR-PDGFR-expressing HEK cells. Figure 6A shows the dependence of labeling density on dose of 405 nm uncaging light. The optical dose to uncage 50% of the HTL-pcDTz molecules was 198 ± 20 J / cm2(mean ± s.e.m., n = 5 experiments). Fig. 6B shows dependence of labeling density on TCO-dye exposure. At a concentration of 10 pM, labeling reached 50% of saturation in 451 ± 22 s (mean ± s.e.m., n =28 / 62#14793713v24 experiments). These results show that HTL-pcDTz combines the genetic specificity of the HaloTag system with the spatial specificity of photo-click.Nonspecific protein targeted photo-click in cultured cells

[0098] Nonspecific staining of cell surface proteins is often accomplished using N-hydroxysuccinimide (NHS) ester or other reactive esters, which are very reactive to primary amines. A method of introducing light-gating to this useful protein-targeting approach is described. NHS ester groups hydrolyze readily, precluding long term storage, and complicating the synthesis. Instead, pcDTz was functionalized with a second click handle, azide, and attached the labile ester at the time of the experiment, using a commercially available linker.

[0099] A sulfonated NHS (sulfoNHS) ester was first used to attach the strained alkyne dibenzocyclooctyne (DBCO) to amines on the extracellular surface (the sulfonate charge prevents membrane permeation). Azide-pcDTz (Figure IB) was then added, which reacted with DBCO via copper- free strain-promoted azide-alkyne cycloaddition

[0013] . pcDTz was uncaged with patterned 405 nm light, and exposed the region to TCO-dyes (Figure ID). This approach achieved subcellular labeling via nonspecific protein staining (Figure 2C).

[0100] Due to the non-specific nature of the sulfoNHS labeling, this strategy also labels proteins coating the glass coverslip, used to promote cell adhesion. Thus, this approach provides a general strategy for photochemical patterning of surface chemistry. In fluorescent cell-labeling applications, one must keep in mind that the label is attached to the substrate below the cell in addition to the cell surface.Antibody mediated photo-click in cultured cells

[0101] Live cell immuno-targeting uses labeled antibodies to target cells that express a particular antigen. The high biomolecular specificity of antibodies makes the strategy useful for biological imaging, as well as in biotechnology and drug delivery. A primary advantage of antibody-based labeling is that the target cells do not need to be genetically modified, so the technique is compatible with primary tissues and even use in humans. For example, antibodydrug conjugates are widely used in cancer therapies

[0014] , However, one may wish to target a label based on the conjunction of an epitope and some other visually identifiable feature; or to target in a temporally or spatially restricted pattern. Photo-control was introduced to enable these features.

[0102] Primary amines were labeled on antibodies by treating first with tetrafluorophenyl ester-DBCO (plus a polyethylene glycol linker, to improve solubility) and then with azide- 29 / 62#14793713v2pcDTz (Methods). This procedure was applied to commercially available biotinylated antihuman CD44 (Figure IE). CD44 is a membrane receptor involved in cell adhesion, growth, motility, and metastasis

[0015] . A CD44-HaloTag fusion

[0016] was expressed in HEK cells, which have very low endogenous expression of CD44 (Figure 7A). The intracellularly expressed HaloTag was stained with cell-permeant HTL-JF635 to confirm expression and membrane trafficking. In the same sample, extracellularly accessible membrane-localized CD44 was confirmed by staining with biotinylated anti-CD44 and streptavidin- AF488 (Figure 7A). It was confirmed that the pcDTz-modified antibody bound to cells expressing CD44 (as indicated by streptavidin- AF488 staining) and did not bind to cells that did not express CD44 (Figures 7B, 7C).

[0103] It was demonstrated that antibody mediated photo-tagging of a subcellular region of a CD44-expressing HEK cell (Figure 2D). Both light and CD44 expression were required for labeling (Figure 7C). Successive rounds of illumination and exposure to different TCO-dyes enabled multicolor tagging (Figure 7C). As in the HaloTag system, CD44 expression was variable between cells. To control for this variability, either co-staining for the biotin group on the antibody using fluorescently tagged streptavidin (Figure 2D), or labeling the intracellular HaloTag (Figure 7C) was performed. PcDTZ-antibody conjugates combine the biological specificity of antibody targeting with the spatial specificity of light. Applications of intersectional optical and chemical targeting were explored next.Photo-click on live zebrafish embryos

[0104] The spatiotemporal control, multicolor capability, and live cell compatibility of pcDTz labeling make this approach attractive for imaging flows in biology. This application area was explored by tracking flows on the surface of a live zebrafish embryo.

[0105] Zebrafish embryos develop rapidly in the first 24 hours after fertilization and are transparent during this stage

[0017] . Tracking all individual cells is possible [18,19], but becomes technically challenging and computationally expensive as the embryo grows, motivating the search for simple methods to track tissue flows without the need for continuous imaging. Recent efforts have explored methods to pattern a registration grid onto live tissue, for tracking or downstream omics

[0020] . It was shown that pcDTz photo-click could be similarly useful. The embryo surface was first functionalized with sulfoNHS ester-DBCO, then azide-pcDTz was added. TCO-dyes were iteratively uncaged and added to pattern the embryo tail with perpendicular stripes in two colors (Figure 3 A, B, D). Embryos tolerated this process well and continued to develop at a similar rate to untreated control embryos (Figure 8). The patterned embryos were imaged 12 hours after staining. The patterns 30 / 62#14793713v2were still clearly visible, but the stripes had been distorted by embryo growth, showing greater broadening on the ventral side of the tail compared to the dorsal side, as the tail straightened (Figure 3C, E). Three-dimensional reconstructions confirmed that the labeling was limited to a superficial layer, as expected for the non-cell permeant sulfoNHS ester.

[0106] Multi-color photo-click patterning is a useful approach to observe surface tissue flows in development without the need for continuous imaging. This technique can also be useful in studies of wound healing or disease progression (e.g., tumor growth), where one wishes to track the motion of large ensembles of cells. The genetically encoded HaloTag approach can also be used to track a genetically specified population of cells.Photo-click labeling for cell sorting

[0107] Photo-click chemistry can be used for combinatorial labeling. With N distinguishable dyes and a binary labeling scheme (present or absent) one can distinguish 2N-1 distinct populations (assuming the all-absent condition is not detectable). This may be useful for cell sorting. In principle, the labeling can be targeted based on any observable feature of the cells. Cells with distinct spectral barcodes can then be sorted into different wells for downstream characterization, e.g., via sequencing, proteomics, or other biochemical analyses. The ability to sort cells based on arbitrary features into multiple buckets would be a powerful enabling capability for optical pooled screens or for characterizing heterogeneous cell populations [21-25],

[0108] Here, three TCO-dyes (Cy3, Cy5, and AZDye488) were used to distinguishably label seven subpopulations of cells within a culture dish. pcDTz was attached to a near-confluent layer of live HEK cells (either via sulfoNHS-DBCO + azide-pcDTz, Figure 4, or via HTR-PDGFR expression + HTL-pcDTz, Figures 9A, 9B). pcDTz was iteratively uncaged in partially overlapping circular regions and incubated with TCO-dyes. For the uncaging step, a light dose of 50 J / cm2was used, well below saturation, so that caged pcDTz remained available for subsequent uncaging steps. For the labeling, a dose of 10 pM, 12 minutes, was used, which came close to saturating the labeling reaction, so that subsequent labeling steps would not follow prior uncaging patterns. Figures 4 and 9A-9B show the resulting cells on the surface. The labeled cells were then dissociated and separated with a fluorescence-activated cell sorter (FACS, Figure 4B). Clearly distinct populations corresponding to each of the seven labeling conditions were detected (Figure 4C). These data demonstrate combinatorial labeling with pcDTz photo-click chemistry.

[0109] Figures 9A-9B shows results from a similar combinatorial labeling approach in cells expressing HTR-PGDFR, with cells categorized in the dish by image analysis. Due to cell-to- 31 / 62#14793713v2cell variation in expression levels, entirely separable populations were not obtained, either with FACS or in-dish imaging. A cell intensity threshold which separated patterned and HaloTag-expressing cells from the dark cells, however, was established (Figures 9A, 9B). Presumably, in a monoclonal cell line expressing HT-PGDFR the expression would be more uniform across cells.

[0110] With careful control of illumination dose and labeling rates, this technique might be extended to discern multiple levels of each dye (rather than the binary case shown above), expanding the number of selectable groups without the need for additional fluorophores. Combinatorial labeling of antibody-pcDTz-labeled cells is presumably possible too.Discussion

[0111] Previous work established that pcDTz leverages the advantages of click chemistry (fast, easy, biocompatible), light-gated reactions (spatial control), and modular probes. Here, intersectional levels of control were enabled by combining pcDTz with chemically and genetically specific cell attachment strategies. Two new pcDTz structures, chloroalkane “HaloTag ligand”-pcDTz and click-capable azide-pcDTz, were presented. With these structures, light-control capabilities were added to three popular cell- attachment strategies: HaloTag, NHS ester amine chemistry, and antibody labeling. Two potential applications for the technique were also explored: tracking tissues flows on the surface of the live zebrafish embryo, and multicolor, combinatorial live cell labeling and sorting.

[0112] The methods described herein can also be used in tracking dynamics of proteins within cells. Here, tracking of tissue flows was demonstrated in embryos on a millimeter scale, with non-specific protein labeling. Light control also enables patterns to be targeted subcellularly (<10 pm), and targeted to specific proteins via a HaloTag linker. These approaches can be used to track the motion of specific protein sub-populations within cells. Super-resolution imaging techniques may enable very high-resolution tracking. The cellsorting application described herein is also useful for optical phenotypic screening and can be combined with subsequent omics.Methods

[0113] Synthesis: The functionalized pcDTz structures used here were prepared from l-(2-nitrophenyl)ethyl 6-(but-3-yn- l-yl)-3-phenyl- 1 ,2,4,5-tetrazine- 1 (4H) -carboxylate and 2-(6-phenyl-l,2,4,5-tetrazin-3-yl)ethan-l-ol, whose syntheses are described previously [8,26].

[0114] Procedure to prepare l-(2-nitrophenyl)ethyl6-(2-(l-(2-(2-(2-((6chlorohexyl) oxy)ethoxy)ethoxy)ethyl)- 1H- 1 ,2,3-triazol-4-yl)ethyl)-3-phenyl- 1 ,2,4,5-tetrazine- 1 (4H) -carboxylate (HTL-pcDTz):32 / 62#14793713v2

[0115] Under argon, 12 mL of CH3CN / H2O (v / v = 2 / 1) was added to a mixture of photocaged dihydrotetrazine l-(2-nitrophenyl)ethyl 6-(but-3-yn-l-yl)-3-phenyl-l,2,4,5-tetrazine-l(4H)-carboxylate (127 mg, 0.31 mmol), l-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6-chlorohexane (125 mg, 0.42 mmol), Cui (106 mg, 0.56 mmol), and sodium L-ascorbate (110 mg, 0.56 mmol) at room temperature. The reaction mixture was stirred at 37 °C, overnight.

[0116] Upon completion, the reaction solvent was removed under reduced pressure. The residue was purified by column chromatography (4% MeOH / CIUCh), and the desired product HLT-pcDTz was obtained (71 mg, 32% yield).

[0117] Procedure to prepare l-(2-nitrophenyl)ethyl 6-(2-azidoethyl)-3-phenyl-l,2,4,5-tetrazine- 1 (4 / 7)-carboxy I ate (azide-pcDTz) :

[0118] Under argon, triethylamine (617 pL, 4.5 mmol), MsCl (343 pL, 4.5 mmol) and DMAP (182 mg, 1.5 mmol) were added to a solution of 2-(6-phenyl- 1,2,4, 5-tetrazin-3-yl)ethan-l-ol (300 mg, 1.5 mmol) in CH2CI2 (8 mL) at room temperature. Upon completion, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (CH2Q2), and the 2-(6-phenyl-l,2,4,5-tetrazin-3-yl)ethyl methanesulfonate was obtained. Then, NaNs (309 mg, 4.75 mmol) was added to a solution of 2-(6-phenyl-l,2,4,5-tetrazin-3-yl)ethyl methanesulfonate in DMF (6 mL), the mixture stirred at room temperature under argon for 6 h. Then, the reaction mixture was extracted with CH2Q2 and washed with a saturated solution of NaCl in water. The extract was combined, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (50% Hexane / EtOAc), and 3-(2-azidoethyl)-6-phenyl-l,2,4,5-tetrazine was obtained (132 mg, 40% yield). Under argon, the thiourea dioxide (76 mg, 0.7 mmol) was added to a solution of 3-(2-azidoethyl)-6-phenyl-l,2,4,5-tetrazine (80 mg 0.35 mmol) in 15 mL of DMF / H2O (v / v = 1 / 2) at room temperature. The reaction mixture was stirred at 95 °C for 1-2 hours. Upon completion, the color of the reaction mixture changed from pink to light yellow. Under argon, 50 mL of EtOAc was added the reaction mixture, which was washed by 30 mL of H2O. The extract was combined and concentrated by reduced pressure. The residue was purified by column chromatography (CH2Q2 as the eluent), and 3-(2-azidoethyl)-6-phenyl-l,4-dihydro-l,2,4,5-tetrazine was obtained (60 mg, 74% yield). Under argon, the solution of l-(2-nitrophenyl)ethyl carbonochloridate (158 mg, 0.65 mmol) in toluene (1 mL) was added to a solution of 3-(2-azidoethyl)-6-phenyl-l,4-dihydro-l,2,4,5-tetrazine (60 mg 0.26 mmol) in pyridine (5 mL) at 0 °C. The reaction mixture was stirred at 37 °C for 24 h. Upon completion, the reaction solvent was removed under reduced pressure.33 / 62#14793713v2The residue was purified by column chromatography (1% MeOH / CH2CI2), and the azide-pcDTz was obtained (70 mg, 83% yield).

[0119] Full experimental details and characterization of the newly described compounds (1H NMR,13C NMR, mass spectra) are provided further below.

[0120] Cell culture and transfection: HEK293T and MDCK cells (ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% GlutaMax-I, penicillin (100 U / ml), and streptomycin (100 pg / ml). Cells were transfected 24 h after passage using TransIT-293 transfection reagents (Minis Bio) for the HEK cells, and Translt-X2 transfection reagents (Minis Bio) for MDCK cells. Experiments on expressing cells were performed 24 h after transfection.

[0121] Plasmids used: pcDNA5 / FRT / TO_IgKchL_HA_HaloTag9_myc_PDGFRtmb (for expression of HaloTag on the outer membrane) (Addgene plasmid # 175544 ;RRID:Addgene_175544)

[0012] , and pEGFPNl-CD44-SGx3-Halo7 (for expression of CD44 with an intracellular HaloTag)

[0016] . Plasmids were diluted 1:1 and 1:5 respectively in pucl9 vector (New England Biolabs). HaloTag expression was confirmed using HaloTag ligand dyes HTL-AF488, HTL-JF635 (Promega CS3495B06, HT1050), 50 nM in culture medium, 30 min, followed by rinsing and, for membrane permeant JF635, another 30 min incubation in fresh media. CD44 expression was confirmed using CD44 Monoclonal Antibody (IM7)-Biotin, eBioscience (Life Technologies, 13-0441-82) and Streptavidin-AlexaFluor 488 conjugate (Life Technologies, SI 1223).

[0122] Cell surface chemistry: HaloTag-expressing cells were incubated with HTL-pcDTz, 1 pM in 0.1% DMSO in culture medium, 30 min, rinsed 3x, and incubated again in fresh medium for 30 min. For nonspecific surface labeling experiments, sulfonated N-hydroxysuccinimide-DBCO (Click Chemistry Tools) was dissolved in anhydrous DMSO and added to cells, 100 pM in 0.1% DMSO in PBS, 10 min, then rinsed 3x. Azide-pcDTz was then added to cells at 1 pM in 0.1% DMSO in culture medium, incubated 60 min, rinsed 3x, and incubated 30 more min in fresh medium. This reaction time was chosen to minimize changes in cell biology (protein internalization, cell division) during the course of labeling; saturation of all DBCO groups is not expected under these reaction conditions. Unreacted DBCO groups on the cell surface are expected to be inert, and unreacted azide-pcDTz is removed by rinsing. These conditions permit bright labeling on live cells.

[0123] For antibody experiments, cells were first blocked for 60 min with 3% bovine serum albumin (BSA) in Hank’s buffered salt solution (HBSS). Functionalized antibody was added at ~10 nM in HBSS + 1% BSA for 30 min. Dish was rinsed 3x, then stained with HTL dye or 34 / 62#14793713v2streptavidin- AF488. All incubation steps were performed at 37 °C, 5% CO2, and all media and cell buffers were pre- warmed to 37 °C.

[0124] Preparation of functionalized antibody: CD44 Monoclonal Antibody (IM7), Biotin, eBioscience (Life Technologies, 13-0441-82) was used after solvent exchange to PBS, pH 7.2, using BSA-passivated Amicon Ultra 10 kDa spin columns to remove sodium azide. EZ-Link TFP Ester-PEGi2-DBCO (Life Technologies), dissolved in DMSO, was added to the antibody in ~50x molar excess. The reaction proceeded at room temperature for 1 hour on a shaker, and overnight at 4 °C. The reaction was quenched with Tris HC1 buffer, then de-salted again on a freshly BSA-passivated 10 kDa spin column to remove unreacted DBCO reagent.

[0125] Antibody concentration and degree of DBCO labeling were estimated using a NanoDrop spectrophotometer, finding recovered [antibody] ~ 1-2 pM and degree of DBCO labeling ~ 0.7. Azide-pcDTz, dissolved in DMSO, was then added in ~10x molar excess over the DBCO, and reacted at room temperature for 1 hour on a shaker, then overnight at 4 °C. The resulting product was diluted 1:100 in HBSS immediately before addition to cells.Functionalized antibody was stored at 4 °C and used within five days.

[0126] Zebrafish embryo surface chemistry: All vertebrate experiments were approved by the Institutional Animal Care and Use Committee of Harvard University (protocols 10-13-4 and 21-06-391-1). The zebrafish (Danio rerio) AB wild-type strain was used for all experiments. Adult fish were raised at 28.5 °C on a 14 h light / 10 h dark cycle. Embryos were collected by crossing female and male adults (3-24 months old). Embryos were collected and dechorionated at approximately 10 hours post fertilization (hpf) by immersing in 1 mg / ml pronase protease (Sigma) in 0.3 x Danieau’s buffer (17.4 mM NaCl, 0.21 mM KC1, 0.12 mM MgSCL, 0.18 mM Ca(NO? f , 1.5 mM HEPES, pH 7.2) for 7 min at room temperature.Embryos were then treated with 100 p M sulfoNHS-DBCO for 10 min at room temperature, rinsed, and treated with 10 pM azide-pcDTz for 30-60 min at room temperature. They were then rinsed and incubated in Danieu’s buffer for 30-120 minutes at room temperature before photo-uncaging experiments.

[0127] In the zebrafish embryos, the reaction between surface amines and reactive ester-DBCO was adjusted to minimize toxicity. More efficient labeling (via increased concentration or use of a more stable ester) can damage the early embryos. Users may need to titrate this reaction to avoid toxicity. The reaction time between surface DBCO and azide-pcDTz was chosen to be fast enough to minimize fish development during the labeling process. In a slower-developing system, allowing the azide-DBCO reaction to proceed for 2-4 hours would increase labeling efficiency.35 / 62#14793713v2

[0128] Photo-uncaging: Patterning of individual cells or of fish embryos was performed on a home-made inverted epifluorescence microscope. A 405 nm laser (Lasever, LSR405NL-200, 200 mW) was patterned by a digital micromirror device (Texas Instruments, DLP3000) and re-imaged onto the sample via a 60x water immersion objective (Olympus, UPLSAPO60XW) or a 1 Ox air objective (Olympus). Illumination dose was -500 J / cm2for near complete uncaging.

[0129] Cells were plated on Matrigel or poly-D-lysine-coated glass and immersed in an “imaging buffer” containing (in mM): 125 NaCl, 2.5 KC1, 2 CaCh, 1 MgCh, 15 HEPES, 25 glucose (pH 7.3). Individual cells were perfused with TCO-dye using a BioPen single cell application system (Fluicell). TCO-dyes used were TCO-Cy3 (AAT Bioquest), TCO-Cy5 (Click Chemistry Tools), TCO-AZDye488 (Click Chemistry Tools), and TCO-CF640R (Biotium).

[0130] Fish embryos were placed in wells on an agarose-coated glass dish for patterning. Fish embryos were transferred to a separate dish for TCO-dye immersion and rinsing.

[0131] Wide-area patterning of confluent cell layers was performed on a wide-area upright microscope, similar to the “Firefly” design described previously

[0027] . Eight from a 405 nm laser (Changchun New Industries, PSU-H-EED, 1W) was patterned by a digital micromirror device (Vialux V7000). Samples were illuminated at a dose of -50 J / cm2for each round of uncaging. TCO-dye and rinsing steps were performed by hand pipetting.

[0132] Microscopy: Patterning and single-cell imaging were performed on a custom-built inverted epifluorescence microscope. Zebrafish embryos were fixed in 4% paraformaldehyde (Thermo Fisher) before the final imaging step. Zebrafish embryo confocal z-stacks were obtained at the Harvard Center for Biological Imaging, at 10 and 20x using a LSM 980 NLO Multi-Photon Microscope (Zeiss). Wide-area cell images were obtained at the Harvard Center for Biological Imaging, at 10 and 20x using a LSM 900 Live Cell Confocal Microscope (Zeiss).

[0133] Flow cytometry: After patterning, cells were dissociated by treatment with trypsin (Thermo Fisher, 15050057), quenched with culture medium, spun down (300 g, 5 min), and resuspended in imaging buffer supplemented with 2% fetal bovine serum. Flow cytometry and FACS were performed at the Flow Cytometry Core at the Harvard University Bauer Core Facility, using a BD FACS Aria Fusion Cell Sorter with two rounds of 4-way sorting, and laser lines at 488 nm, 561 nm, and 637 nm, for TCO-AZDye488, TCO-Cy3, and TCO-Cy5 dye.36 / 62#14793713v2

[0134] Image analysis: Image processing of confocal z-stacks and wide-area cell images was performed in Zen Blue software (Zeiss). Single cell images were analyzed in MATLAB (Mathworks). Cell segmentation for classification from image (Figures 9A, 9B) was performed using Ilastik: cells were segmented based on a nuclear DAPI stain, and a mask for each cell was exported to MATLAB. Masks were expanded to include the labeled membrane, and average intensity was calculated for each cell to generate cell intensity histograms.Details of preparation and characterization of pcDTz compounds

[0135] Common materials or chemical reagents were purchased commercially and used without further purification. All reactions were monitored by thin-layer chromatography (TLC), or high-resolution mass spectra (HR-MS). TLC was performed using silica gel plate (GF254, 0.23 mm) which were visualized with a UV lamp (254 nm and 365 nm). HR-MS were measured with an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC / MS system equipped with electrospray ionization (ESI). Nuclear magnetic resonance (NMR) spectra were recorded on a Quantum-1400 or AVANCE NEO 700 spectrometer with TMS as the internal standard. Chemical shifts (6) were reported in parts per million (ppm) relative to residual solvent peaks. Column chromatography was carried out using Biotage Rening Cartridge (particle size 0.040-0.063 mm) using technical grade solvents.Scheme SI

[0136] Preparation of l-(2-nitrophenyl)ethy!6-(2-(l-(2-(2-(2- ((6chlorohexyl)oxy)ethoxy)ethoxy)ethyl)- lH-l,2,3-triazol-4-yl)ethyl)-3-phenyl-l,2,4,5-tetrazine- 1 (4H)-carboxy late (HTL-pcDTz):37 / 62#14793713v2

[0137] 1 -(2-nitrophenyl)ethyl6-(but-3 -yn- 1 -y 1) -3 -phenyl- 1 ,2,4, 5 -tetrazine- 1 (4J7)-carboxyl ate (compound SI) was prepared according to the reported literature [1], Under argon, 12 mL of CH3CN / H2O (v / v = 2 / 1) was added to a mixture of photocaged dihydrotetrazine compound SI (127 mg, 0.31 mmol), l-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6-chlorohexane (125 mg, 0.42 mmol), Cui (106 mg, 0.56 mmol), and sodium L-ascorbate (110 mg, 0.56 mmol) at room temperature. The reaction mixture was stirred at 37 °C overnight. Upon completion, the reaction solvent was removed under reduced pressure. The residue was purified by column chromatography (4% MeOH / CH2Cl2), and the desired product HLT-pcDTz was obtained (71 mg, 32% yield).

[0138] 1H NMR (700 MHz, Chloroform^ / ) 88.02 (s, 1H), 7.98 (d, J= 8.2 Hz, 1H), 7.82 -7.78 (m, 3H), 7.65 (t, J= 7.6 Hz, 1H), 7.53 (q, J= 8.3, 7.8 Hz, 1H), 7.48 (d, J= 7.6 Hz, 2H), 7.43 (t, J= 7.8 Hz, 1H), 6.46 (q, J= 6.5 Hz, 1H), 4.44 (t, J= 5.0 Hz, 2H), 3.77 (t, J= 5.0 Hz, 2H), 3.61 (dd, J = 5.9, 3.1 Hz, 2H), 3.59 - 3.54 (m, 4H), 3.50 (dd, J = 8.1, 5.5 Hz, 4H), 3.42 (t, J= 6.7 Hz, 2H), 3.08 (s, 2H), 2.98 (s, 2H), 1.77 (d, J= 6.6 Hz, 3H), 1.73 (p, J= 6.8 Hz, 2H), 1.54 (p, J= 6.8 Hz, 2H), 1.43 - 1.37 (m, 2H), 1.32 (tt, = 9.9, 5.7 Hz, 2H).13C NMR (176 MHz, CDC13) 6155.47, 149.98, 147.45, 146.21, 143.76, 138.12, 133.89, 131.74, 128.90, 128.84, 128.43, 127.40, 126.76, 124.56, 122.62, 71.26, 70.61, 70.54, 70.49, 70.42, 70.04, 69.44, 53.72, 50.16, 45.06, 32.49, 30.91, 29.36, 26.64, 25.37, 22.33, 22.25. HR-MS: m / z = 699.30103 [M+H]+, calc’d for C33H44O7N8C1: 699.30160.Scheme S2S2 S3 S4 azide-pcDTz

[0139] Reagents and conditions: a) compound S2 (1 eq), MsCl (3 eq), triethylamine (3 eq), DMAP (1 eq), CH2C12, 0 °C to r.t., under argon, then NaN3 (5 eq), DMF, yield 40%; b) compound S3 (1 eq), Thiourea dioxide (2 eq), DMF / H2O (v / v = 1 / 2), 95 °C, yield: 74%; c)38 / 62#14793713v2compound S4 (1 eq), l-(2- nitrophenyl)ethyl carb onochlori date (1.5 eq), Tol / Py (v / v =1 / 5) , 37 °C, under argon, yield: 83%.

[0140] Preparation of 3-(2-azidoethyl)-6-phenyl-l,2,4,5-tetrazine (compound S3)

[0141] 2-(6-phenyl-l,2,4,5-tetrazin-3-yl)ethan-l-ol (compound S2) was prepared according to the reported literature [2], Under argon, triethylamine (617 pL, 4.5 mmol), MsCl (343 pL 4.5 mmol) and DMAP (182 mg, 1.5 mmol) were added to a solution of 2-(6-phenyl-l,2,4,5-tetrazin-3-yl)ethan-l-ol (300 mg, 1.5 mmol) in CH2C12(8 mL) at room temperature. Upon completion, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (CH2C12 as the eluent), and the 2-(6-phenyl-l,2,4,5-tetrazin-3-yl)ethyl methanesulfonate was obtained. Then, NaN3(309 mg, 4.75 mmol) was added to a solution of 2-(6-phenyl-l,2,4,5-tetrazin-3-yl)ethyl methanesulfonate in DMF (6 mL), the mixture stirred at room temperature under argon for 6 h. Then, the reaction mixture was extracted with CH2C12and washed with a saturated solution of NaCl in water. The extract was combined, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (50% Hexane / EtOAc), and the desired product S3 was obtained (132 mg, 40% yield).

[0142] XH NMR (400 MHz, Chloroforms / ) 88.65 - 8.58 (m, 2H), 7.69 -7.56 (m, 3H), 4.0313(t, J= 6.7 Hz, 2H), 3.64 (t, J= 6.7 Hz, 2H). C NMR (101 MHz, chloroform- / / ) 6167.26, 164.73, 132.97, 131.63, 129.41, 128.22, 48.84, 34.54. HR-MS: m / z = 228.09908 [M+H]+, calc’d for C10H10N7: 228.09922.

[0143] Preparation of 3-(2-azidoethyl)-6-phenyl-l,4-dihydro-l,2,4,5-tetrazine (compoundS4):

[0144] Under argon, the thiourea dioxide (76 mg, 0.7 mmol) was added to a solution of S3 (80 mg, 0.35 mmol) in 15 mL of DMF / H2O (v / v = 1 / 2) at room temperature. The reaction39 / 62#14793713v2mixture was stirred at 95 °C for 1-2 hours. Upon completion, the color of the reaction mixture changed from pink to light yellow. Under argon, 50 mL of EtOAc was added the reaction mixture, which was washed by 30 mL H2O. The extract was combined and concentrated by reduced pressure. The residue was purified by column chromatography (CH2C12as the eluent), and the desired product compound S4 was obtained (60 mg, 74% yield).

[0145] XH NMR (400 MHz, Chloroform^ / ) 8 7.62 (dq, J = 6.6, 1.4 Hz, 1H), 7.43 (dddd, J 13 = 11.7, 8.9, 5.4, 1.5 Hz, 1H), 3.66 - 3.58 (m, 1H), 2.47 (td, J = 6.6, 1.1 Hz, 1H). C NMR (101 MHz, CHLOR.OFOR.M-Z1) 6 148.56, 148.35, 130.85, 130.19, 128.99, 126.07, 48.04, 30.36. HR-MS: m / z = 230.11452 [M+H]+, calc’d for C10H12N7: 230.11487.

[0146] Preparation of 1 -(2-nitrophenyl)ethyl 6-(2-azidoethyl)-3-phenyl-l,2,4,5-tetrazine- 1(47 / )- carboxylate (azide-pcDTz)

[0147] Under argon, the solution of l-(2-nitrophenyl)ethyl carbonochloridate (158 mg, 0.65 mmol) in toluene (1 mL) was added to a solution of compound S4 (60 mg, 0.26 mmol) in pyridine (5 ml) at 0 °C. The reaction mixture was stirred at 37 °C for 24 h. Upon completion, the reaction solvent was removed under reduced pressure. The residue was purified by column chromatography (1% MeOH / CH2C12), and the azide-pcDTz was obtained (70 mg, 83% yield).

[0148] H NMR (700 MHz, Chloroform- ) 6 8.00 (dd, J= 8.2, 1.4 Hz, 1H), 7.80 (dd, J = 7.9, 1.5 Hz, 1H), 7.76 - 7.74 (m, 2H), 7.66 (td, J= 7.6, 1.5 Hz, 1H), 7.58 - 7.55 (m, 1H), 7.50 (dd, J= 8.4, 6.9 Hz, 2H), 7.48 - 7.44 (m, 2H), 3.59 - 3.39 (m, 2H), 2.98 (td, J= 6.5, 1.9 Hz, 2H), 1.78 (d, J= 6.6Hz, 3H).13C NMR (176 MHz, CDC13) 6 154.95, 150.05, 147.47, 142.25, 138.02, 133.87, 131.96, 129.10, 128.66, 128.52, 127.31, 126.57, 124.67, 70.76, 47.78, 31.01, 22.26. HR-MS: m / z =423.15198 [M+H]+, calc’d for CI9H19O4N8: 423.15238.Example 2. Infrared-Caged Tetrazine

[0149] Violet light (e.g., 405 nm) can be toxic to some cells, such as neurons. Analogous labeling strategies were therefore developed using a new compound, PDX-496, which is uncaged by near infrared (635-800 nm) light (Figures 22A-22B). As with pcDTZ, PDX-49640 / 62#14793713v2can be targeted to cells by appending a HaloTag ligand (in which the azide is replaced with HaloTag (Figure 23A)), by nonspecific linkage with NHS-DBCO chemistry (Figure 23B), or by reaction with DBCO-functionalized antibodies.

[0150] Targeted fluorescent labeling of HEK293 cells using PDX-496 was conducted (Figure 24). The cells were first labeled with NHS-DBCO, then reacted with PDX-496. The cells were illuminated with 637 nm light (25 W / cm2, 30 seconds), then reacted with TCO-Cy5. The cells in the illuminated region were labeled with the dye, and the cells in the nonilluminated region were not.Example 3. Targeting Macromolecules using PcDTZ and PDX-496

[0151] Previously, photocaged tetrazines were used to target fluorescent dyes to cells. This Example demonstrates that the compounds provided herein (uncaged at either violet or near IR wavelengths) can also target biological macromolecules to cells. An example labeling stack where an anti-CD44 antibody is targeted to HEK293 cells is provided in Figure 25. Fluorescence of the AF488 dye, demonstrating patterning of the antibody in a checkerboard pattern, is shown in Figure 26.REFERENCES(1) Chien, M.-P; Werley, C. A.; Farhi, S. L.; Cohen, A. E. Photostick: A Method for Selective Isolation of Target Cells from Culture. Chem. Sci. 2015, 6 (3), 1701-1705. doi.org / 10.1039 / C4SC03676J.(2) Binan, L.; Mazzaferri, J.; Choquet, K.; Lorenzo, L.-E.; Wang, Y. C.; Affar, E. B.; De Koninck, Y; Ragoussis, J.; Kleinman, C. L.; Costantino, S. Live Single-Cell Laser Tag. Nat. Commun. 2016, 7 (1), 11636. doi.org / 10.1038 / ncommsl 1636.(3) Kuo, C.-T.; Thompson, A. M.; Gallina, M. 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[0152] The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.EQUIVALENTS AND SCOPE

[0153] In the articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Embodiments or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0154] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in 44 / 62#14793713v2any other claims that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0155] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.

[0156] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.45 / 62#14793713v2

Claims

CLAIMSWhat is claimed is:

1. A compound comprising the structure: [photocaged moiety] -[click chemistry handle].

2. The compound of claim 1, wherein the compound comprises the structure:[photocaged tetrazine] -[click chemistry handle].

3. The compound of claim 1 or 2, wherein the compound comprises the structure:[photocaged tetrazine] -[azide].

4. The compound of claim 1 or 2, wherein the compound comprises the structure:wherein:R1is a photocaging moiety;R2is an alkyl group substituted with a click chemistry handle, wherein the alkyl group may optionally be substituted with one or more further substituents; andR3is an optionally substituted aryl group.

5. The compound of claim 4, wherein the photocaging moiety comprises a substituted ester.

6. The compound of claim 4 or 5, wherein the click chemistry handle comprises an azide.

7. The compound of any one of claims 4-6, wherein R2is Ci-Ce alkyl substituted with the click chemistry handle.46 / 62#14793713v28. The compound of any one of claims 4-6, wherein R2is Ci-Ce alkyl substituted with an azide.

9. The compound of any one of claims 4-8, wherein R3is an unsubstituted aryl group.

10. The compound of any one of claims 4-8, wherein R3is an optionally substituted benzyl group.

11. The compound of any one of claims 4-8, wherein R3is an unsubstituted benzyl group.

12. The compound of claim 4, wherein the compound comprises the structure:

13. The compound of claim 4, wherein the compound comprises the structure:

14. The compound of claim 4, wherein the compound comprises the structure:47 / 62#14793713v215. The compound of claim 4, wherein the compound comprises the structure:wherein R4is an optionally substituted alkyl group.

16. The compound of any one of claims 1-15, wherein the compound comprises the structure:

17. The compound of any one of claims 1-15, wherein the compound comprises the structure:

18. A compound comprising the structure: [photocaged moiety]-[halotag ligand],19. The compound of claim 18, wherein the compound comprises the structure: [photocaged tetrazine]-[halotag ligand].48 / 62#14793713v220. The compound of claim 19 or 20, wherein the compound comprises the structure:wherein:R1is a photocaging moiety;R2is an alkyl group substituted with a halotag ligand, wherein the alkyl group may optionally be substituted with one or more further substituents; andR3is an optionally substituted aryl group.

21. The compound of claim 20, wherein the photocaging moiety comprises a substituted ester.

22. The compound of claim 20 or 21, wherein R2is Ci-Ce alkyl substituted with the halotag ligand.

23. The compound of any one of claims 20-22, wherein the halotag ligand comprises the structure:

24. The compound of any one of claims 20-23, wherein R3is an unsubstituted aryl group.

25. The compound of any one of claims 20-23, wherein R3is an optionally substituted benzyl group.

26. The compound of any one of claims 20-23, wherein R3is an unsubstituted benzyl group.

27. The compound of claim 20, wherein the compound comprises the structure:49 / 62#14793713v228. The compound of claim 20, wherein the compound comprises the structure:

29. The compound of claim 20, wherein the compound comprises the structure:

30. The compound of claim 20, wherein the compound comprises the structure:wherein R4is an optionally substituted alkyl group.50 / 62#14793713v231. The compound of any one of claims 18-30, wherein the compound comprises the structure:

32. The compound of any one of claims 18-30, wherein the compound comprises the structure:

33. A composition comprising the compound of any one of claims 1-17 and / or the compound of any one of claims 18-32.

34. A system comprising the compound of any one of claims 1-17 and / or the compound of any one of claims 18-32, or the composition of claim 33, wherein the system optionally further comprises one or more cells, microscopes, computers, and / or light sources.

35. A kit comprising the compound of any one of claims 1-17 and / or the compound of any one of claims 18-32, or the composition of claim 33.51 / 62#14793713v236. A method comprising:attaching one or more compounds comprising one or more photocaged moieties to the surface of one or more cells, wherein the one or more compounds are attached to the cell surface using a cell-targeting chemical method;uncaging the one or more photocaged moieties; andcontacting the uncaged moieties with one or more small molecules or biological macromolecules capable of reacting with the uncaged moieties.

37. A method comprising:attaching one or more compounds comprising one or more photocaged moieties to the surface of one or more cells, wherein the one or more compounds are attached to the cell surface using a cell-targeting chemical method;uncaging the one or more photocaged moieties;contacting the uncaged moieties with one or more visualizable dyes capable of reacting with the uncaged moieties; andimaging the one or more cells to determine the presence and / or locations of the one or more visualizable dyes.

38. The method of claim 36, wherein the one or more small molecules or biological macromolecules comprise one or more visualizable dyes.

39. The method of claim 36, wherein the one or more small molecules or biological macromolecules comprise one or more antibodies, antibody fragments, or antibody variants.

40. The method of any one of claims 36, 38, or 39 further comprising a step of imaging the one or more cells to determine the presence and / or locations of the one or more small molecules or biological macromolecules.

41. The method of any one of claims 36-40, wherein the cell-targeting chemical method comprises a halotag-chloroalkane system, non-specific reaction with amines on cell surface proteins, and / or reaction with an antibody that binds a cell surface protein.

42. The method of any one of claims 36-41, wherein at least one of the photocaged moieties comprises a tetrazine.52 / 62#14793713v243. The method of any one of claims 36-42, wherein the one or more photocaged moieties is uncaged by contacting the cell with visible light.

44. The method of any one of claims 36-42, wherein the one or more photocaged moieties is uncaged by contacting the cell with visible light at a wavelength of about 405 nm.

45. The method of any one of claims 36-42, wherein the one or more photocaged moieties is uncaged by contacting the cell with infrared or near infrared light.

46. The method of any one of claims 36-42, wherein the one or more photocaged moieties is uncaged by contacting the cell with infrared or near infrared light at a wavelength of about 635-800 nm.

47. The method of any one of claims 36-46, wherein the one or more photocaged moieties are uncaged at different times or at the same time.

48. The method of any one of claims 36-47, wherein the one or more small molecules or biological macromolecules are fused to a click chemistry handle.

49. The method of claim 48, wherein the click chemistry handle comprises a transcyclooctene (TCO).

50. The method of claim 49, wherein at least one of the photocaged moieties comprises a tetrazine, and wherein the TCO reacts with the tetrazine.

51. The method of any one of claims 36-50, wherein at least one of the one or more compounds comprising a photocaged moiety is attached to the cell surface using a halotag-chloroalkane system, wherein the method further comprises expressing in the cell a halotag fused to one or more proteins of interest, and wherein the method is used to monitor the expression and / or subcellular location of the one or more proteins of interest.

52. The method of any one of claims 36-51, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:53 / 62#14793713v2[photocaged moiety] -[halotag ligand], [photocaged tetrazine] -[halotag ligand], or53. The method of any one of claims 36-51, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:wherein:R1is a photocaging moiety;R2is an alkyl group substituted with a halotag ligand, wherein the alkyl group may optionally be substituted with one or more further substituents; andR3is an optionally substituted aryl group.

54. The method of claim 53, wherein the photocaging moiety comprises a substituted ester.

55. The method of claim 53 or 54, wherein R2is Ci-Ce alkyl substituted with the halotag ligand.

56. The method of any one of claims 53-55, wherein the halotag ligand comprises the structure:54 / 62#14793713v257. The method of any one of claims 53-56, wherein R3is an unsubstituted aryl group.

58. The method of any one of claims 53-56, wherein R3is an optionally substituted benzyl group.

59. The method of any one of claims 53-56, wherein R3is an unsubstituted benzyl group.

60. The method of claim 53, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:

61. The method of claim 53, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:

62. The method of claim 53, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:55 / 62#14793713v263. The method of claim 53, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:wherein R4is an optionally substituted alkyl group.

64. The method of any one of claims 53-63, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:

65. The method of any one of claims 53-63, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:

66. The method of any one of claims 36-65, wherein at least one of the one or more compounds comprising a photocaged moiety is attached to the cell surface by reaction with56 / 62#14793713v2an antibody that binds a cell surface protein, wherein the method is used to monitor the expression and / or subcellular location of the cell surface protein.

67. The method of any one of claims 36-66, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:[photocaged moiety] -[click chemistry handle], [photocaged tetrazine] -[click chemistryhandle], [photocaged tetrazine] -[azide], or68. The method of any one of claims 36-67, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:wherein:R1is a photocaging moiety;R2is an alkyl group substituted with a click chemistry handle, wherein the alkyl group may optionally be substituted with one or more further substituents; andR3is an optionally substituted aryl group.

69. The method of claim 68, wherein the photocaging moiety comprises a substituted ester.

70. The method of claim 68 or 69, wherein the click chemistry handle comprises an azide.

71. The method of any one of claims 68-70, wherein R2is Ci-Ce alkyl substituted with the click chemistry handle.57 / 62#14793713v272. The method of any one of claims 68-70, wherein R2is Ci-Ce alkyl substituted with an azide.

73. The method of any one of claims 68-72, wherein R3is an unsubstituted aryl group.

74. The method of any one of claims 68-72, wherein R3is an optionally substituted benzyl group.

75. The method of any one of claims 68-72, wherein R3is an unsubstituted benzyl group.

76. The method of claim 68, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:<77. The method of claim 68, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:

78. The method of claim 68, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:58 / 62#14793713v279. The method of claim 68, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:wherein R4is an optionally substituted alkyl group.

80. The method of any one of claims 68-79, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:59 / 62#14793713v281. The method of any one of claims 68-79, wherein one of the one or more compounds comprising one or more photocaged moieties comprises the structure:

82. The method of any one of claims 66-81, wherein the compound comprising a photocaged moiety is attached to the cell surface using non-specific reaction with amines on cell surface proteins and / or reaction with an antibody that binds a cell surface protein.

83. The method of claim 82, wherein attaching the compound comprising a photocaged moiety to the cell surface using non-specific reaction with amines on cell surface proteins comprises contacting the amines with a second compound comprising an electrophile and an alkyne moiety, wherein the alkyne moiety reacts with an azide in the compound comprising a photocaged moiety via click chemistry.

84. The method of claim 82, wherein attaching the compound comprising a photocaged moiety to the cell surface by reaction with an antibody that binds a cell surface protein comprises contacting the cell with the antibody, and contacting a primary amine group on the antibody with a second compound comprising an electrophile and an alkyne moiety, wherein the alkyne moiety reacts with an azide in the compound comprising a photocaged moiety via click chemistry.

85. A method comprising:attaching one or more compounds comprising one or more photocaged moieties to the surface of one or more cells, wherein the one or more compounds are attached to the cell surface using a cell-targeting chemical method comprising a halotag-chloroalkane system, non-specific reaction with amines on cell surface proteins, and / or reaction with an antibody that binds a cell surface protein;uncaging the one or more photocaged moieties;60 / 62#14793713v2contacting the uncaged moi eties with one or more visualizable dyes capable of reacting with the uncaged moieties; andimaging the one or more cells to determine the presence and / or locations of the one or more visualizable dyes;wherein the one or more compounds comprising one or more photocaged moieties61 / 62#14793713v2

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