Fluorogenic bifunctional probe of protease activity

Abstract

Disclosed herein are aspects of a compound according to Formula I or Formula II. Also disclosed are aspects of a composition and kit comprising the compound. The compounds are useful as probes for protease activity, such as cathepsin B activity. Additionally, the compounds are useful for identifying and / or monitoring cancers, for example, colorectal, prostate, ovarian, or breast cancer. Also disclosed are aspects of methods for making the compounds and using the compounds to monitor protease activity in vitro or in vivo.

Description

FLUOROGENIC BIFUNCTIONAL PROBE OF PROTEASE ACTIVITYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application no. 63 / 689,102, filed August 30, 2024, and U.S. Provisional Application no. 63 / 599,926. filed November 16, 2023. The entire contents of the aforementioned applications are incorporated by reference herein.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] Tire application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 25, 2024, is named “TP386526WOl .xml” and is 9,418 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD

[0003] Disclosed herein are aspects of a compound suitable for use as a Anorogenic probe. Also disclosed are methods for making and using the compounds.BACKGROUND

[0004] Proteases, such as cathepsin B. are involved in degradation of proteins and organelles, antigen presentation, and execution of cell death pathways. However, there is a lack of tools for real-time monitoring of lysosomal degradation of proteins in live cells, such as using an imaging platform. Additionally, aberrant protease activity can be associated with numerous cancers, including colorectal, prostate, ovarian, and breast cancers. Significant effort has gone into the development of molecular and medical imaging technologies that center on protease activity in general and on cathepsin B in particular.SUMMARY

[0005] Disclosed herein are aspects of a compound according to Fonnula IR— L1— X— L2peptide L3Formula I or a salt thereof. With respect to Formula I, X is -dye- or -N(-dye)-, where dye is a fluorescent dye or phosphorescent dye, quencher is an excited state energy quenching moiety, peptide is a protease-cleavable peptide moiety and may be in either the forward (N-tenninal-to-C-terminal) or reverse (C-terminal-to-N- terminal) orientation, R is a reactive group suitable to conjugate the compound to a target molecule, and each of L1, L2, and L3independently is a linker selected from a covalent bond or a moiety comprising two or more covalent bonds and at least one atom selected from C, N, 0, P, or S.

[0006] In some aspects, the compound has a structure according to Formula I-A or I-BR — L1— dye — L2- peptide— L3-Fonnula I-A dyeL3-Formula I-B.

[0007] Also disclosed herein are aspects of a compound according to Formula IIR— L1peptide L2Formula II.

[0008] Aspects of a composition comprising a compound according to any one of Formulas I, I-A, I-B, or II also are disclosed herein. In some aspects, the composition comprises the compound conjugated to a biomolecule, such as an antibody, enzyme, protein, oligonucleotide, or dextran. Tire composition may further comprise a solvent, a buffer, a surfactant, or a combination thereof. Further disclosed are aspects of a kit comprising the compound or the composition.

[0009] Also disclosed herein are aspects of a method, comprising contacting a cell with a compound disclosed herein, exposing the cell to light, and determining tire presence or absence of a signal, such as a fluorescent or phosphorescent signal.

[0010] The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a schematic drawing illustrating an exemplary mechanism of action of the disclosed compounds.

[0012] FIG. 2 is a graph of mean average intensity versus antibody, illustrating the signal intensity after Her-2 positive SKBR3 cells were treated with Compound 1-1 conjugated to trastuzumab (trade name Herceptin) (anti-Her2 antibody) or EGFR positive A431 cells treated with Compound 1-1 conjugated to cetuximab (anti-EGFR antibody).

[0013] FIG. 3 is a graph of mean average intensity versus antibody, illustrating tire signal intensity after Her-2 and EGFR negative MCF7 cells were treated w ith Compound 1-1 conjugated to either trastuzumab (trade name Herceptin) (anti-Her2 antibody) or cetuximab (anti-EGFR antibody).

[0014] FIG. 4 is a graph of the ratio of SKBR3 / MCF7 (trastuzumab) and A431 / MCF7 (cetuximab) signal versus antibody, illustrating the ratio of the mean fluorescence intensity (MFI) of positive to negative signal from FIGS. 2 and 3.

[0015] FIG. 5 is a digital image demonstrating the signals observed in Her-2 positive SKBR3 cells, EGFR positive A431 cells, and Her-2 and EGFR negative MCF7 cells after treatment with cetuximab-Compound 1-1 or trastuzumab-Compound 1-1 conjugates.

[0016] FIG. 6 is a graph illustrating flow cytometry analysis of a rituximab-Compound 1-1 conjugate, and providing the signal to background ratio of Ramos and Jurkat cells that are both treated w ith rituximab- Compound 1-1 conjugate, as determined by the ratio of the mean fluorescence intensity (MFI) of the Ramos population to the Jurkat population.

[0017] FIG. 7 is a graph illustrating flow cytometry analysis of a rituximab-Compound 1-1 conjugate, and providing the signal to background of Ramos cells that are treated with rituximab-Compound 1-1 conjugate, as detennined by the ratio of the mean fluorescence intensity (MFI) of the stained versus unstained populations.

[0018] FIG. 8 is a graph of cell counts vs DAPI staining showing the populations of live and dead cells of cells treated with rituximab-Compound 1-1 and CD19-FITC.

[0019] FIG. 9 A is a graph of CD19-FITC staining vs rituximab-Compound 1-1 staining, showing Ramos cells that are positive for CD20 and CD 19, while the control Jurkat cell population is negative for both CD20 and CD 19.

[0020] FIG. 9B is a graph of CD19-Alexa Fluor™ 700 staining vs rituximab (anti-CD20)-Compound 1-7 conjugate staining showing Ramos cells are positive for both CD19 and CD20. Hu IgGl Isotype control- Compound 1-7 conjugate is used as a negative control to show the background staining due to Compound 1-7.

[0021] FIG. 9C is a graph illustrating the mean fluorescence intensity (MFI) of the flow cytometry analysis of FIG. 9B.

[0022] FIG. 10 is a digital image showing the signal from SKBR3 cells treated with trastuzumab- Compound 1-1 at different concentrations of cathepsin B inhibitor, CA-074 (Me).

[0023] FIG. 11A is a graph of mean average fluorescence intensity versus concentration, illustrating the reduction in intensity of trastuzumab-Compound 1-1 conjugate with increasing amounts of CA-074 (Me), a cathepsin B inhibitor.

[0024] FIG. 1 IB is a graph of mean average fluorescence intensity versus concentration, illustrating tire reduction in intensity of trastuzumab-Compound 1-7 conjugate in SKBR3 cells with increasing amounts of CA-074 (Me).

[0025] FIG. 11C is a graph of mean average fluorescence intensity versus concentration, illustrating the reduction in intensity of cetuximab-Compound 1-7 conjugate in A431 cells with increasing amounts of CA- 074 (Me).

[0026] FIG. 12 is a digital image illustrating co-localization of trastuzumab-Compound 1-1 conjugate with LysoTracker™ Red (top) and trastuzumab - pHrodo™ Red conjugate (middle), or co-localization of trastuzumab-Compound 1-1 conjugate with BacMam™ Lampl-GFP (bottom) indicating that the conjugates localize to the lysosome.

[0027] FIG. 13A is a digital image of SKBR3 cells and MCF7 cells stained with trastuzumab-Compound 1-7 conjugate and illustrating that the HER2 positive SKBR3 cells produced a signal while the HER2 negative MCF7 cells did not.

[0028] FIG. 13B is a graph of Her-2 positive SKBR3 cells and EGFR-positive A431 cells treated with trastuzumab-Compound 1-7 conjugate, cetuximab-Compound 1-7 conjugate, or human IgGl (hlgGl)- Compound 1-7 conjugate (control), indicating specific internalization of the trastuzumab-Compound 1-7 conjugate in the SKBR3 cells and the cetuximab-Compound 1-7 conjugate in the A431 cells.

[0029] FIG. 14 is a digital image of SKBR3 cells and MCF7 cells stained with trastuzumab-Compound I- 3 conjugate and illustrating that the HER2 positive SKBR3 cells produced a signal while the HER2 negative MCF7 cells did not.

[0030] FIG. 15 is a graph of signal to noise versus Cathepsin B concentration, illustrating the fluorescence response to Cathepsin B of Compound II- 1 (Res) and Compound 1-1 (DQ) from 30 minutes to 120 minutes.

[0031] FIG. 16 is a graph of signal to noise versus Cathepsin B concentration, illustrating the fluorescence response to Cathepsin B of Compound 1-1 (DQ) from 30 minutes to 120 minutes.

[0032] FIG. 17 is a graph of absorbance versus wavelength, illustrating the absorbance and degree of antibody labeling of various batches on Compound 1-1.

[0033] FIGs. 18A and 18B are graphs of signal and signal to noise versus concentration of cathepsin B, comparing the signals from Compound II- 1 (FIG. 18 A) and a coumarin-based cathepsin B substrate (Z-LR- AMC) (FIG. 18B).

[0034] FIGs. 19A and 19B are graphs illustrating the fluorescence spectra (FIG. 19A) and response of the Z-LR-AMC substrate to cathepsin B (FIG. 19B).

[0035] FIGs. 20A and 20B are graphs illustrating the fluorescence spectra (FIG. 20A) and response of Compound II-l to cathepsin B (FIG. 20B).

[0036] FIG. 21 is a graph of signal versus concentration of cathepsin B, comparing the signals from Compound II- 1 and a chloro-rhodamine substrate (PEG-LR-Chloro-Rliod).

[0037] FIGs. 22A and 22B are graphs of signal versus concentration of cathepsin B comparing the fluorescence signals of Compound II-l (FIG. 22A) and Compound II-2 (FIG. 22B).

[0038] FIG. 23 is a graph illustrating the reactivity of Compound 1-1 free acid with various cathepsin enzymes (cathepsin B. cathepsin D, and cathepsin L).

[0039] FIG. 24 is a graph illustrating the inhibition of cathepsin B and cathepsin L by Compound 1-1.

[0040] FIG. 25 is a digital image of SKBR3 cells treated with trastuzumab-compound 1-7 conjugate or Hu IgGl Isotypc control-compound 1-7 conjugate followed by fixation with 4% PFA. Images were captured immediately following fixation and 24 hours post fixation.

[0041] FIG. 26 is a graph illustrating the quantification of signal / background (trastuzumab / Hu IgGl Isotype control) of the images in FIG 25.DETAILED DESCRIPTIONI. Terms and Definitions[0042J The following explanations of terms and methods are provided to better describe tire present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms “a,” “an,” and “the” refer to one or more than one, unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B. or A and B.” without excluding additional elements. All references, including patents and patent applications cited herein, are incorporated by reference.

[0043] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or limits of detection under standard test conditions / methods. When directly and explicitly distinguishing embodiments from discussed prior art, tire embodiment numbers are not approximates unless the word “about” is recited.

[0044] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in tire practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0045] “Alkyl” refers to a saturated aliphatic hydrocarbyl moiety having from 1 to 25 (C 1-25) carbon atoms, typically 1 to 10 (Cmo) carbon atoms such as 1 to 6 (Cue) carbon atoms or 1 to 4 (C1.4) carbon atoms. This tenn includes, by way of example, linear, branched and cyclic (cycloalkyl) hydrocarbyl groups. Cycloalkyl groups may contain from three to twenty-five carbon atoms, for example, from three to fifteen, from three to ten, or from three to six carbon atoms. Exemplary alkyl groups include, but are not limited to, methyl (CH3), ethyl (-CH2CH3), n-propyl (-CH2CH2CH3), isopropyl (-CH(CH3)2), n-butyl (-CH2CH2CH2CH3), isobutyl (-CH2CH2(CH3)2), sec-butyl (-CEXCEL CEECEfi), t-butyl (-C(CH3)3), n-pentyl (- CH2CH2CH2CH2CH3), neopentyl (-CEE CHsX), cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl.

[0046] “Alkenyl” refers to an unsaturated aliphatic hydrocarbyl moiety having one or more double bonds and from 2 to 25 (C2-25) carbon atoms, typically 2 to 10 (C2-10) carbon atoms such as 2 to 6 (C2-6) carbon atoms or 2 to 4 (C2-4) carbon atoms. This term includes, by way of example, linear, branched and cyclic (cycloalkenyl) hydrocarbyl groups. Cycloalkenyl groups may contain from three to twenty-five carbon atoms, for example, from three to fifteen, from three to ten, or from three to six carbon atoms.

[0047] “Alkynyl” refers to an unsaturated aliphatic hydrocarbyl moiety having one or more triple bonds and from 2 to 25 (C2-25) carbon atoms, typically 2 to 10 (C2-10) carbon atoms such as 2 to 6 (C2-e) carbon atoms or 2 to 4 (C2-4) carbon atoms. This tenn includes, by way of example, linear, branched and cyclic (cycloalkynyl) hydrocarbyl groups. Cycloalkynyl groups may contain from three to twenty-five carbon atoms, for example, from three to fifteen, from three to ten, or from three to six carbon atoms.

[0048] “Amine” refers to the moiety -NR’ 2 where each R’ independently is H, or C1-10 alkyl, or both R’ groups together with the nitrogen to which they are attached form a 5- to 8-membered heterocyclyl.

[0049] “Aminooxy” refers to the group -0NH2.

[0050] “Aryl” refers to an aromatic carbocyclic group of. unless specified otherwise, from 6 to 15 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., 1,2,3,4-tetrahydroquinoline, benzodioxole, and the like). If any aromatic ring portion contains a heteroatom, the group is heteroaryl and not aryl. Aryl groups may be, for example, monocyclic, bicyclic, tricyclic or tetracyclic.

[0051] “Azide” refers to the moiety -N3.

[0052] “Cathepsin B probe” refers to the compounds of Formula I, Formula I-A, Formula I-B, and Formula II disclosed herein.

[0053] “Cyclooctyne” refers to a strained-ring alkyne containing a ring of eight carbon atoms connected by seven single bonds and one triple bond. Exemplary cyclooctynes include but are not limited to

[0054] “Ester” refers to the moiety -CO2R’, where R’ is CMO alkyl.

[0055] “Free acid” refers to a compound comprising an acidic moiety as the reactive group, such as a carboxylic acid or a hydroxy moiety.

[0056] “Heteroaryl” refers to an aromatic ring system comprising from 5 to 15 ring atoms comprising at least one carbon atom, and typically plural carbon atoms, and at least one, such as from one to five, heteroatoms. The heteroatom(s) may be nitrogen, phosphorus, oxygen, silicon or sulfur atom(s). The heteroaryl moiety may be a monocyclic moiety, or may comprise multiple rings, such as in a bicyclic or tricyclic ring system, provided that at least one of the rings contains a heteroatom.

[0057] “Heterocyclyl” refers to a non-aromatic ring system comprising from 3 to 15 ring atoms comprising at least one carbon atom, and typically plural carbon atoms, and at least one, such as from one to five, heteroatoms. Tire heteroatom(s) may be nitrogen, phosphorus, oxygen, silicon or sulfur atom(s). The heterocyclyl moiety may be a monocyclic moiety, or may comprise multiple rings, such as in a bicyclic or tricyclic ring system, provided that at least one of the rings contains a heteroatom. Such a multiple ring moiety can include fused or bridged ring systems as well as spirocyclic systems; and any nitrogen, phosphorus, carbon, silicon or sulfur atoms in the heterocyclyl moiety can be optionally oxidized to various oxidation states.

[0058] “Hydrazide” refers to the moiety -C(O)NHNH2.

[0059] “Hydrazine” refers to the moiety -NHNH2.

[0060] “Hydroxyl” refers to the moiety -OH.

[0061] “Hydroxylamine” refers to the moiety -NH20H.

[0062] “Glyoxal” refers to the moiety -C(O)C(O)H.

[0063] “Isocyanate” refers to the moiety -NCO.

[0064] “Isothiocyanate” refers to the moiety -NCS.

[0065] “Maleimide” refers to the moiety

[0066] “Oxaziridine” refers to the moiety

[0067] “Peptide” refers to a compound comprising amino acid residues connected by peptide bonds. As used herein, a peptide compound has from 2 to 10 or more amino acid residues. In certain aspects, a peptide compound has 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[0068] “Sulfonyl amide” refers to the moiety -SO2NR 2 where each R’ independently is H, or CMO alkyl, or both R’ groups together with the nitrogen to which they are attached form a 5- to 8-membered heterocyclyl or a 5 - to 6-membered heteroaryl.

[0069] “Sulfonyl halide” refers to the moiety -SO2X where X is a halide, such as F, Br, Cl or I, preferably F, Br or Cl.

[0070] “Thiacycloalkyne” refers to a strained-ring alkyne that has a sulfur atom replacing a carbon atom and includes 3,3,6,6-tetramethylthiacycloheptyne (TMTH), TMTH-sulfoximine (TMTHSI) and the compounds described in de Almeida et al, Angew. Chem. Int. Ed. Engl. 51:2443 (2012).

[0071] “Thiol” refers to the moiety -SH.

[0072] “Vinyl” refers to the moiety -CFtyCEF

[0073] Unless otherwise defined herein, the term “biomolecule” refers to any biological molecule. In some aspects, the biomolecule is chosen from an amino acid, a peptide, a protein, an antibody, an antibody fragment, an enzyme, a receptor, a monosaccharide, a polysaccharide, a carbohydrate, a lectin, an ioncomplexing moiety, a nucleotide, an oligonucleotide, a nucleic acid, an aptamer, a hapten, a drug, a toxin, a lipid, a phospholipid, a lipoprotein, a glycoprotein, a honnone, a lipopolysaccharide, a liposome, a lipophilic polymer, a non-biological organic polymer, a polymeric microparticle, a bioparticle, an animal cell, a plant cell, a bacterium, a yeast, virus, virus-like particle, and ligand. In certain aspects, the biomolecule is an antibody.

[0074] Unless otherwise defined herein, the term “linker” refers to a single covalent bond or a moiety comprising series of stable covalent bonds, tire moiety often incorporating 1-40 plural valent atoms selected from the group consisting of C, N, O, S and P that covalently attach the Anorogenic compounds to another moiety such as a chemically reactive group or a biological and non-biological component. The number of plural valent atoms in a linker may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 20, 25, 30 or a larger number up to 40 or more. A linker may be linear or non-linear; some linkers have pendant side chains or pendant functional groups, or both. Examples of such linkers include water solubilizing groups such as polyethylene glycol (PEG) groups. Examples of such pendant moieties are hydrophilicity modifiers, for example solubilizing groups like, e.g. sulfo. In certain aspects, a linker is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds.

[0075] Unless otherwise defined herein, the term "PEG or “polyethylene glycol” refers to any polyethylene glycol moiety, -[CH2-CH2-O]n-, wherein n is an integer. In certain aspects, n is chosen from 3, 4, and 5.

[0076] Unless otherwise defined herein, the term “reactive group,” refers to chemical moieties that are reactive as one of ordinary skill in the art would understand and generally represents a point of attachment for another substance. The reactive group is a moiety, such as a carboxylic acid or succinimidyl ester, on the compounds of the present disclosure that is capable of chemically reacting with a functional group on a different compound to form a covalent linkage. Reactive groups generally include nucleophiles, electrophiles and photoactivatable groups. Examples of reactive groups include, but are not limited to, olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, aminooxy. oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbonates, carbamates, carbamic halides, imines, azides, azo compounds, azoxy compounds, nitroso compounds, acrylamides, activated esters of a carboxylic acid, acyl azides, acyl halides, hydroxy, alkyl halides, sulfonates, amines, anilines, aryl halides, aziridines, boronates, diazoalkancs, epoxides, glycols, haloacetamides, halotriazines, imido esters, sulfonyl halides, and thiols. Reactive functional groups also include those used to prepare bioconjugates, for example, a succinimidyl ester (SE), a N- hydroxysuccinimide ester, a sulfo-succinimidyl ester and other substituted succinimidyl esters, a maleimide, reactive phenyl esters such as a dibromophenyl ester, a nitrophenyl ester, a thiophenyl ester, a substituted thiophenyl ester, a sulfodichlorophenyl (SDP) ester, a sulfotetrafluorophenyl (STP) ester, a tetrafluorophenyl (TFP) ester, a pentafluorophenyl (PFP) ester, a nitrilotriacetic acid (NTA), an aminodextran, an acetoxymediyl ester (AM), an isocyanate, a cyanate, an isothiocyanate, a thiocyanate, thiacycloheptynes, and a cyclooctyne, such as dibenzocyclooctyne (DIBO) or dibenzoazacyclooctyne (DBCO). Methods to prepare each of these functional groups are well known in the art and their application to or modification for a particular purpose is within the ability of one of skill in the art (see, for example, Sandler and Karo, eds., Organic Functional Group Preparations. Academic Press, San Diego, 1989).

[0077] Selected examples of reactive groups and moieties and linkages that result therefrom are shown in Table 1, where the reaction of an electrophilic group and a nucleophilic group yields a covalent linkage.Table 1: Examples of certain reactive groups that form useful covalent linkagesII. Compounds

[0078] The disclosed compounds are useful as probes for protease activity, for example, to monitor lysosomal protein degradation pathway by conjugation of the probe to a protein of interest. The probe may also function as an activity-based lysosomal tracker in live cells and may be used to visualize lysosomallocalization and degradation of antibodies and proteins. The disclosed compounds comprise a protease- cleavable peptide moiety which can be cleaved by enzymes such as cathepsin B, which is a lysosomal specific enzyme. Outside of the lysosome, the disclosed compounds have little to no observable background fluorescence, and only upon localization to the protease-rich environment of the lysosome, the protease -cleavable peptide moiety is cleaved which separates the dye from the quencher resulting in a fluorescent signal from the unquenched dye.

[0079] In some aspects, the compound has a structure according to a general formula IR — L1— X — L2- peptide— L3Formula I or a salt thereof.

[0080] With respect to Formula I, X is -dye- or -N(-dye)-. In some aspects, X is -dye- and the compound has a structure according to Formula I-A.Formula I-A

[0081] In other aspects, X is -N(-dye)- and the compound has a structure according to Formula I-B.Formula I-B

[0082] In alternative aspects, the compound has a structure according to a general Formula IIR — L1— peptide — L2—Formula II or a salt thereof.

[0083] With respect to Formulas I, I-A, I-B and II: peptide is a protease -cleavable peptide moiety and may be in either the forward (N-terminal-to-C-terminal) or reverse (C-terminal-to-N-tenninal) orientation; dye is a fluorescent dye or phosphorescent dye; R is a reactive group suitable to conjugate the compound to atarget molecule: if present, quencher is an excited state energy quenching moiety; and each of L1, L2. and L3independently is a linker selected from a covalent bond or a moiety comprising two or more covalent bonds and at least one atom selected from C, N, O, P, or S.

[0084] With particular respect to Formula II, L2is a covalent bond or a self-immolative linker.A. Quencher

[0085] The quencher may have an absorption of from 250 nm to 1,000 nm. In some aspects, the quencher comprises a quenching moiety selected from an azo dye-based quencher, a cyanine-based quencher, a rhodamine-based quencher, an azaphthalocyanine-based quencher, a hemin quencher, malachite green, a malachite green analogue, or a combination thereof. In some aspects, the quencher comprises one quenching moiety, but in other aspects, the quencher comprises two or more quenching moictics, such as 2, 3, 4, or 5 quenching moieties.

[0086] In some aspects, the quencher is derived from an azobenzene quencher, a Xanthylium dye, a nitrobenzooxadiazole amine compound, or malachite green. In some aspects, the quencher is or comprises a moiety derived fromMalachite green Q-1B. Dye

[0087] The dye may be any fluorescent or phosphorescent dye suitable for use with a quencher as disclosed herein. In some aspects, the dye is a cyanine dye, a rhodamine or rhodamine analogue dye, a fluorescein or fluorescein analogue dye, a xanthine or xanthine analogue dye, a BODIPY dye, a coumarin dye, a phthalocyanine dye, a porphyrin dye, a pyrene dye, a fluorene or polyfluorene dye, an excited-state intramolecular proton-transfer (ESIPT) dye, an aggregation-induced emission dye, a metal complex dye, or a combination thereof In certain aspects, the dye is a fluorescein dye, rhodamine, Group 14 Rhodamine dye, Phospha-rhodamine, a cyanine dye with 2, 3, 5, 7 or 9-methine structure, or coumarin.

[0088] In certain aspects, tire dye is derived from

[0089] In some aspects of Formula II, dye is an acridone dye, a fluorescein dye or an analogue thereof, coumarin, xanthine-hybrid cyanine dye. a phenol-containing dye, or an aniline-containing dye. In certain aspects, dye is selected fromR2 = Cl, NH2, OH, ether bond, or ester bondR2 = Cl, NH2, OH, ether bond, or ester bond R1 = F, or a carbamate group R1 = F, or a carbamate groupC. Peptide

[0090] The peptide is any peptide that is cleaved by the protease of interest. In some aspects, the peptide is a cathepsin B cleavable peptide. In certain aspects, the peptide is selected from Gly-Gly-Phe-Gly (SEQ ID NO: 1), Val-Cit, Val-Cit-PAB, Gly-Gly, Gly-Gly-Gly, Phe-Lys, Vai-Ala, Val-Gly, Val-Val, Ala-Ala- Asn, Gly-Phe-Leu-Gly (SEQ ID NO: 2), Arg-Arg, Phe-Arg-Arg-Gly (SEQ ID NO: 3), Gly-Phe-Leu-Gly- Lys (SEQ ID NO: 4), Lys-Lys, Lys-Lys-PAB, Gly-Arg-Arg-Gly-Lys-Gly-Gly (SEQ ID NO: 5), Gly-Glu-Leu-Gly (SEQ ID NO: 6). Val-Arg, Glu-Arg, or Gly-Ile-Val-Arg-Ala-Lys (SEQ ID NO: 7). where PAB is a para-aminobenzyl group.

[0091] In one aspect, the peptide is Val-Cit.

[0092] In one aspect, the peptide is Val-Cit-PAB.

[0093] It was unexpectedly found that the orientation of the protease-cleavable peptide moiety in the compounds provided herein affects the ability of the cleaved compound to be fixed with fixative agents such as formaldehyde. For conjugates with compounds comprising the protease-cleavable peptide moiety in the forward orientation (i.e. the dye is located at the N-terminal side of the peptide moiety and the quencher is located at the C-terminal side of the peptide moiety), after protease cleavage the remaining peptide portion remains with the dye as shown in Scheme 1 below.Scheme 1However, for compounds comprising the protease-cleavable peptide moiety in the reverse orientation (z.e. the quencher is located at the N-terminal side of the peptide moiety and the dye is located at the C-terminal side of the peptide moiety), both the peptide moiety and the quencher are removed from the conjugate after cleavage leaving the dye and a free amine group which is available to react with fixatives such as formaldehyde as shown in Scheme 2 below.Scheme 2D. Reactive Group R

[0094] With respect to Formula I, R is a reactive group suitable for conjugating the compound to a target molecule. R may be an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a photoactivatable group, a reactive platinum complex, a silyl halide, a sulfonyl halide, and a thiol. In certain embodiments, the reactive group is chosen from an acrylamide, a carboxylic acid, an activated ester of a carboxylic acid, an acyl azide, an acyl halide, hydroxy, an aldehyde, an alkyl halide, a sulfonate, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carbodiimide, a carbonate, a carbamate, a carbamic halide, a diazoalkane, an epoxide, a glycol, a haloacetamide, a halomethyl, a halotriazine, a hydrazine, a hydroxylamine, aminooxy, an imido ester, an iodoacetamide, an isothiocyanate, a ketone, a maleimide, a sulfonyl halide, a thiol group, a succinimidyl ester, a substituted succinimidyl ester, a sulfo-succinimidyl ester, a reactive phenyl ester, a dibromophenyl ester, a nitrophenyl ester, a sulfodichlorophenyl ester, a sulfotetrafluorophenyl ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, a thiophenyl ester, a substituted thiophenyl ester, a nitrilotriacetic acid, an isocyanate, a cyanate, an aminodextran, an acetoxymethyl ester, a thiacycloheptyne, and a cyclooctyne, such as a dibenzocyclooctyne (DIBO) or dibcnzoazacyclooctync (DBCO). In certain aspects, R is an ester, isocyanate, isothiocyanate, maleimide, vinyl moiety, thiol, amine, hydroxyl, hydrazine, hydrazide, hydroxy lamine, aminooxy. clickable handle for click chemistry (for example, an azide, alkyne, tetrazine, or trans-cyclooctene (TCO)), sulfonyl halide (for example a sulfonyl bromide, sulfonyl chloride, or sulfonyl fluoride), sulfonyl amide (for example, a sulfonyl triazole), glyoxal, oxaziridine, or a photoreactive moiety (for example, benzophenone, aziridine or an azido-compound).aromatic group or a substituted aromatic group, such as a phenyl or substituted phenyl,E. Linkers

[0096] Each of linkers L1, L2and L3independently is a covalent bond or a moiety comprising two or more covalent bonds and at least one atom selected from C, N, O, P, or S. In some aspects, each of L1, L2. and L3independently is a linker selected from polyethylene glycol (PEG), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted unsaturated alkyl, alkenyl, alkynyl,alkoxy, alkanoyl, alkylamino, aryloxy, arylamino, aralkyl, aralkoxy. aralkanoyl. aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, alkanoylamino. aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, thiourea, phosphoryl, sulfonyl, sulfonamido, or ketone.

[0097] In some aspects, each of L1, L2and L3independently are selected from polyethylene glycol (PEG), substituted or unsubstituted C„ aryl, substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms selected from N. 0 or S, substituted or unsubstituted Ci-ealkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl. Cue alkoxy, C2-6 alkanoyl (C2-6 alkyl- C(=O)-), Cue alkylamino, Ce aryloxy, Ce arylamino, C7-9 aralkyl, C7-9 aralkoxy, C7-9 aralkanoyl, C7-9 aralkamino, (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-O-, (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms selected from N, O or S)-NH-, (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2. or 3 heteroatoms selected from N. O or S)-Ci-6 alkyl-, (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1 , 2, or 3 heteroatoms selected from N, 0 or S)-Ci6alkyl-O-, (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-Ci-e alkyl-C(=O)-, (substituted or unsubstituted 5- to 10-mcmbcrcd hctcroaryl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-Ci-6 alkyl-NH-, C3-7 cycloalkyl, C3-7 cycloalkenyl, C3-7 cycloalkylalkyl, C3-7 cycloalkoxy, C3-7 cycloalkanoyl, C3-7 cycloalkamino, substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S, (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, O or S)- O-, (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, O or S)-NH-, (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-Ci-6alkyl-, (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2. or 3 heteroatoms selected from N, O or S)-Ci-e alkyl-O-, (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, O or S)- Ci-6 alkyl-C(=O)-, (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-C .(!alkyl-NH-. Cue alkanoylamino (-Ci-e alkyl-C(=O)NH-), -C. aryl- C(=O)NH-, aralkanoylamino (-C6 aryl-Ci.6alkyl-C(=O)NH-), alkylcarboxy (-Ci-e alkyl-C(=O)-), carbonate (-OC(=O)O-), carbamate (-OC(=O)NH-), guanidinyl (-NHC(=NH)NH-), urea (-NHC(=O)NH-), thiourea (-NHC(=S)NH-), phosphoryl (-P(=O)(OH)-), sulfonyl (-SO2-). sulfonamido (-SO2NH-), or ketone (-C(=O)- )■

[0098] In some aspects, each of L1, L2and L3independently are selected from -K-L-J-, where each of K and J independently is a bond, -C(=O)-, -NRa-, -OC(=O)-, -C(=O)O-, -OC(=O)NH-, -NHC(=O)O-, -SO2-, -SO2NH-, -NHSO2-, -NHC(=S)NH-, -P(=O)(OH)-, -NHC(=O)NH-, -NHC(=NH)NH-, -OC(=O)O-, or -O- ; andL is -Ci-20 alkyl-, -[(CH2CH2)O]x(CH2CH2)-, -(CH2)y-(substituted or unsubstituted Cs aryl)-(CH2)y- , -(CH2)y-(substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, O or S)-(CH2)y-, -(CH2)y-(substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2. or 3 heteroatoms selected from N, O or S)-(CH2)y-, -(CH2)y-(substituted or unsubstituted C3-8 cycloaliphatic)-(CH2)y-. With respect to each L independently, x is from 1 to 6, and each y independently is from 0 to 6, and Rais H, Ci^alkyl, 2,4-disulfonatobenzylcystic acid

[0099] In some aspects, at least one of L1, L2. or L3is or comprises PEG.

[0100] In some aspects, at least one of L1, L2, or L3is -K-L-J- where L is -[(CH2CH2)O]X(CH2CH2)-. In some aspects, at least two of L1, L2, or L3are -K-L-J- where each L independently is - [(CH2CH2)O]X(CH2CH2)-.

[0101] In certain aspects of Formula II, L2is a self-immolative linker and may be selected from, in any orientation,

[0102] Exemplar) compounds according to Formula I include:Compound 1-2;Compound 1-5 “free acid”;Compound 1-7 “free acid”:Compound 1-9: andCompound I- 10.

[0103] Exemplary compounds according to Formula II include, but are not limited to,Compound II-2.Salts

[0104] In some aspects of Formulas I, I-A. I-B, or II, the disclosed compound is a free base compound, that is, not a salt and therefore does not comprise one or more charged moieties and one or more counter ions. However, in other aspects of Formulas I, I-A, I-B, or II, the disclosed compound is a salt. In such aspects, the salt may comprise one or more than one counter ion, such as 1, 2, 3, 4, 5 or more counter ions, such as 1, 2, 3, 4, or 5 counter ions. The counter ion(s) may be any suitable counter ion, and where the salt comprises more than one counter ion, the counter ions may be the same or different. In some aspects, the counter ion(s) is or comprise a basically charged organic compound, for example, a trialkylammonium ion,such as a triethylammonium ion; an alkaline metal ion, such as a sodium ion, lithium ion and / or potassium ion; and / or an alkaline earth metal ion, such as a magnesium ion and / or a calcium ion.Target Molecule

[0105] In any aspects of Formulas I, I-A, I-B, or II. the target molecule may be any molecule suitable for conjugating to the disclosed compound. In some aspects, the target molecule is a biomolecule, and may be a biomolecule comprising an amine functional group. In some aspects, the biomolecule is chosen from an amino acid, a peptide, a protein, an antibody, an antibody fragment, an enzyme, a receptor, a monosaccharide, a polysaccharide, a carbohydrate, a lectin, an ion-complexing moiety, a nucleotide, an oligonucleotide, a nucleic acid, an aptamer, a hapten, a drug, a toxin, a lipid, a phospholipid, a lipoprotein, a glycoprotein, a hormone, a lipopolysaccharide, a liposome, a lipophilic polymer, a non-biological organic polymer, a polymeric microparticle, a bioparticle, an animal cell, a plant cell, a bacterium, a yeast, virus, virus-like particle, and ligand. In some aspects, the biomolecule is an antibody, antibody fragment, antibody-drug conjugate, enzyme, protein, peptide, aptamer, lectin, oligonucleotide, carbohydrate, hapten, drug, toxin, or dextran. In some aspects, the biomolcculc is an antibody.III. Compositions and Kits

[0106] Also disclosed herein are aspects of a composition comprising a compound as disclosed herein that is conjugated to a biomolecule. The biomolecule may be any biomolecule suitable for conjugating to the disclosed compound. In some aspects, tire biomolecule has an amine moiety suitable for conjugation to the compound. Tire biomolecule may be selected to be taken up by a cell or tissue that contains a protease of interest, so that the conjugated compound is available to interact with the protease. In some aspects, the biomolecule is an antibody, enzyme, protein, oligonucleotide, or dextran. And in any aspects, the composition may further comprise one or more of a solvent, a buffer, a surfactant, or a combination thereof.

[0107] Kits comprising the disclosed compounds or compositions thereof also are disclosed herein. In some aspects, the kit comprises one or more of the disclosed compounds, and / or compound(s) conjugated to a biomolecule. The kit may further comprise one of more of a solvent, a buffer, a surfactant, a base suitable for conjugation, a purification column, a collection tube, or a combination thereof. In some aspects, the kit may include a compound that performs two or more functions in the kit. For example, tire kit may comprise sodium bicarbonate that can act as both a buffer and a base.

[0108] The surfactant may be any surfactant suitable for use with the disclosed compounds and compositions thereof. In some aspects, the surfactant is selected from Polyoxyethylene Lauryl Ether (Brij1M-35), Polyoxyethylene (20) sorbitan monolaurate (Tween1M-20), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy] ethanol (Triton™ X-100). octylphenoxypolyethoxyethanol (Nonidet™ P-40) or a combination thereof.

[0109] The buffer may be any buffer suitable for use with the disclosed compounds and compositions thereof, such as an aqueous buffer, bicarbonate buffer, phosphate buffer, carbonate buffer, borate buffer, or a combination thereof. In some aspects, the buffer is selected from phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane-HCl (Tris-HCl), 2-[4-(2-Hydroxyethyl)piperazin- 1 -yl]ethane- 1 - sulfonic acid (HEPES), or a combination thereof. For example, a bicarbonate buffer (pH about 8.3) may be useful for disclosed aspects of the compounds comprising a succinimidyl ester; a phosphate buffer (pH about 7.2 to about 8) may be useful for aspects of the disclosed compounds comprising a thiol -reactive functional group; and / or a carbonate or borate buffer (pH about 9) may be useful for disclosed aspects of the compound comprising an isothiocyanate or dichlorotriazine reactive group.[001 10] The solvent may be any solvent suitable for use with the disclosed compounds and compositions thereof. In some aspects, the solvent is selected from water, DMSO, methanol, or a combination thereof.

[0111] In a certain aspect, a kit comprises one or more of: a surfactant selected from Polyoxyethylene Lauryl Ether (Brij™-35), Polyoxyethylene (20) sorbitan monolaurate (Tween™-20), 2-[4-(2,4,4- trimcthylpcntan-2-yl)phcnoxy]cthanol (Triton™ X-100), octylphenoxypolyethoxyethanol (Nonidet™ P- 40) or a combination thereof; a buffer selected from phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane-HCl (Tris-HCl), 2-[4-(2-Hydroxyethyl)piperazin- 1 -yl]ethane- 1 - sulfonic acid (HEPES), or a combination thereof; and / or a solvent selected from water, DMSO, methanol, or a combination thereof.IV. Synthesis

[0112] Disclosed compounds can be prepared as exemplified below, and as will be understood by a person of ordinary skill in the art of organic synthesis. An exemplary synthesis may include the following first reaction step according to Scheme 3.PG— L3-RG + RG— quencher - ► PG— L3Scheme 3

[0113] With respect to Scheme 3, PG is a protecting group suitable to prevent the L3compound from reacting at both ends, and RG is a reactive group suitable for conjugating to the quencher. Protecting groups and reactive groups are disclosed herein and are known to persons of ordinary skill in the art. Exemplary groups are provided herein in the Examples, and include BOC groups, amines, and carboxylic acid groupsand activated analogues thereof. Additional information concerning protecting groups can be found in Wuts and Greene, '‘Greene's Protective Groups in Organic Synthesis,” 4thedition, published by Wiley.

[0114] In some aspects, the L3compound and the quencher are combined in a solvent and in the presence of a base, such as an organic base, for example a trialkyl amine base. Suitable solvents include, but are not limited to, DMF, THF, acetonitrile, or a combination thereof.[001 15] An exemplary second reaction step is shown in Scheme 4.PG— L3Scheme 4

[0116] With respect to Scheme 4, the L3-quencher compound is deprotected and conjugated to the peptide moiety. Methods of deprotection are disclosed herein and are known to persons of ordinary skill in the art. Additional information concerning specific deprotection methods for different protecting groups can be found in Wuts and Greene, ‘'Greene's Protective Groups in Organic Synthesis.”

[0117] The peptide is conjugated to the L3moiety by any suitable technique. Typically, the peptide contains either a reactive (such as unprotected) amine or carboxylic acid moiety and this conjugates to a suitable reactive group, such as a carboxylic acid or amine, on the L3moiety. Methods of conjugation are disclosed herein and are known to persons of ordinary skill in the art. In some aspects, a carboxylic acid moiety is activated and treated with an amine moiety in a suitable solvent and in the presence of a base, such as an organic base (for example, a trialkylamine or pyridine) or an inorganic base (for example, a carbonate base such as potassium carbonate or sodium carbonate). Suitable activation methods include, but are not limited to: treatment with O-(N-Succinimidyl)-N,N,N\N’-tetramethyluronium tetrafluoroborate (TSTU); forming the acid chloride, such as by treatment with thionyl chloride; treatment with l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and a base such as diisopropylethylamine (DIPEA); treatment with carbonyldiimidazole (CDI); or treatment with a carbodiimide, such as dicyclohexylcarbodiimide (DCC) or l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) .

[0118] A third exemplary reaction step is shown in Scheme 5.Scheme 5

[0119] With respect to Scheme 5, each PG independently is a protecting group suitable to prevent L1and L2from reacting with the dye. Typically, the dye comprises a reactive moiety suitable for conjugating to an amine, such as a carboxylic acid moiety. The dye is conjugated to the L'-NH-L2moiety by any suitable technique. In some aspects, the dye comprises a carboxylic acid moiety which forms an amide bond with the NH moiety . Hie carboxylic acid may be activated prior to conjugation, such as by using an activating agent disclosed here. In other aspects, the carboxylic acid reacts with the amine in the presence of ((7- Azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate) (PyAOB) and a suitable base, such as an organic base, for example a trialkylamine base.

[0120] An exemplary fourth reaction step is shown in Scheme 6.Scheme 6

[0121] With respect to Scheme 6, the product from Scheme 3 is first deprotected by a suitable technique. Methods of deprotection are disclosed herein and are known to persons of ordinary skill in the art. Hie deprotected compound is then treated with the quencher-L3-peptide compound from Scheme 4. In some aspects, L2comprises an acid moiety and this is first activated and then reacted with an amine on the peptide moiety. In certain aspects, the acid moiety is activated by treatment with 3,5-dichloro-4- ((dimethylamino)(dimethyliminio)methoxy)benzenesulfonate and N,N-dimethyl aminopyridine (DMAP). Alternative activating techniques are disclosed herein.

[0122] Hie activated compound then is treated with the quencher-L3-peptide compound in the presence of a suitable base, such as an organic base, for example a trialkylamine base, such as triethylamine.

[0123] In sonic cases, the disclosed compounds can be prepared as exemplified below in Scheme 7 and as will be understood by a person of ordinary skill in the art of organic synthesis. An exemplary synthesis may include the following three reaction steps according to Scheme 7. Coupling a reactive dye with the linker containing protected L1and L2groups, followed by deprotection reaction, generates the bifiinctional dye-linker (step (1)). In step (2), the bifunctional dye-linker reacts with the reactive protected peptide to form an intermediate, which is further deprotected to form the bifiinction dye-peptide conjugate. In step (3), the bifunctional dyc-pcptidc conjugate can be coupled with a reactive quencher to produce a dye-peptide-quencher intermediate with reactive group Ri . Transformation of Ri of the intermediate to reactive group R generates the desired compound, which is conjugatable to biomolecules and other targets.Scheme 7V. Applications

[0124] The disclosed compounds are useful as fluorescent probes and can be used for real-time monitoring of endosomal sorting and lysosomal degradation of proteins in live cells, and also as indicators of protease activity. Real-time monitoring of endosomal sorting and also lysosomal degradation of proteins both can be usefill, for example, in drug development. Typically, a target protein (such as an antibody-drug conjugate or ADC) binds to a degrader which binds to a cell surface receptor and the complex is internalized into the cell. The receptor then recycles back to the cell surface and the protein is ferried to the lysosome which contains enzymes to degrade the protein. But often questions remain concerning where the degrader goes and whether the target protein is really degraded in the lysosome, whether it goes to another compartment, or whether it get recycled back to the cell surface.

[0125] The compounds disclosed herein can be used to provide a robust indication of lysosomal entry with a bright fluorogenic signal upon protease cleavage, such as cathepsin B cleavage, which occurs in the lysosome. The compound typically is selected or synthesized to have a peptide moiety that is suitable for use as a substrate for a protease, such as cathepsin B. The disclosed compounds are easily conjugated to biomolcculcs, such as antibodies and other proteins, and then can provide live-cell, dynamic assessment ofendosomal sorting and lysosomal degradation, and also provide a sensitive, specific, bright indicator with no genetic engineering required. These compounds fill an unmet need for real-time monitoring of endosomal sorting and lysosomal degradation of proteins in live cells. The disclosed compounds, or a composition comprising the compounds optionally conjugated to a biomolecule, are suitable for use in in vitro, ex vivo and in vivo assays. The data provided herein demonstrates that the compounds work in both imaging and flow cytometry with high sensitivity, and they are specific to cellular uptake and lysosomal degradation.

[0126] Typically, after contacting a sample, such as a cell or tissue, with a disclosed compound or composition thereof, the presence or absence of fluorescence is monitored. Typically, an absence of fluorescence indicates an absence of protease activity, and the presence of fluorescence indicates protease activity. In some examples, different samples are tested using the same compound, and the sample with the higher protease activity yields a Anorogenic signal with a faster rate and overall higher intensity than the sample with less activity. In other examples where two different substrates are tested, relative fluorescence intensity is used to investigate the protease activities. The data also is used for tracking the delivery of the disclosed compound or composition thereof, or of the disclosed compound conjugated to a biomolecule.

[0127] The disclosed compounds, or compositions thereof, are useful for detecting diseases and / or conditions for which aberrant protease activity is indicated. Such diseases and / or conditions include, but are not limited to, cancers. In some aspects, the disclosed compounds are useful as a cathepsin B probe, and therefore can be used to identify and / or monitor cells and / or tissues for cathepsin B activity. In some aspects, disclosed compounds useful as cathepsin B probes are used to identify and / or monitor cancers such as colorectal, prostate, ovarian, or breast cancer.

[0128] In some aspects, a method for using the disclosed compounds may comprise contacting a cell with a compound or composition disclosed herein, exposing the cell to light, such as visible light or UV light, and determining the presence or absence of a fluorescent or phosphorescent signal. Exposing the cell to light and determining the presence or absence of the fluorescent or phosphorescent signal may comprise exposing the cell to light at a first wavelength and detecting the presence or absence of light having a second wavelength that is different from the first wavelength. Tire cell may be an in vitro cell or an ex vivo cell. In some aspects, the cell is in vivo.

[0129] Contacting the cell may comprise incubating the cell for a time interval sufficient to facilitate entry of the compound or composition into the cell. The time interval may be from 1 hour or less to 24 hours or more, such as from 2 to 16 hours.

[0130] In certain aspects, the method comprises conjugating a compound disclosed herein to a biomolecule to form a biomolecule-compound conjugate, contacting a cell with the biomolecule-compound conjugate, incubating tire cell for a time interval adequate to allow entry of the biomolecule-compound conjugate into the cell, illuminating the cell with an appropriate w avelength of light, and detecting fluorescent emissions from the biomolecule-compound conjugate.

[0131] In certain aspects, the method comprises contacting a sample with a compound disclosed herein or biomolecule-compound conjugate disclosed herein. Hie sample may comprise live cells, intracellular fluids, extracellular fluids, biological fluids, sera, biological fermentation media, environmental samples, industrial samples, proteins, peptides, buffer solutions, biological fluids or chemical reactors, blood fluids, saliva, urine, water, soil, wastewater, sea water, pharmaceuticals, foodstuffs, or beverages.

[0132] In certain aspects, the sample or medium in which a compound, conjugate or composition provided herein is present is illuminated with a wavelength of light selected to give a detectable optical response, and observed with a means for detecting the optical response. Equipment that is useful for illuminating the compounds, conjugates and compositions disclosed herein includes, but is not limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laser diodes. These illumination sources are optically integrated into laser scanners, fluorescence microplate readers or standard or micro-fluorometers, or chromatographic detectors.

[0133] In certain aspects, methods are provided for using a compound, conjugate, or composition provided herein for analysis or detection. More particularly, the detection may be performed by optical means. In certain embodiments, the fluorescence emission is optionally detected by visual inspection, or by use of any of the following devices: CCD cameras, video cameras, photographic film, laser scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow' cytometers, fluorescence microplate readers, or by means for amplifying the signal such as photomultiplier tubes. When the sample is examined using a flow cytometer, a fluorescence microscope or a fluorometer, the instrument is optionally used to distinguish and discriminate between the fluorescent compounds, conjugates or compositions provided herein from that of a second fluorophore. Where a sample is examined using a flow' cytometer, examination of the sample optionally includes isolation of particles within the sample based on tire fluorescence response by using a sorting device.

[0134] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but tire scope of the claims should not be limited to those features exemplified.EXAMPLESExample 1: Synthesis of Compound 1-6[00135 J 1. Synthesis of Compound 4

[0136] Compound 1 (1.400 g) and O-(N-Succinimidyl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TSTU) (741 mg) were dissolved in DMF (30 mL). To the solution was added TEA (1.400 mL) and the solution was stirred at room temperature until TLC showed the disappearance of Compound 1 (about 1 hour) and formation of a new compound (2). Compound 3 (493 mg) was then added to the solution under stirring, and the mixture was stirred until TLC showed the disappearance of Compound 2 (30-60 minutes). The solvent was evaporated with rotary evaporation and the residue was dissolved in dichloromethane (DCM) (150 mL). The DCM solution was washed with water twice (2 x 150mL). The organic layer was dried over Na2SO4, filtered, and evaporated with rotary evaporation. The residue was purified by silica gel column chromatography eluting with methanol in DCM. The desired fractions were combined and evaporated with rotary evaporation to give Compound 4 as a blue solid (1.35 g). Proton NMR of the product was consistent with the structure.

[0137] 2. Synthesis of Compound 7

[0138] Compound 4 (50 mg) was dissolved in a mixture of DCM (1 mL) and TFA (trifluoroacetic acid) (1 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 4 (about 30 minutes). The solution was evaporated with rotary evaporation and the residue was co-evaporated with DCM-Toluene (1: 1) twice. Hie residue was further dried under high vacuum for 1 hour and then used for the next step without purification. The residue was dissolved in DMF (1 mL) and to the solution were added Compound 6 (28 mg) and compound HATU (27 mg). DIEA (0.1 mL) was added, and the solution was stirred at room temperature under argon for 1 hour. The solvent was evaporated under high vacuum. The residue was purified by silica gel column chromatography with eluant of DCM-Methanol. Evaporation of the solvent gave Compound 7 as a blue solid. Yield: 60 mg (80% for two steps).

[0139] 3. Synthesis of Compound 8

[0140] Compound 7 (10 mg) was dissolved in a mixture of DCM ( 1 mL) and TFA (trifluoroacetic acid) ( 1 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 7 (about 30 minutes). The solution was evaporated with rotary evaporation and the residue was co-evaporatedwith DCM-Toluene (1: 1) twice. The residue was further dried under high vacuum for 1 hour and then used for the next step without purification.

[0141] 4. Synthesis of Compound 11

[0142] Compound 9 (174 mg, 1.5 equivalent), Compound 10 (300 mg, 1 equivalents) and PyAOP (7- Azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate) (203 mg, 1.5 equivalents) were dissolved in 12 mL DMF. To the solution was added triethylamine (TEA) (0.18 mL, 5 equivalents) under stirring and inert atmosphere. The mixture was stirred at room temperature until TLC showed the disappearance of Compound 10 (about 45 minutes). The solvent was evaporated with rotary evaporation to give a blue solid. The solid was purified by reverse phase column chromatography eluting with tri ethylammonium acetate buffer and then water / methanol. Evaporation of the solvent followed by lyophilization gave Compound 11 as a light blue solid (330 mg).

[0143] 5. Synthesis of Compound 12

[0144] Compound 11 (330 mg) was dissolved in TFA (trifluoroacetic acid) (5 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 11 (about 2 hours). TFA was evaporated with rotary evaporation and the residue was co-evaporated with CH3CN-Toluene (1 : 1) twice. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. Evaporation of tire solvent followed by lyophilization gave Compound 12 as a blue solid. Yield: 280 mg (68% for two steps).

[0145] 6. Synthesis of 14

[0146] Compound 12 (23 mg), Compound 13 (13 mg) and N,N-dimethyl aminopyridine (DMAP) (4.5 mg) were dissolved in anhydrous DMF (1 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 12 (about 1 hour). The solvent was evaporated with rotary evaporation. The residue was purified by reverse phase column chromatography eluting with water / acetonitrile. Evaporation of the solvent followed by lyophilization gave Compound 14 as a blue solid. Yield: 27 mg (79%).

[0147] 7. Synthesis of Compound 1-6

[0148] Compound 14 (25 mg, 1.7 equivalents), Compound 8 (9 mg, 1 equivalent) and triethyl amine (16 pL) were dissolved in anhydrous DMF (1 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 8 (about 1 hour). The solvent was evaporated with rotary evaporation. The residue was purified by reverse phase column chromatography eluting with tri ethylammonium acetate buffer and then water / acetonitrile. Evaporation of the solvent followed by lyophilization gave Compound 1-6 as a blue solid. Yield: 9 mg (46%). LCMS ofthe product was consistent with the structure. MS: 808 [M+3]3+, 1212 [M+3]2+.Example 2: Synthesis of Compound 1-1

[0149] 1. Synthesis of Compound 16

[0150] Compound 5 was synthesized using the procedure described in Example 1, starting with 1.2 g of Compound 4. To the flask containing Compound 5 were added compound 15 (845 mg), anhydrous DMF (60 mL) and triethyl amine (550 pL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 5 (about 1 hour). The solvent was evaporated with rotary' evaporation. The solid was washed with ethyl acetate twice (2 x 250 mL) by sonication and stirring. The solid was collected by filtration and dried under high vacuum to give Compound 16 as a blue solid. Compound 16 was used for the next step without further purification.

[0151] 2. Synthesis of Compound 17

[0152] Compound 16 (900 mg) was dissolved in DCM (15 mL) and the solution was placed in an ice-bath.Cold trifluoroacetic acid (15 mL) was added to the solution under stirring and Argon. The solution wasstirred in ice-bath for about 1 hour). The reaction mixture was added dropwise into vigorously stirred diethyl ether (about 800 mL) over 7-10 minutes. A blue precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with diethyl ether. Tire solid was purified by silica gel column chromatography eluting with DCM-methanol. The desired fractions were combined and evaporated with rotary’ evaporation. Tire solid was dissolved in 15 mL of a mixture of 10% methanol in DCM. The solution was filtered to remove silica gel. The solution was evaporated and dried under high vacuum to give Compound 17 as a blue solid (450 mg, 50%).

[0153] 3. Synthesis of Compound 1-1

[0154] Compound 12 (240 mg), Compound 13 (160 mg) and N,N-dimethyl aminopyridine (DMAP) (37 mg) were dissolved in anhydrous DMF (10 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 12 (about 1.5 hours). Hie reaction mixture was added dropwise into vigorously stirred ethyl acetate (about 400 mL). A blue precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with ethyl acetate. The solid was transferred to a 250-mL round bottom flask and dried under high vacuum for 1 hour to give crude Compound 14. Compound 14 was used for the next step without purification.

[0155] Compound 17 (143 mg), anhydrous DMF (10 mL), and triethyl amine (103 pL) were added to the round bottom flask containing the crude Compound 14. The solution was stirred at room temperature until TLC showed the disappearance of Compound 17 (about 1 hour). The reaction mixture was added dropwise into vigorously stirred diethyl ether (about 300 mL) over 2-3 minutes. A blue precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with diethyl ether. After being dried, the solid was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 1-1 as a blue solid. Yield: 140 mg (45% for two steps). LCMS of the product was consistent with tire structure. MS: 837 [M+3]J+, 1256 [M+3]2+.Example 3: Synthesis of Compound 1-2

[0156] 1 . Synthesis of Compound 19

[0157] Compound 18 (150 mg). Compound 3 (46 mg) and triethyl amine (132 pL) were dissolved in 3 mL DMF. The solution was stirred under inert atmosphere at room temperature until TLC showed the disappearance of Compound 18 (about 1 hour). The solvent was evaporated with rotary evaporation. The residue was dissolved in dichloromethane (DCM) (50 mL). The DCM solution was washed with water twice (2 x 50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated with rotary evaporation. The residue was purified by silica gel column chromatography eluting with methanol in DCM. The desired fractions were combined and evaporated with rotary evaporation to give Compound 19 as a red solid (110 mg, 69%). Proton NMR of the product was consistent with the structure.

[0158] 2. Synthesis of Compound 22

[0159] Compound 19 (75 mg) was dissolved in a mixture of DCM (2 mL) and TFA (trifluoroacetic acid) (2 mL). Tire solution was stirred at room temperature until TLC showed the disappearance of Compound 19 (about 30 minutes). The solution was evaporated with rotary evaporation and the residue was coevaporated with DCM-Toluene (1: 1) twice. The residue was further dried under high vacuum for 1 hour and then used for the next step without purification.

[0160] To the flask containing Compound 19 were added Compound 15 (as shown in Example 2) (58 mg), anhydrous DMF (2 mL) and triethyl amine (63 uL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 19 (about 1 hour). The solvent was evaporated with rotary evaporation. The solid was washed with ethyl acetate twice (2 x 250 mL) by sonication and stirring. The solid was collected by filtration and dried under high vacuum to give Compound 21 as a red solid. Compound 21 was used for the next step without further purification.

[0161] Tire crude compound 21 was dissolved in DCM (2 mL) and the solution was placed in an icc-bath. Cold trifluoroacetic acid (2 mL) was added to the solution under stirring and Argon. Hie solution was stirred in ice-bath for about 1 hour). The reaction mixture was added dropwise into vigorously stirred diethyl ether (about 100 mL) over 7-10 minutes. Red precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with diethyl ether. The solid was purified bysilica gel column chromatography eluting with DCM-methanol. The desired fractions were combined and evaporated with rotary evaporation. The solid was dissolved in 10 mL of a mixture of 10% methanol in DCM. The solution was filtered to remove silica gel. The solution was evaporated and dried under high vacuum to give Compound 22 as a red solid (50 mg, 42% for 3 steps).

[0162] 3. Synthesis of compound 24

[0163] Compound 9 (18 mg), Compound 23 (30 mg) and PyAOP (7-Azabenzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate) (27 mg) were dissolved in 1.5 mL DMF. To the solution was added triethylamine (TEA) (18 pL) under stirring and inert atmosphere. The mixture was stirred at room temperature until TLC showed the disappearance of Compound 23 (about 45 minutes). The solvent was evaporated with rotary evaporation to give a dark purple solid. To the residue was added TFA (trifluoroacetic acid) (1 mL). The solution was stirred at room temperature for 2 hours. TFA was evaporated with rotary evaporation and the residue was co-evaporated with CHsCN-Toluene (1: 1) twice. Hie residue was purified by reverse phase column chromatography. Evaporation of the solvent followed by lyophilization gave Compound 24 as a dark purple solid. Yield: 35 mg (79% for two steps).

[0164] 4. Synthesis of Compound 25

[0165] Compound 24 (20 mg), Compound 13 (13 mg) and N,N-dimethyl aminopyridine (DMAP) (4.0 mg) were dissolved in anhydrous DMF (1.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 24 (about 1 hour). The reaction mixture was added dropwise into vigorously stirred ethyl acetate (about 40 mL). Dark purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with ethyl acetate. The solid was transferred to a 50-rnL round bottom flask and dried under high vacuum for 1 hour to give crude Compound 25. Compound 25 was used for the next step without purification.

[0166] 5. Synthesis of Compound 1-2Compound 1-2

[0167] Compound 22 (9.3 mg), anhydrous DMF (1 mL), and tncthyl amine (18 pL) were added to the round bottom flask containing the crude Compound 25. The solution was stirred at room temperature until TLC showed the disappearance of Compound 22 (about 1 hour). The reaction mixture was added dropwise into vigorously stirred diethyl ether (about 12 mL) over 2-3 minutes. A dark purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifuge and washed with diethyl ether. After being dried, the solid was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 1-2 as a dark purple solid. Yield: 9 mg (41% for two steps). LCMS of the product was consistent with the structure. MS: 820 M3+, 1230 M3+.Example 4: Synthesis of Compound 1-3

[0168] 1. Synthesis of Compound 27

[0169] Compound 26 (12 mg), Compound 23 (from Example 3) (20 mg) and PyAOP (7-Azabcnzotriazol- l-yloxy)tripyrrolidinophosphonium hexafl uorophosphate) (13 mg) were dissolved in 1 mL DMF. To the solution was added triethylamine (TEA) (15 pL) under stirring and inert atmosphere. The mixture was stirred at room temperature until TLC showed the disappearance of Compound 23 (about 45 minutes). The solvent was evaporated with rotary evaporation to give a dark purple solid. To the residue was added TFA (trifluoroacetic acid) (1 mL). The solution was stirred at room temperature for 2 hours. TFA was evaporated with rotary evaporation and the residue was co-cvaporatcd with CLLCN-Tolucnc (1: 1) twice. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / methanol. Evaporation of the solvent followed by lyophilization gave Compound 27 as a dark purple solid. Yield: 25 mg (79% for two steps).

[0170] 2. Synthesis of Compound 29

[0171] Compound 27 (24.4 mg), Compound 28 (12 mg), anhydrous DMF (0.5 mL), and N,N- Diisopropylethylamine (DIEA) (5 pL) were added to a small vial. The solution was stirred at room temperature until TLC showed the disappearance of Compound 27. Piperidine (0. 1 mL) was added to the reaction vial and the solution was stirred for 30 minutes. The reaction mixture was added dropwise into vigorously stirred ethyl acetate (about 12 mL) over 2-3 minutes. A dark purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifuge and washed with ethyl acetate. After being dried, the solid was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / methanol. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 29 as a dark purple solid. Yield: 24 mg (80% for two steps).

[0172] 3. Synthesis of Compound 30

[0173] Compound 29 (20.2 mg). Compound 18 (13 mg), anhydrous DMF (0.5 mL), and triethylamine (TEA) (10 pL) were mixed in a small vial. The solution was stirred at room temperature until TLC showed the disappearance of Compound 29 (about 1 hour). The reaction mixture was added dropwise into vigorously stirred ethyl acetate (about 12 mL) over 2-3 minutes. A dark purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifuge and washed with ethyl acetate. After being dried, the solid was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / methanol. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 30 as a dark purple solid. Yield: 20 mg (80%).

[0174] 4. Synthesis of Compound 1-3

[0175] Compound 30 (10.5 mg), compound 13 (3 mg) and N,N-dimethyl aminopyridine (DMAP) (1.1 mg) were dissolved in anhydrous DMF (0.5 mb). The solution was stirred at room temperature until TLC showed the disappearance of Compound 30 (about 1 hour). Tire solvent was evaporated with rotary evaporation. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 1-3 as a dark purple solid. Yield: 11 mg (93%). LCMS of tire product was consistent with the structure. MS: 855 [M+2]3+, 1284 [M+4]2+.Example 5: Synthesis of Compound 1-4

[0176] 1. Synthesis of Compound 3333

[0177] Compound 31 (54 mg), compound 32 (20 mg) and triethyl amine (40 pL) were dissolved in anhydrous DMF (2 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 31 (about 1 hour). The solvent was evaporated with rotary evaporation. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / m ethanol. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 33 as a brown solid. Yield: 44 mg (80%).

[0178] 2. Synthesis of Compound 34

[0179] Compound 33 (40 mg), Compound 13 (18 mg) and N,N-dimethyl aminopyridine (DMAP) (6.5 mg) were dissolved in anhydrous DMF (1.2 mb). The solution was stirred at room temperature until TLC showed the disappearance of Compound 33 (about 1 hour). The solvent was evaporated with rotary evaporation. Tire residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 34 as a brown solid. Yield: 40 mg (73%).

[0180] 3. Synthesis of Compound 37

[0181] Compound 34 (12 mg), Compound 22 (from Example 3) (12 mg) and triethyl amine (10 pL) were dissolved in anhydrous DMF (0.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 34 (about 1 hour). PyAOP (7-Azabenzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate) (8 mg) and Compound 36 (8 mg) were added and the solution was stirred at room temperature for one more hour. Tire solvent was evaporated with rotary evaporation to give a dark purple solid. To the residue was added TFA (trifluoroacetic acid) (1 mL). The solution was stirred at room temperature for 2 hours. TFA was evaporated with rotary evaporation and the residue was co-evaporated with CHsCN-Toluene (1: 1) twice. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then 'ater / methanol. Evaporation of the solvent followed by lyophilization gave Compound 37 as a dark purple solid. Yield: 7.5 mg (30% for three steps).

[0182] 4. Synthesis of Compound 1-4

[0183] Compound 37 (6 mg), Compound 13 (3 mg) and N,N-dimethyl aminopyridine (DMAP) (2 mg) were dissolved in anhydrous DMF (0.5 m ). The solution was stirred at room temperature until TLC showed the disappearance of Compound 37 (about 1 hour). The solvent was evaporated with rotary evaporation. Tire residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 1-4 as a dark purple solid. Yield: 6 mg (88%). LCMS of the product was consistent with the structure. MS: 819 [M+3]3+, 1229 [M+4]2+.Example 6: Synthesis of Compound 1-5

[0184] 1. Synthesis of Compound 38

[0185] Compound 31 (30 mg), Compound 26 (26 mg) and triethyl amine (25 pL) were dissolved in anhydrous DMF (1.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 31 (as shown in Example 5) (about 2 hours). The solvent was evaporated with rotary evaporation to give a brown solid. To the residue was added TFA (trifluoroacetic acid) (1 mL). The solution was stirred at room temperature for 2 hours. TFA was evaporated with rotary evaporation and the residue was co-evaporated with CFLCN-Toluene (1: 1) twice. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / methanol. Evaporation of the solvent followed by lyophilization gave Compound 38 as a brown solid. Yield: 29 mg (72% for two steps).

[0186] 2. Synthesis of Compound 39

[0187] Compound 38 (13.6 mg), Compound 28 (13 mg), anhydrous DMF (0.5 mL), and N,N- Diisopropylethylamine (DIEA) (5 pL) were added to a small vial. The solution was stirred at room temperature until TLC showed the disappearance of Compound 38. Piperidine (0.1 mL) was added to the reaction vial and the solution was stirred for 30 minutes. Tire reaction mixture was added dropwise into vigorously stirred ethyl acetate (about 12 mL) over 2-3 minutes. A brown precipitate formed. Hie mixture was stirred for 15 minutes, and the precipitate was collected by centrifuge and washed with ethyl acetate. After being dried, the solid was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / methanol. The desired fractions were combined.Evaporation of the solvent followed by lyophilization gave Compound 39 as a brown solid. Yield: 10.8 mg (60% for two steps).

[0188] 3. Synthesis of Compound 40

[0189] Compound 39 (10 mg), compound A (8 mg), anhydrous DMF (0.5 mL), and triethylamine (TEA) (10 pL) were mixed in a small vial. The solution was stirred at room temperature until TLC showed the disappearance of Compound 39 (about 1 hour). The reaction mixture was added dropwisc into vigorously stirred ethyl acetate (about 12 mL) over 2-3 minutes. A dark purple precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifuge and washed with ethyl acetate. After being dried, tire solid was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / methanol. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 40 as a dark purple solid. Yield: 9.5 mg (71%).

[0190] 4. Synthesis of Compound 1-5

[0191] Compound 40 (8.7 mg). Compound 13 (1.7 mg) and N,N-dimethyl aminopyridine (DMAP) (0.61 mg) were dissolved in anhydrous DMF (0.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 40 (about 1 hour). The solvent was evaporated with rotary evaporation. Tire residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 1-5 as a dark purple solid. Yield: 7 mg (70%). LCMS of the product was consistent with the structure. MS: 756 [M]3+, 1134 [M+2]2+.Example 7: Synthesis of Compound 1-7

[0192] 1. Synthesis of Compound 41

[0193] Compound 39 (19 mg), Compound B (17 mg), anhydrous DMF (0.5 mL), and triethylamine (TEA) (10 pL) were mixed in a small vial. The solution was stirred at room temperature until TLC showed the disappearance of Compound 39 (about 1 hour). The reaction mixture was added dropwise into vigorously stirred ethyl acetate (about 12 mL) over 2-3 minutes. A dark purple precipitate fonned. The mixture was stirred for 15 minutes, and the precipitate was collected by centrifuge and washed with ethyl acetate. After being dried, the solid was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / methanoL The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 40 as a dark purple solid. Yield: 24 mg (71%).

[0194] 4. Synthesis of Compound 1-7

[0195] Compound 41 (23.9 mg), Compound 13 (3.9 mg) and N,N-dimethyl aminopyridine (DMAP) (1.4 mg) were dissolved in anhydrous DMF (0.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 41 (about 1 hour). Tire solvent was evaporated with rotary evaporation. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 1-7 as a dark purple solid. Yield: 19 mg (70%). LCMS of the product was consistent with the structure. MS: 809 [M+3]3+, 1215 [M+6]2+.Example 8: Synthesis of Compound 1-8

[0196] 1. Synthesis of Compound 42

[0197] Compound 26 (83 mg), Compound 10 (100 mg) and PyAOP (7-Azabenzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate) (68 mg) were dissolved in 3 mL DMF. To the solution was added triethylamine (TEA) (60 pL) under stirring and inert atmosphere. The mixture was stirred at room temperature until TLC showed the disappearance of Compound 10 (about 45 minutes). The solvent was evaporated with rotary evaporation to give a blue solid. To the residue was added TFA (trifluoroacetic acid) (2 mL). The solution was stirred at room temperature for 2 hours. TFA was evaporated with rotary evaporation and tire residue was co-evaporated with CH3CN-Toluene (1: 1) twice. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / methanol. Evaporation of the solvent followed by lyophilization gave Compound 42 as a blue solid. Yield: 100 mg (70% for two steps).

[0198] 2. Synthesis of Compound 43

[0199] Compound 42 (88 mg), Compound 28 (50 mg), anhydrous DMF (4.5 mL), and N,N- Diisopropylethylamine (DIEA) (28 pL) were added to a small vial. The solution was stirred at room temperature until TLC showed the disappearance of Compound 42. Piperidine (0.75 mL) was added to the reaction vial and the solution was stirred for 30 minutes. The reaction mixture was added dropwise into vigorously stirred ethyl acetate (about 150 mL) over 2-3 minutes. Blue precipitate formed. The mixture was stirred for 15 minutes, and tire precipitate was collected by filtration and washed with ethyl acetate. After being dried, the solid was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / methanoL The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 43 as a blue solid. Yield: 80 mg (74% for two steps).

[0200] 3. Synthesis of Compound 44

[0201] Compound 43 (80 mg), Compound 2 (48 mg), anhydrous DMF (2 mL), and triethylamine (TEA) (28 pL) were mixed in a small vial. Tire solution was stirred at room temperature until TLC showed tire disappearance of Compound 43 (about 1 hour). The solvent was evaporated to dry. The solid was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / m ethanol. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 44 as a blue solid. Yield: 95 mg (92%).

[0202] 4. Synthesis of Compound 1-8

[0203] Compound 44 (42 mg), compound 13 (11 mg) and N,N-dimethyl aminopyridine (DMAP) (2.9 mg) were dissolved in anhydrous DMF (1.5 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 44 (about 1.5 hour). The reaction mixture was added dropwise into vigorously stirred ethyl acetate (about 50 mL) over 2-3 minutes. A blue precipitate formed. The mixture was stirred for 15 minutes, and the precipitate was collected by filtration and washed with ethyl acetate. After being dried, the residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 1-3 as a blue solid. Yield: 36 mg (77%). LCMS of the product was consistent with the structure. MS: 872 [M]3+, 1308 [M]2+.Example 9: Synthesis of Compound 1-9

[0204] Compound 1-1 (20 mg), Compound 45 (6 mg) and triethylamine (10 pL) were dissolved in anhydrous DMF (1 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 45 (about 4 hours). The solvent was evaporated to dry. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. The desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 1-9 as a blue solid. Yield: 12 mg (57%). LCMS of the product was consistent with the structure. MS: 898 [M]3+, 1348 [M]2+.Example 10: Synthesis of Compound 1-10

[0205] Compound 1-7 (20 mg), Compound 45 (6 mg) and triethylamine (10 pL) were dissolved in anhydrous DMF (1 mL). The solution was stirred at room temperature until TLC showed the disappearance of Compound 1-7 (about 4.5 hours). The solvent was evaporated to dry. The residue was purified by reverse phase column chromatography eluting with triethylammonium acetate buffer and then water / acetonitrile. Tire desired fractions were combined. Evaporation of the solvent followed by lyophilization gave Compound 1-10 as a purple solid. Yield: 14.8 mg (70%). LCMS of the product was consistent with the structure. MS: 653 [M]4+.Example 11Conjugation to a Biomolecule

[0206] 20 pg of lyophilized Compound 1-1 was dissolved in 10 pL of DMSO to make a 2 mg / mL solution. To 100 pg of either trastuzumab (trade name Herceptin), cetuximab, or rituximab was added 10 pL of 1 M sodium bicarbonate and the volume was adjusted to 90 pL with lx PBS in a 1.5 mL Eppendorf tube. 10 pL of a 2 mg / mL solution of Compound 1-1 was added and the mixture was vortexed briefly, and then incubated for 2 hours at 20 °C with gentle shaking on an orbital shaker. Following conjugation, material was purified using a 0.5 mL Zeba™ dye and biotin removal column (Thermo Fisher Scientific), pre-equilibrated with lx PBS. The purified material was collected in a clean 1.5 mL Eppendorf tube and spun at 18k x g for 10 minutes to remove any precipitate.Quantification

[0207] The conjugated labeled antibody was diluted to 1:4 in 100% DMSO and the average of 3 Nanodrop™ measurements at OD280 and OD.,,,2 was taken. The extinction coefficient for the dye in 80% DMSO was 344,500 M ’cm1and the correction factor for the dye at OD280 was 0. 187.Internalization Assay - Imaging

[0208] Cells were seeded at a density of 5,000 cells / well in cell culture media in a total volume of 100 L in a 96 well plate allow to recover overnight. SKBR3 cells were used as a positive cell line for Her-2, A431 cells are positive for EGFR, and MCF7 cells are negative for both Her-2 and MCF7.

[0209] After overnight recovery, lOx stocks of trastuzumab-Compound 1-1 (SKBR3 +) or cetuximab- Compound 1-1 (A431 +) were prepared at 20 pg / mL in PBS, and 10 pL / well were added to each well in triplicate and incubated for various time points from 2-16 hours at 37 °C, 5% CO2. 15 minutes before imaging, Hoechst nuclear stain was added at a final concentration of 0. 1 pg / mL.

[0210] For co-localization experiments with pHrodo™ Red, cells were co-incubated with trastuzumab- pHrodo™ Red (Thermo Fisher Scientific) followed by wash and Hoechst staining. For co-localization experiments with LysoTracker™ Red (Thenno Fisher Scientific), following 16 hour incubations, cells were treated with 500 nM LysoTracker™ red diluted in cell culture media for 2 hours prior to wash and Hoechst staining.

[0211] Images were captured on the EVOS™ M7000 Imaging System (Thermo Fisher Scientific) using transmitted light, DAPI filter for nuclear staining, RFP filter for pHrodo™ Red or LysoTracker™ Red staining, and Cy5 filter cube for Compound 1-1 staining after 16 hours.

[0212] Quantification of Signal (SKBR3 or A431 cells) and background (MCF7 cells) was determined by high content on a Cellinsight™ CX7 LED Pro High Content Screening Platform (Thermo Fisher Scientific) using CircSpot thresholding and analyzing for mean CircSpot intensity in order to determine signal / background.

[0213] Conjugation and internalization using other compounds disclosed herein followed the methods described in Example 11 with respect to Compound 1-1.Internalization Assay — Flow Cytometry

[0214] Ramos (positive for CD20 and CD 19) or Jurkat (negative for CD20 and CD 19) cells were spun down at 500xg for 5 minutes then re-suspend in RPMI at a density of 1 x 106cells / mL. Cells were seeded at a density of 100,000 cells / well in a final volume of 100 pL / well. A lOx stock of rituximab-Compound1-1 was prepared at 20 pg / mL in lx PBS, pH 7.4 and added at 10 pL / well to give a final concentration of 2 pg / mL. Cells were incubated overnight at 37 °C, 5% COi. Following incubation, cells were washed in flow stain buffer. For dual staining, 5 pL of anti-CD19 - FITC was added for 30 minutes. Cells were washed 3x in lx PBS, and resuspended in 200 pL PBS + 3 pM DAPI. Cells were analyzed on the Attune™ NxT Flow Cytometer (Thermo Fisher Scientific).Inhibition of Cathepsin B

[0215] SKBR3 cells were seeded at a density of 5,000 cell / well in a 96 well plate as above. Following overnight recovery, cells were treated with either the cathepsin B inhibitor CA-074 (Me) or DMSO control titrated from 100 pM to 98 nM for 2 hours before treatment with trastuzumab-Compound 1-1. After incubation at 37 °C and 5% CO2, Hoechst nuclear stain was added at a concentration of 0.1 pg / mL for 15 minutes prior to imaging. Images were captured on the EVOS™ M7000 using transmitted light, DAPI filter for nuclear staining and Cy5 filter cube for Cathepsin B probe staining.

[0216] Quantification of signal was determined by high content on a Celllnsight™ CX7 LED using CircSpot thresholding and analyzing for mean CircSpot intensity.Results

[0217] Trastuzumab and cetuximab are therapeutic antibodies targeted to the Her-2 receptor and EGFR respectively. Upon binding, these antibodies are internalized and eventually degraded by the lysosome. The cathepsin B probe Compound 1-1 has amine reactive functionality and was conjugated to both of these antibodies in order to monitor degradation by the lysosome. SKBR3 cells are positive for Her-2 while A431 cells are positive of EGFR. As a negative control, MCF7 cells were used as they are negative for both membrane receptors. Upon initial treatment, there was no fluorescent signal observed, but over time (data not shown) fluorescence in the Cy5 channel began to accumulate in bright puncta throughout the cell. After 16 hours, bright signal was observed in the positive cell lines (FIGS. 2 and 5), while no fluorescence was observed in the MCF7 negative cell line (FIGS. 3 and 5). Quantification was done on the Cellinsight™ CX7 LED Pro High Content Screening Platform (Thermo Fisher Scientific), thresholding on tire punctate staining, yielding a high signal to background (FIG. 4). Importantly, these experiments were perfonned in cell culture media and were not washed prior to imaging allowing for real time monitoring of cathepsin B activity and degradation of the target.

[0218] Numerous cathepsins exist in the endosomes and lysosomes so in order to confirm that the probe is indeed specific to cathepsin B, the cathepsin B specific inhibitor CA-074 (Me) was used to inhibit its activity. SKBR3 cells were treatment with various concentrations of CA-074 (Me) for 2-hour prior to treatment with trastuzumab-Compound 1-1. Following a 16-hour incubation at 37 °C / 5% CO2,fluorescence intensity was quantified under matched exposures on the Celllnsight™ CX7 LED thresholding on the punctate staining. Quantification of the mean fluorescence intensity demonstrated a dose response to CA-074 (Me) inhibition (FIG. 10). Representative brightfield images were captured on the EVOS™ M7000 Imaging System showed punctate staining in non-treated cells while at higher concentrations, cathepsin B inhibition led to negative cathepsin B probe staining (FIGs. 11A-11C).

[0219] SKBR3 cells were stained with LysoTracker™ Red (green) (FIG. 12, top) or co-stained with trastuzumab - pHrodo™ Red (green) (FIG. 12, middle) and co-localization with Trastuzumab-Compound 1-1 (red) was observed. Merged images show overlapping fluorescence in yellow.

[0220] SKBR3 cells, A431 cells or MCF7 cells were stained with either trastuzumab-Compound 1-7, human IgGl isotype control-Compound 1-7. (FIGs. 13A and 13B ) or trastuzumab-Compound 1-3 conjugates (FIG. 14). Fluorescence punctate staining was observed in SKBR3 cells in both the FITC channel (green) (FIG. 13 A, left), and the RFP channel (FIG. 14, left), while no signal was observed in either channel in tire MCF7 negative cell line (FIGs. 13A and 14, right). Quantification of the mean CircSpot average intensity of SKBR3 and A431 cells treated with Compound 1-7 antibody conjugates demonstrated specific internalization of the Trastuzumab-Compound 1-7 conjugate in the SKBR3 cells and the Cetuximab-Compound 1-7 conjugate in the A431 cells (FIG. 13B).Example 12

[0221] Cathepsin probes were tested against Cathepsin B enzyme in a plate-based assay to compare activity. One probe was Compound II-2 that contained an LR cleavage site. The other probe was Compound I- 1.Materials & Methods

[0222] Reaction buffer: base buffer = phosphate buffered saline, pH 7.4 adjust pH to pH 6 with concentrated phosphoric acid1 mg / mL cysteine = 8.25 mM0.1 % Brij -35 surfactant

[0223] Enzyme: recombinant cathepsin B, human; BioLegend cat#557704 activity = 2.5 U per pg; dilute prior to performing assayenzyme prepared in 2X serial dilutions

[0224] Probe: prepared 2X solution @ 12 LIM. so final concentration was 6 pM

[0225] Plate reader:Tecan SPARKResults

[0226] FIGs. 15 and 16 provide the results. Tire resorufin-based probe (Compound II-l) had much higher signal than the dye-quencher probe (Compound 1-1), but both reagents showed increasing signal over time and with higher enzyme concentration.Example 13

[0227] Extinction coefficients for compounds containing either the fluorescent dye from Compound 1-1 or the quenching moiety from Compound 1-1 were measured in various solvent systems, in order to find a solvent system that would not cause spectral distortion of either moiety. This experiment was designed to establish extinctions of the individual components, which should be additive for the cathepsin B probe.Materials & Methods

[0228] A phalloidin conjugate of the dye moiety of Compound 1-1 and the free acid of the quencher moiety of Compound 1-1 were dissolved in DMSO at unknown high concentrations, then diluted into various solvents. For both dyes, the reference solvent was methanol. For non-reference solvents, the extinctions were determined by comparison with the values in reference solvent. All measurements were made in a Tecan SPARK plate reader in quartz cuvettes (1 cm path length).Conclusions

[0229] Tire dye moiety of Compound 1-1 had an estimated extinction of 220,700 in DMSO, and 228,000 in 90:10 DMSO:water.

[0230] Tire quencher moiety of Compound 1-1 had an estimated extinction of 64,300 in DMSO, and 69,000 in 90: 10 DMSO:water.

[0231] Tire Compound 1-1 cathepsin B probe containing both the dye moiety and quencher moiety is expected to have an extinction of 285,000 in DMSO, and 297,000 in 90: 10 DMSO:water, if the dye spectra are not distorted.Example 14

[0232] Three lots of tire fluorogenic cathepsin B probe. Compound 1-1. were conjugated to trastuzumab (trade name = Herceptin) by reaction of an SDP ester of the substrate with the antibody. Free dye was removed with a spin column, and the degree of labeling quantitated.Materials & Methods

[0233] The SDP ester of Compound 1-1 was dissolved in DMSO. then reacted with antibody in PBS buffer containing 100 mM sodium bicarbonate (pH about 9-10). After incubation for >1 hour, the crude conjugates were purified using Zeba™ biotin and dye removal spin columns (Thermo Fisher Scientific). The purified conjugates were diluted 10X into DMSO, so the final solution contained 10% aqueous. Correction factors were determined in tire same buffer system, as well as extinction coefficients for the substrate and antibody.Table 21 2 3Conclusions

[0234] FIG. 17 and Table 2 above provide the results. For all three lots of the reactive cathepsin B probe, the degree of labeling was between 1 to 2 Compound 1-1 cathepsin B probes per antibody.Example 15

[0235] Extinction coefficients were determined for Compound 1-1 in various solvent mixtures.Concentration of Compound 1-1 was determined by weigh-out (mass). The purpose of this experimentwas to determine degree of labeling on antibody conjugates, which cannot be diluted into methanol because they precipitate.Materials & Methods

[0236] About 5 mg of Compound 1-1 was weighed into a glass vial. Compound 1-1 was dissolved in DMSO. then diluted into various solvent mixtures including methanol, PBS-Tween-20, and mixtures of DMSO with water or PBS. Extinctions were determined from the solution concentration and the dye maximum absorbance using Beer's law.Conclusions

[0237] In PBS-Tween, the spectrum of Compound 1-1 was highly distorted, such that the maximum absorbance occurred at 608 nm rather than the expected about 660 nm. In methanol, DMSO, and high- DMSO aqueous mixtures, the spectrum was minimally distorted with minor changes in the maximum absorbance wavelength. For DMSO:water mixtures containing less than 70% DMSO, the spectrum became distorted (data not shown). The conclusion was that any aqueous solution with > 70% DMSO is suitable for determination of degree of labeling, but the extinction coefficient will vary from about 300- 380,000 depending on w ater content.Example 16

[0238] Coumarin-based AMC-LR (7-amino-4-methylcoumarin-Leu-Arg) cathepsin B probe was selected as a positive control and w as tested against Cathepsin B enzyme in a plate-based assay to compare activity. The test probe was Compound II- 1 .Materials & Methods

[0239] Reaction buffer: base buffer = phosphate buffered saline. pH 7.4 adjust pH to pH 6 with concentrated phosphoric acid1 mg / mL cysteine = 8.25 mM0.1 % Brij -35 surfactant

[0240] Enzyme: recombinant cathepsin B, human; BioLegend cat#557704 activity = 2.5 U per pg; diluted prior to perfonning assay enzyme prepared in 2X serial dilutions, then mixed 1 : 1 with substrate

[0241] Probe: prepared 2X solution @ 12 pM, so final concentration was 6 pM

[0242] Plate reader:Tecan SPARKConclusions

[0243] FIGS. 18A-20B provide the results, which demonstrated that Compound II- 1 exhibited more than double the signal of the coumarin-based AMC-LR probe, and slightly faster rate.Example 17

[0244] Cathepsin B probe candidate based on chloro-rhodamine (Cl-Rhod) was tested against Cathepsin B enzyme in a plate-based assay to compare activity. The probe was compared with Compound II- 1 that also contained an LR cleavage site. However, in Cl-Rhod the peptide is attached to the xanthene ring directly, whereas in compounds according to Formula II, such as Compound II- 1, the peptide is attached to the dye through a self immolative linker. Additionally, the peptide is attached to a bulkier polycyclic moiety, for example, in contrast to coumarin.Cl-RhodMaterials & Methods

[0245] Reaction buffer: base buffer = phosphate buffered saline. pH 7.4 adjust pH to pH 6 with concentrated phosphoric acid 1 mg / mL cysteine = 8.25 mM0.1% Brij-35 surfactant

[0246] Enzyme: recombinant cathepsin B, human; BioLegend cat#557704 activity = 2.5 U per pg; dilute prior to performing assay enzyme prepared in 2X serial dilutions, then mixed 1 : 1 with substrate

[0247] Probe: prepared 2X solution @ 12 pM, so final concentration was 6 pM

[0248] Plate reader:Tecan SPARKConclusions

[0249] The Cl-Rhod had no measurable activity with cathepsin B compared with the resorufin probe (FIG. 21).Example 18

[0250] Compound II-2 was tested against Cathepsin B enzyme in a plate-based assay to compare activity. The probe was compared with Compound II- 1 that also contained an LR cleavage site.Materials & Methods

[0251] Reaction buffer: base buffer = phosphate buffered saline. pH 7.4 adjust pH to pH 6 with concentrated phosphoric acid1 mg / mL cysteine = 8.25 mM0.1 % Brij -35 surfactant

[0252] Enzyme: recombinant cathepsin B, human; BioLegend cat#557704 activity = 2.5 U per pg; diluted prior to performing assay enzyme prepared in 2X serial dilutions, then mixed 1 : 1 with substrate

[0253] Probe: prepared 2X solution @ 12 pM. so final concentration was 6 pM

[0254] Plate reader:Tecan SPARKConclusions

[0255] Compound II-2 was active against cathepsin B but had lower activity with cathepsin B compared with Compound 1-1. and much lower signal-to-noise ratio (FIGS. 22A-22B).Example 19

[0256] The Val-Cit linker was initially thought to be specific for Cathepsin B; however, recent studies have demonstrated that it can also be cleaved by other cathepsin enzymes (Caculitan et al. Cancer Res. 77:7027 2017). To assess the promiscuity of the Cathepsin B probes disclosed herein, the activity of Cathepsin B, Cathepsin L and Cathepsin D was compared using Compound 1-1. The free acid form of Compound 1-1 was titrated with recombinant Cathepsin B, D and L for 2 hours and the fluorescence was measured on a Tecan Spark plate reader. Surprisingly, Cathepsin L exhibited approximately 4x greater activity towards Compound 1-1 than Cathepsin B, while Cathepsin D had little to no activity under tire assay conditions (FIG. 23). Thus, it appears that the Cathepsin B probes provided herein may not be specific for Cathepsin B alone, but the probes do show specificity for certain lysosomal cathepsins and prevent proteolytic release outside the lysosome.

[0257] The impact of inhibition on Cathepsin B and Cathepsin L activity was investigated using Compound 1-1 as a measure of enzymatic activity. Recombinant Cathepsin B and L were titrated with the Cathepsin B inhibitor CA-074 for 1 hour followed by incubation with the free acid form of Compound 1-1 for 1 hour and then fluorescence was measured on a Tecan Spark. While CA-074 potently inhibited Cathepsin B (IC50 = 20 pM), it had little inhibitor effect on Cathepsin L (IC50 = 1.875 nM), indicating it to be a good inhibitor for live cell inhibition (FIG. 24).Example 20

[0258] SKBR3 cells were treated with trastuzumab-compound 1-7 conjugate or Hu IgGl Isotype control- Compound 1-7 conjugate followed by fixation with 4% PFA. Images were captured immediately following fixation and 24 hours post fixation using the EVOS™ M7000 Imaging System (FIG. 25). Quantification of signal was determined by high content on a Cellinsight™ CX7 LED using CircSpot thresholding and analyzing for mean CircSpot intensity and shown in FIG. 26. These results indicate that conjugates of compounds comprising the “reverse” orientation of the peptide, such as Compound 1-7, can be fixed with 4% PFA.

[0259] In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated aspects are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim as the technology all that comes within the scope and spirit of these claims.INCORPORATION BY REFERENCE

[0260] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A compound according to Formula IR — L1— X — L2- — L3Formula I or a salt thereof, wherein:X is -dye- or -N(-dye)-; peptide is a protease-cleavable peptide moiety;R is a reactive group suitable to conjugate the compound to a target molecule: and each of L1, L2, and L3independently is a linker selected from a covalent bond or a moiety comprising two or more covalent bonds and at least one atom selected from C, N, O, P, or S.

2. The compound of claim 1, wherein the compound has a structure according to Formula 1-AFormula I-A.

3. The compound of claim 1, wherein the compound has a structure according toFormula I-BFormula I-B.

4. The compound of any one of claims 1-3, wherein L2is attached to the N-terminal end of the peptide.

5. Tire compound of any one of claims 1-3, wherein L2is attached to the C-terminal end of the peptide.

6. The compound of any one of claims 1-5, wherein each of L1, L2, and LJindependently is a linker selected from polyethylene glycol (PEG), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted unsaturated alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl. heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy. heterocyclylalkanoyL heterocyclylalkamino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, thiourea, phosphoryl, sulfonyl, sulfonamido, or ketone.

7. Tire compound of any one of claims 1-5, wherein each of L1, L2and L3independently are selected from polyethylene glycol (PEG), substituted or unsubstituted Ce aryl, substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms selected from N, O or S, substituted or unsubstituted Ci.6alkyl, substituted or unsubstituted C2.6alkenyl, substituted or unsubstituted C2.6alkynyl, Ci.6alkoxy, C2.6alkanoyl (C2.6 alkyl-C(=O)-), Ci.6alkylamino, C6aryloxy, C6arylamino, C7-9 aralkyl, C7-9 aralkoxy, C7-9 aralkanoyl, C7-9 aralkamino, (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1. 2, or 3 heteroatoms selected from N, O or S)- O-, (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms selected from N. O or S)-NH-. (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1.

2. or 3 heteroatoms selected from N, 0 or S)-Ci.e alkyl-, (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms selected from N, O or S)-Ci-6 alkyl-O-, (substituted or unsubstituted 5- to 10-mcmbcrcd hctcroaryl comprising 1, 2, or 3 heteroatoms selected from N, O or S)- Ci-6alkyl-C(=O)-, (substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2. or 3 heteroatoms selected from N, 0 or S)-Ci-6 alkyl -NH-, C3-7 cycloalkyl, C3-7 cycloalkenyl, C3-7 cycloalkylalkyl, C3-7 cycloalkoxy, C3-7 cycloalkanoyl, C3-7 cycloalkamino, substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S, (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-O-, (substituted or unsubstituted 5- to 10-mcmbcrcd heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N. 0 or S)-NEI-. (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising1, 2. or 3 heteroatoms selected from N, 0 or S)-Ci.e alkyl-, (substituted or unsubstituted 5- to 10- membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-Ci.6alkyl-O-, (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, O or S)-Ci_6alkyl-C(=O)-, (substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-Ci-6 alkyl-NH-, Ci-6 alkanoylamino (-C1.6alkyl-C(=O)NH-). -Cearyl-C(=O)NH-, aralkanoylamino (-C6 aryl-Ci-6alkyl-C(=O)NH-), alkylcarboxy (- Ci.6alkyl-C(=O)-), carbonate (-OC(=O)O-), carbamate (-OC(=O)NH-), guanidinyl (-NHC(=NH)NH-), urea (-NHC(=O)NH-), thiourea (-NHC(=S)NH-), phosphory l (-P(=O)(OH)-), sulfonyl (-SO2-), sulfonamide (-SO2NH-), or ketone (-C(=O)-).

8. Hie compound of any one of claims 1-5, wherein each of L1, L2and L3independently are selected from -K-L-J-, wherein: each of K and J independently is a bond, -C(=O)-, -NRa-, -OC(=O)-, -C(=O)O-, - OC(=O)NH-, -NHC(=O)O-, -SO2-, -SO2NH-, -NHSO2-, -NHC(=S)NH-, -P(=O)(OH)-, -NHC(=O)NH-, - NHC( NH)NH-. -OC(=O)O-, or -O-;L is -C1.20 alkyl-, -[(CH2CH2)O]X(CH2CH2)-, -(CH2)y-(substituted or unsubstituted Ce aryl)-(CH2)y-, -(CH2)y-(substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-(CH2)y-, -(CH2)y-(substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms selected from N, O or S)-(CH2)y-, -(CH2)y-(substituted or unsubstituted C3-s cycloaliphatic)-(CH2)y-; x is from 1 to 6; each y independently is from 0 to 6 andRais H, C1-4 alkyl, 2,4-disulfonatobenzyl, cystic acid residue, or monosulfonatobenzyl.

9. The compound of claim 8, wherein L is -[(CH2CH2)O]X(CH2CH2)- for at least one of L1, L2, and L3.

10. The compound of claim 8, wherein L is -[(CH2CH2)O]X(CH2CH2)- for at least two of L1, L2, and L3.

11. The compound of any one of claims 1-10, wherein the dye is a cyanine dye, a rhodamine or rhodamine analogue dye, a fluorescein or fluorescein analogue dye, a xanthine or xanthine analogue dye, a BODIPY dye, a coumarin dye, a phthalocyanine dye, a porphyrin dye, a pyrene dye, a fluorene or polyfluorene dye, an excited-state intramolecular proton-transfer (ESIPT) dye, an aggregation-induced emission dye, a metal complex dye, or a combination thereof.

12. The compound of anyone of claims 1-11, wherein the dye is a fluorescein dye, rhodamine, Group 14 Rhodamine dye, Phospha-rhodamine, a cyanine dye with 2, 3, 5, 7 or 9-methine structure, or coumarin.

13. The compound of any one of claims 1-11, wherein the quencher has an absorption of from 250 nm to 1,000 nm.

14. Tire compound of any one of claims 1-12, wherein the quencher comprises an azo dye-based quencher, a cyanine-based quencher, a rhodamine-based quencher, an azaphthalocyanine- based quencher, a hemin quencher, malachite green, a malachite green analogue, or a combination thereof.

15. The compound of claim 14, wherein the quencher comprises 2, 3, 4, or 5 quenching moieties.

16. Tire compound of any one of claims 1-15, wherein the quencher comprises17. The compound of any one of claims 1-16, wherein R is an ester, isocyanate, isothiocyanate, maleimide, vinyl moiety, thiol, amine, hydroxyl, hydrazine, hydrazide, hydroxylamine, aminooxy, clickable handle for click chemistry, sulfonyl halide, sulfonyl amide, glyoxal, oxaziridine, a carbonate, a carbamic halide, or a photoreactive moiety.

18. The compound of claim 17, wherein the clickable handle for click chemistry is chosen from an azide, an alkyne, a strained-ring alkyne, a cyclooctyne, a thiacycloheptyne, a tetrazine, and a trans-cyclooctene (TCO).

19. The compound of any one of claims 1-18, wherein the peptide is a cathepsin B cleavable peptide.

20. The compound of any one of claims 1-19, wherein:the peptide is selected from Gly-Gly-Phe-Gly (SEQ ID NO: 1), Val-Cit, Val-Cit-PAB.Gly-Gly, Gly-Gly-Gly, Phe-Lys, Vai-Ala, Val-Gly, Val-Val, Ala-Ala-Asn, Gly-Phe-Leu-Gly (SEQ ID NO: 2), Arg-Arg, Phe-Arg-Arg-Gly (SEQ ID NO: 3), Gly-Phe-Leu-Gly-Lys (SEQ ID NO: 4), Lys-Lys, Lys-Lys-PAB, Gly-Arg-Arg-Gly-Lys-Gly-Gly (SEQ ID NO: 5), Gly-Glu-Leu-Gly, (SEQ ID NO: 6) Val- Arg, Glu-Arg, or Gly-Ile-Val-Arg-Ala-Lys (SEQ ID NO: 7); andPAB is a para-aminobenzyl group.

21. The compound of any one of claims 1-20, wherein the peptide is Val-Cit or Val- Cit-PAB.

22. Tire compound of any one of clams 1-21, wherein the compound is a salt comprising 1, 2, 3, 4, or 5 counter ions.

23. The compound of claim 22, wherein the 1, 2, 3, 4, or 5 counter ions comprise a trialkylammonium ion.

24. Tire compound of claim 23, wherein the trialkylammonium ion is a triethylammonium ion.

26. A compound having a general Formula II R — L1— peptide — L2Formula II or a salt thereof, wherein: peptide is a protease-cleavable peptide moiety;R is a reactive group suitable to conjugate the compound to a target molecule; andL1is a linker selected from a covalent bond or a moiety comprising two or more covalent bonds and at least one atom selected from C, N, O, P, or S; andL2is a covalent bond or a self-immolative linker.

27. The compound of claim 26, wherein L1is a linker selected from polyethylene glycol (PEG), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted unsaturated alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl,heteroaralkoxy, heteroaralkanoyl, heteroaralkamino. cycloalkyl. cycloalkenyk cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, thiourea, phosphoryl, sulfonyl, sulfonamide, or ketone.

28. Tire compound of claim 26. wherein L1is a linker selected from -K-L-J-, wherein: each of K and J independently is a bond, -C(=O)-, -NRa-, -OC(=O)-, -C(=O)O-, - OC( O)NH-. -NHC(=O)O-, -SO2-, -SO2NH-, -NHSO2-, -NHC(=S)NH-, -P(=O)(OH)-, -NHC(=O)NH-, - NHC(=NH)NH-, -OC(=O)O-, or -O-;L is -Ci-20 alkyl-, -[(CH2CH2)O]X(CH2CH2)-, -(CH2)y-(substituted or unsubstituted Ce aryl)-(CH2)y-, -(CH2)y-(substituted or unsubstituted 5- to 10-membered heterocyclyl comprising 1, 2, or 3 heteroatoms selected from N, 0 or S)-(CH2)y-, -(CH2)y-(substituted or unsubstituted 5- to 10-membered heteroaryl comprising 1, 2, or 3 heteroatoms selected from N, O or S)-(CH2)y-, -(CH2)y-(substituted or unsubstituted C3-s cycloaliphatic)-(CH2)y-; x is from 1 to 6; each y independently is from 0 to 6: andRais H. Cwalkyl. 2,4-disulfonatobenzyl, cystic acid residue, or monosulfonatobenzyl.

29. The compound of any one of claims 26-28, wherein the dye is an acridone dye, a fluorescein dye or an analogue thereof, coumarin, xanthine-hybrid cyanine dye, a phenol -containing dye, or an aniline-containing dye.

30. The compound of any one of claims 26-29, wherein the dye is resorufin, DDAQ, fluorescein, or coumarin.

31. The compound of any one of claims 26-30, wherein R is an ester, isocyanate, isothiocyanate, maleimide, vinyl moiety, thiol, amine, hydroxyl, hydrazine, hydrazide, hydrox lamine, aminooxy. clickable handle for click chemistry, sulfonyl halide, sulfonyl amide, glyoxal, oxaziridine, a carbonate, a carbamic halide, or a photoreactive moiety.

32. Tire compound of claim 31. wherein the clickable handle for click chemistry is chosen from an azide, an alkyne, a strained-ring alkyne, a cyclooctyne, a thiacycloheptyne, a tetrazine, and a trans-cyclooctene (TCO).

33. The compound of any one of claims 26-32, wherein the peptide is a cathepsin B cleavable peptide.

34. The compound of any one of claims 26-33, wherein: the peptide is selected from Gly-Gly-Phe-Gly (SEQ ID NO: 1), Val-Cit, Val-Cit-PAB, Gly-Gly, Gly-Gly-Gly, Phe-Lys, Vai-Ala, Val-Gly, Val-Val, Ala-Ala-Asn, Gly-Phe-Leu-Gly (SEQ ID NO: 2), Arg-Arg, Phe-Arg-Arg-Gly (SEQ ID NO: 3), Gly-Phe-Leu-Gly-Lys (SEQ ID NO: 4). Lys-Lys, Lys-Lys-PAB, Gly-Arg-Arg-Gly-Lys-Gly-Gly (SEQ ID NO: 5), Gly-Glu-Leu-Gly (SEQ ID NO: 6), Val- Arg. Glu-Arg. or Gly-Ile-Val-Arg-Ala-Lys (SEQ ID NO: 7); andPAB is a para-aminobenzyl group.

35. The compound of any one of claims 26-34, wherein the peptide is Val-Cit or Val-Cit-PAB.

36. Tire compound of any one of claims 26-35. wherein L1is - [(CH2CH2)O]X(CH2CH2)-.

37. The compound of any one of claims 26-36, wherein L2is selected from38. The compound of any one of claims 26-37, wherein the compound is selected from:

39. A composition, comprising a compound according to any one of claims 1-38 conjugated to a biomolecule.

40. The composition of claim 39, wherein the biomolecule is an antibody, enzyme, protein, oligonucleotide, or dextran.

41. Tire composition of any one of claims 38-40, further comprising one or more of a solvent, a buffer, a surfactant, or a combination thereof.

42. A kit, comprising: a compound of any one of claims 1-38 or a composition of any one of claims 39-41; and one or more of a solvent, a buffer, a surfactant, a purification column, a collection tube, sodium bicarbonate, or any combination thereof.

43. The kit of claim 42, wherein the surfactant is Polyoxyethylene Lauryl Ether (Brij™-35), Polyoxyethylene (20) sorbitan monolaurate (Tween™-20), 2-[4-(2,4,4-trimethylpentan-2- yljphcnoxy] ethanol (Triton™ X- 100), octylphcnoxypolycthoxycthanol (Nonidct™ P-40) or a combination thereof.

44. The kit of claim 42 or 43. wherein the buffer is phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane-HCl (Tris-HCl), 2-[4-(2-Hydroxyethyl)piperazin- 1 -yl]ethane- 1 - sulfonic acid (HEPES), or a combination thereof.

45. Tire kit of any one of claims 42-44, wherein the solvent comprises water, DMSO. methanol, or a combination thereof.

46. A method, comprising: contacting a cell with a compound of any one of claims 1-38; exposing the cell to light; and detemiining the presence or absence of a signal.

47. A method, comprising: contacting a sample with a compound of any one of claims 1-38; exposing the sample to light; and determining tire presence or absence of a signal.

48. The method of claim 46 or claim 47, wherein exposing the cell or sample to light and determining the presence or absence of the signal comprises exposing the cell or sample to light at a first wavelength and detecting the presence or absence of light having a second wavelength that is different from the first wavelength.

49. Tire method of claim 46 or claim 48, wherein the cell is an in vitro cell or an ex vivo cell.

50. The method of claim 46 or claim 48, wherein the cell is in vivo.

51. A method, comprising: conjugating a compound according to any one of claims 1-38 to a biomolecule to form a biomolecule-compound conjugate .

52. The method according to claim 51 further comprising: incubating the cell for a time interval adequate to allow entry of tire biomoleculecompound conjugate into the cell;illuminating the cell with an appropriate wavelength of light to excite the dye to generate a signal; and detecting the signal from the biomolecule-compound conjugate.

53. A method comprisin : contacting a cell with the biomolecule-compound conjugate of claim 51; incubating the cell for a time interval adequate to allow entry of the biomoleculecompound conjugate into the cell; illuminating tire cell with an appropriate wavelength of light to excite the dye to generate a signal; and detecting the signal from the biomolecule-compound conjugate.

54. A method, comprising: contacting a cell with a composition according to any one of claims 39-41; incubating the cell for a time interval adequate to allow entry of the composition into the cell; illuminating the cell with an appropriate wavelength of light to excite the dye to generate a signal; and detecting the signal from the composition.

55. A method, comprising : contacting a sample with a compound according to any one of claims 1-38, or a composition according to any one of claims 39-41, or the biomolecule-compound conjugate of claim 51; incubating the sample for a time interval adequate to allow for enzymatic cleavage of the peptide; illuminating tire sample with an appropriate wavelength of light that will excite the dye to generate a signal; and detecting the signal from the sample.

56. The method of any one of claims 46-55, wherein the time interval is from 1 hour to 24 hours.

57. The method of any one of claims 46-56, wherein the wavelength of light is a wavelength of visible light.

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