Coumarin-based crosslinking reagents

Abstract

To provide a novel coumarin-based reagent, and more specifically provide a conjugate including at least one coumarin-based reagent disclosed.SOLUTION: In some embodiments, the existence of the coumarin in a coumarin-based reagent allows detection of a label difficult to detect in general, for example, specific hapten.SELECTED DRAWING: None

Description

[Background technology]

[0001] Immunohistochemistry (IHC) refers to the process of detecting, localizing, and / or quantifying antigens, such as proteins, in biological samples using antibodies specific for a particular antigen. IHC offers the substantial advantage of pinpointing the precise location of a particular protein within a tissue sample. It is also an effective method for examining the tissue itself. In situ hybridization (ISH) refers to the process of detecting, localizing, and quantifying nucleic acids. Both IHC and ISH can be performed on a variety of biological samples, including tissues (fresh-frozen, formalin-fixed, paraffin-embedded, etc.) and cytological samples. Target recognition can be detected using a variety of labels (e.g., chromogenic, fluorescent, luminescent, radioactive), regardless of whether the target is a nucleic acid or an antigen. The ability to reliably detect low-abundance cellular markers for diagnostic purposes is increasingly important for reliable target detection, localization, and quantification in clinical settings, making amplification of the recognition event desirable. For example, deposition of hundreds or thousands of labeled molecules at the site of a marker in response to a single antigen detection event enhances the ability to detect that recognition event through amplification.

[0002] The use of small molecules, such as haptens, to detect antigens and nucleic acids in tissues has become a primary method of IHC. Haptens, in combination with anti-hapten antibodies, serve to detect specific molecular targets. For example, specific binding moieties, such as primary antibodies and nucleic acid probes, can be labeled with one or more hapten molecules. When these specific binding moieties bind to their molecular targets, they can be detected using colorimetric detection systems or anti-hapten antibody conjugates containing an enzyme as part of a detectable label, such as a fluorescent label. Binding of the detectable anti-hapten antibody conjugate to a sample indicates the presence of the target in the sample.

[0003] It is believed that some haptens are difficult to detect, resulting in a general overestimation of hapten loading and an underestimation of conjugate concentration. Summary of the Invention

[0004] In one aspect of the disclosure, a compound of formula (IA) or (IB): TIFF0007743561000001.tif45170

[0005] (In the formula,

[0006] A and B are independently a reactive functional group, a detectable label, or an enzyme-reactive moiety;

[0007] L 1 and L 2 is the linker,

[0008] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin;

[0009] Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0010] m and n are independently integers ranging from 1 to 4. It is a compound defined by any one of the structures:

[0011] In some embodiments, both A and B are reactive functional groups (e.g., carboxylic acid groups). In some embodiments, A is a reactive functional group and B is a detectable label. In some embodiments, the detectable label is a hapten, a chromogen, or a fluorophore. In some embodiments, B is a hapten. In some embodiments, the hapten is selected from the group consisting of oxazole, pyrazole, thiazole, benzofurazan, triterpene, urea, thiourea other than rhodamine thiourea, nitroaryl other than dinitrophenyl or trinitrophenyl, rotenoid, cyclolignan, heterobiaryl, azoaryl, and benzodiazepine. In some embodiments, the hapten is selected from the group consisting of benzofuran hapten and thiazole sulfonamide hapten. In some embodiments, the hapten is selected from the group consisting of 5-nitro-3-pyrazolecarbamide (NP), 2-acetamido-4-methyl-5-thiazolesulfonamide (TS), 7-(diethylamino)-2-oxo-2H-chromene-3-carboxyylic acid (DCC), and 2,1,3-benzoxadiazole-5-carbamide (BF). In some embodiments, A is an enzyme-reactive moiety selected from the group consisting of a tyramide moiety, a moiety that is a derivative of a tyramide moiety, and a quinone methide precursor moiety.

[0012] In some embodiments, a compound of either Formula (IA) or (IB) has an extinction coefficient of at least 10,000 M-1 cm-1. In some embodiments, a compound of either Formula (IA) or (IB) has an extinction coefficient of at least 15,000 M-1 cm-1. In still other embodiments, a compound of either Formula (IA) or (IB) has an extinction coefficient of at least 20,000 M-1 cm-1. In some embodiments, a compound of either Formula (IA) or (IB) has an extinction coefficient of at least 25,000 M-1 cm-1.

[0013] In some embodiments, the enzyme-reactive moiety has formula (VIIA): TIFF0007743561000002.tif44170

[0014] (In the formula, each R 11 groups are independently selected from H or lower alkyl groups having 1 to 4 carbon atoms, and wherein R x is H or a C1-C4 alkyl group) It has the following structure.

[0015] In some embodiments, the enzyme-reactive moiety has formula (VIIIA): TIFF0007743561000003.tif37170 (in the formula,

[0016] R 2 is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or sugar;

[0017] R 13 is a halide,

[0018] R 15 , R 16 , R 17 , and R 18 are independently selected from hydrogen or an aliphatic group having 1 to 4 carbon atoms;

[0019] R 14 is -(CH2) w NH-, -O(CH2) w NH, -N(H)C(O)(CH2) w NH-, -C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -, -N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(o)N(H)(CH2CH2O) w -, -(CH2) w S-, -O(CH2) wS-, -N(H)C(O)(CH2) w S-, -C(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) w NH- or -N(H)(CH2) w NH-, and w is an integer ranging from 1 to 12. The structure is given by:

[0020] In some embodiments, B is a reactive group (e.g., an azide group) capable of participating in a click chemistry reaction. In some embodiments, the group capable of participating in a click chemistry reaction is a DBCO group, an azide, a TCO group, an alkene group, or a tetrazine group.

[0021] In some embodiments, W has formula (IVA) or (IVB): TIFF0007743561000004.tif15170

[0022] (wherein Q is a bond or a substituted or unsubstituted straight or branched chain C1-C 16 Alkyl group, -[(CH2CH2) j -O] k -CH2-, -[(CH2) j -O] k -CH2- (wherein j is an integer ranging from 1 to 4 and k is an integer ranging from 1 to 16); -NR x -;-C(O)-NR x -; or -NR x -C(O)- and R xis H or a C1-C4 alkyl group, and Y is a substituted or unsubstituted coumarin moiety, or a moiety that is a substituted or unsubstituted derivative or analog of coumarin. In some embodiments, the substituted or unsubstituted coumarin moiety, or a moiety that is a substituted or unsubstituted derivative or analog of coumarin, is selected from the group consisting of a substituted or unsubstituted straight or branched chain C1-C6 alkyl group, a substituted or unsubstituted straight or branched chain C1-C6 alkoxy group, a substituted or unsubstituted straight or branched chain C1-C6 heteroalkyl group, a trifluoromethyl group, a hydroxyl group, a sulfate group, a cyano group, a halogen, a phosphate group, a sugar, a carboxylic acid group, a nitro group, or —C(O)NR x R y ;-SR x ;-SO2;-SO2Cl;-SO3H;-SO4H;-SO2NR x R y ;-N(H)-NR x R y ; and -NR x R y and wherein R x and R y are independently H or a C1-C4 alkyl group. The structure is either of the following:

[0023] In some embodiments, W has formula (VA) or (VB): TIFF0007743561000005.tif77170

[0024] (Wherein, the portion of formula (VA) is 0, 1, 2, 3, or 4 R t groups, and the compounds of formula (VB) may contain 0, 1, 2, 3, 4, or 5 R t groups, and each R t is a substituted or unsubstituted straight or branched chain C1-C 12 Alkyl groups; substituted or unsubstituted straight or branched chain C1-C 12 Alkoxy groups, substituted or unsubstituted, straight or branched chain C1-C 12Heteroalkyl groups; Trifluoromethyl groups; Hydroxyl groups; Sulfate groups; Cyano groups; Halogens; Phosphate groups; Sugars; Carboxylic acid groups; Nitro groups; -C(O)NR x R y ;-SR x ;-SO2;-SO2Cl;-SO3H;-SO4H;-SO2NR x R y ;-N(H)-NR x R y ;-NR x R y and wherein R x and R y are independently H or a C1-C4 alkyl group,

[0025] R 1 is C1~C 10 substituted or unsubstituted branched or unbranched alkyl groups, -O-, -O-CH2-, -N(R x )-, or -S-,

[0026] R 5 is C1~C 10 a substituted or unsubstituted branched or unbranched alkyl group, —C(O)—O—, —C(O)—N(H)—;

[0027] Each X is independently a bond or a substituted or unsubstituted straight or branched chain C1-C 16 Alkyl group; -[(CH2) j -O] k -CH2- (j is an integer ranging from 1 to 4, and k is an integer ranging from 1 to 16); -NR x -;-C(O)-NR x -; or -NR x -C(O)-(R x is H or a C1-C4 alkyl group. The structure is either of the following:

[0028] In some embodiments, W has formula (VC) or (VD): TIFF0007743561000006.tif94170

[0029] (In the formula,

[0030] R 2 , R 3 , R 4 , R 6 , and R 7 is a substituted or unsubstituted straight-chain or branched C1-C6 alkyl group; a substituted or unsubstituted straight-chain or branched C1-C6 alkoxy group; a substituted or unsubstituted straight-chain or branched C1-C6 heteroalkyl group; a trifluoromethyl group; a hydroxyl group; a sulfate group; a cyano group; a halogen; a phosphate group; a sugar; a carboxylic acid group; a nitro group; -C(O)NR x R y ;-SR x ;-SO2;-SO2Cl;-SO3H;-SO4H;-SO2NR x R y ;-N(H)-NR x R y ;-NR x R y are independently selected from

[0031] R 5 is C1~C 10 a substituted or unsubstituted branched or unbranched alkyl group, —C(O)—O—, —C(O)—N(H)—;

[0032] R 1 is C1~C 10 substituted or unsubstituted branched or unbranched alkyl groups, -O-, -O-CH2-, -N(R x )-, or -S-,

[0033] Each X is independently a bond or a substituted or unsubstituted straight or branched chain C1-C 16 Alkyl group; -[(CH2) j -O] k -CH2- (j is an integer ranging from 1 to 4, and k is an integer ranging from 1 to 16); -NR x -;-C(O)-NR x -; or -NR x -C(O)-,

[0034] In the formula, each R x and R y are independently H or a C1-C4 alkyl group. The structure is either of the following:

[0035] In some embodiments, each L 1 and / or L 2 The groups are independently of the formula (VIA): TIFF0007743561000007.tif41170

[0036] (In the formula,

[0037] f is 0 or an integer ranging from 1 to 24;

[0038] j is an integer ranging from 1 to 24,

[0039] R 8 is a bond or O, S, -N(R c )(R d ), or -N + (R c )(R d )(R e ) and

[0040] R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or -N(R c )(R d ) and

[0041] R c , R d , and R e are independently selected from H or a C1-C4 alkyl group;

[0042] R 9 and R 10 are independently a bond or a group having up to 6 carbon atoms and including a carbonyl, amide, imide, ester, ether, amine, thione, or thiol. It has.

[0043] In another embodiment of the present disclosure, compounds of formula (IXA) or (IXB): TIFF0007743561000008.tif65170

[0044] (In the formula,

[0045] T is a substituent selected from a specific binding entity, an amino acid, a peptide, a protein, a saccharide, a nucleoside, a nucleotide, an oligonucleotide, a drug, a lipid, or a nanoparticle;

[0046] R z is a detectable label,

[0047] o is an integer ranging from 1 to 10;

[0048] L 1 and L 2 is the linker,

[0049] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin;

[0050] Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0051] m and n are independently integers ranging from 1 to 4. It is a compound defined by any one of the structures:

[0052] In some embodiments, T is an antibody. In some embodiments, the antibody is a primary antibody. In some embodiments, the antibody is a secondary antibody. In some embodiments, T is a nucleic acid. In some embodiments, R z is a hapten. In some embodiments, R z is an enzyme. In some embodiments, the enzyme is selected from the group consisting of peroxidases and phosphatases.

[0053] In some embodiments, W has formula (VC) or (VD): TIFF0007743561000009.tif94170

[0054] (In the formula,

[0055] R 2 , R 3 , R 4 , R 6 , and R 7 is a substituted or unsubstituted straight-chain or branched C1-C6 alkyl group; a substituted or unsubstituted straight-chain or branched C1-C6 alkoxy group; a substituted or unsubstituted straight-chain or branched C1-C6 heteroalkyl group; a trifluoromethyl group; a hydroxyl group; a sulfate group; a cyano group; a halogen; a phosphate group; a sugar; a carboxylic acid group; a nitro group; -C(O)NR x R y ;-SR x ;-SO2;-SO2Cl;-SO3H;-SO4H;-SO2NR x R y ;-N(H)-NR x R y ;-NR x R y are independently selected from

[0056] R 5 is C1~C 10 a substituted or unsubstituted branched or unbranched alkyl group, —C(O)—O—, —C(O)—N(H)—;

[0057] R 1 is C1~C 10 substituted or unsubstituted branched or unbranched alkyl groups, -O-, -O-CH2-, -N(R x )-, or -S-,

[0058] Each X is independently a bond or a substituted or unsubstituted straight or branched chain C1-C 16 Alkyl group; -[(CH2) j -O] k-CH2- (j is an integer ranging from 1 to 4, and k is an integer ranging from 1 to 16); -NR x -;-C(O)-NR x -; or -NR x -C(O)-,

[0059] In the formula, each R x and R y are independently H or a C1-C4 alkyl group. The structure is either of the following:

[0060] In some embodiments, each L 1 and / or L 2 The groups are independently of the formula (VIA): TIFF0007743561000010.tif41170

[0061] (In the formula,

[0062] f is 0 or an integer ranging from 1 to 24;

[0063] j is an integer ranging from 1 to 24,

[0064] R 8 is a bond or O, S, -N(R c )(R d ), or -N + (R c )(R d )(R e ) and

[0065] R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or -N(R c )(R d ) and

[0066] R c , R d , and R e are independently selected from H or a C1-C4 alkyl group;

[0067] R 9and R 10 are independently a bond or a group having up to 6 carbon atoms and including a carbonyl, amide, imide, ester, ether, amine, thione, or thiol. It has.

[0068] In another aspect of the present disclosure, there is provided a method for detecting one or more targets in a biological sample, comprising: (a) labeling the sample with a primary antibody specific for one of the targets in the biological sample; and (b) labeling the sample with a primary antibody having a structure represented by formula (IXC) or (IXD): TIFF0007743561000011.tif66170

[0069] (In the formula,

[0070] Ab is the secondary antibody

[0071] R z is a detectable label,

[0072] o is an integer ranging from 1 to 10;

[0073] L 1 and L 2 is the linker,

[0074] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin;

[0075] Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0076] wherein m and n are independently integers ranging from 1 to 4; and

[0077] (c) contacting the sample with one or more reagents to identify a detectable label R z and detecting The method includes:

[0078] In some embodiments, the Ab is an anti-primary antibody. In some embodiments, the primary antibody is conjugated to a hapten and the Ab is an anti-hapten antibody.

[0079] Another aspect of the present disclosure is a method for detecting one or more targets in a biological sample, comprising: (a) labeling the sample with an enzyme by contacting the sample with a conjugate comprising the enzyme and a primary antibody specific for one of the targets in the biological sample; and (b) contacting the sample with a conjugate comprising a primary antibody of formula (IA) or (IB): TIFF0007743561000012.tif45170

[0080] (In the formula,

[0081] A is an enzyme-reactive moiety,

[0082] B is a detectable label,

[0083] L 1 and L 2 is the linker,

[0084] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin; Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0085] wherein m and n are independently integers ranging from 1 to 4; and

[0086] (c) contacting the sample with one or more reagents to detect detectable label B; The method includes:

[0087] In some embodiments, a compound of either Formula (IA) or (IB) has an extinction coefficient of at least 10,000 M-1 cm-1. In some embodiments, a compound of either Formula (IA) or (IB) has an extinction coefficient of at least 15,000 M-1 cm-1. In still other embodiments, a compound of either Formula (IA) or (IB) has an extinction coefficient of at least 20,000 M-1 cm-1. In some embodiments, a compound of either Formula (IA) or (IB) has an extinction coefficient of at least 25,000 M-1 cm-1.

[0088] In some embodiments, W is of formula (VC) or (VD): TIFF0007743561000013.tif94170

[0089] (In the formula,

[0090] R 2 , R 3 , R 4 , R 6 , and R 7 is a substituted or unsubstituted straight-chain or branched C1-C6 alkyl group; a substituted or unsubstituted straight-chain or branched C1-C6 alkoxy group; a substituted or unsubstituted straight-chain or branched C1-C6 heteroalkyl group; a trifluoromethyl group; a hydroxyl group; a sulfate group; a cyano group; a halogen; a phosphate group; a sugar; a carboxylic acid group; a nitro group; -C(O)NR x R y ;-SR x ;-SO2;-SO2Cl;-SO3H;-SO4H;-SO2NR x R y ;-N(H)-NR x R y ;-NR x R y are independently selected from

[0091] R 5 is C1~C 10 a substituted or unsubstituted branched or unbranched alkyl group, —C(O)—O—, —C(O)—N(H)—;

[0092] R 1 is C1~C 10 substituted or unsubstituted branched or unbranched alkyl groups, -O-, -O-CH2-, -N(R x )-, or -S-,

[0093] Each X is independently a bond or a substituted or unsubstituted straight or branched chain C1-C 16 Alkyl group; -[(CH2) j -O] k -CH2- (j is an integer ranging from 1 to 4, and k is an integer ranging from 1 to 16); -NR x -;-C(O)-NR x -; or -NR x -C(O)-,

[0094] In the formula, each R x and R y are independently H or a C1-C4 alkyl group. The structure is either of the following: [Brief explanation of the drawings]

[0095] For a general understanding of the features of the present disclosure, reference is made to the drawings, wherein like reference numerals are used throughout to identify identical elements.

[0096] [Figure 1] FIG. 1 shows the staining of a sample with a coumarin-based compound conjugated to a nucleic acid probe, which is linked to digoxigenin ("DIG"), which can be detected by application of an appropriate detection reagent, as known in the art.

[0097] [Figure 2]Figure 2 shows the staining of samples with a goat anti-rabbit antibody and a coumarin-based conjugate containing the DIG hapten. Two tonsil tissue samples were labeled with a primary antibody specific for PD-L1, followed by the introduction of a coumarin-based conjugate to label the PD-L1 in the samples with DIG. DIG was then detected by the introduction of an anti-DIG antibody conjugated to peroxidase, which reacts with the introduced 3,3'-diaminobenzidine ("DAB").

[0098] [Figure 3] Figure 3 shows the staining of samples with goat anti-rabbit antibodies and a coumarin-based conjugate containing the DIG hapten. Here, two tonsil tissue samples were labeled with primary antibodies specific for the estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67. The coumarin-based conjugate was then introduced to label the targets within the samples with DIG. DIG was then detected by the introduction of an anti-DIG antibody conjugated to peroxidase, which reacts with the introduced 3,3'-diaminobenzidine ("DAB").

[0099] [Figure 4] FIG. 4 shows a flow chart illustrating the steps of a multiplex detection strategy according to some embodiments.

[0100] [Figure 5] Figure 5 shows the use of a coumarin-based moiety of any of formulas (IIIA), (IIIB), (IIIC), and (IIID) conjugated to a tyramide moiety and a hapten, such as DIG, and compares the staining performance with a tyramide moiety conjugated to DIG.

[0101] [Figure 6] Figure 6 provides a scheme for covalent immobilization of proteins on an AR2G biosensor followed by analyte binding. After activation, immobilization, and quenching, the kinetics of association and dissociation between the immobilized ligand and the analyte are measured.

[0102] [Figure 7] Figure 7 shows a representative cross-reactivity screen of the association and dissociation steps of the mouse anti-hapten antibodies shown above with the BSA-CL-BF immunogen. Recognition of MS anti-BF mAbs (H1, H6), MS anti-PPT mAbs (C1, C6), and MS anti-DABSYL mAbs (D1, D6) is shown. No significant interaction with anti-PPT or anti-DABSYL mAbs was observed.

[0103] [Figure 8] Figure 8 shows a representative cross-reactivity screen of the association and dissociation steps of mouse anti-hapten antibodies with the BSA-CL-BF immunogen. MS anti-NP mAb (B1, B6; C1, C6) and MS anti-TS mAb (H1, H6; G1, G6) BSA-CLBF recognition is shown at the top.

[0104] [Figure 9] Figure 9 shows a representative cross-reactivity screen of the association and dissociation steps of the mouse anti-hapten antibodies shown above with the BSA-CL-BF immunogen. MS anti-NCA mAb (B1, B6; C1, C6) and MS anti-HQ mAb (H1, H6; G1, G6) BSA-CLBF recognition is shown above.

[0105] [Figure 10] Figure 10 shows a representative cross-reactivity screen of the association and dissociation steps of the mouse anti-hapten antibodies shown above with the BSA-CL-BF immunogen. MS anti-DCC mAb (B1, B6; D1, D6) and MS anti-ROT mAb (F1, F6; G1, G6) BSA-CLBF recognition is shown above.

[0106] [Figure 11] Figure 11 shows a representative cross-reactivity screen of the association and dissociation steps of the mouse anti-hapten antibodies shown above with the BSA-CL-BF immunogen. MS anti-DIG mAb (B1, B6; C1, C6) and MS anti-DNP mAb (F1, F6; G1, G6) BSA-CLBF recognition is shown above.

[0107] [Figure 12] Figure 12 shows a representative cross-reactivity screen of the association and dissociation steps of the mouse anti-hapten antibodies shown above with the BSA-CL-BF immunogen. MS anti-BD mAb (C1, C6; D1, D6) and MS anti-DNP mAb (F1, F6; G1, G6) BSA-CLBF recognition is shown above.

[0108] [Figure 13] Figure 13 shows the branched chromogenic linkers GAR-lys(DCC)dPEGTS (Compound A) and GAR-dPEG4-lys(DCC)dPEGTS (Compound B).

[0109] [Figure 14] Figure 14 shows the BLI ELISA assay profiles performed with Rb mAb immobilization (600-1800 s), GAR-Rb recognition (2400-4200 s), and anti-TS label recognition (6000-7800 s). GAR-lys(DCC)dPEG8TS conjugate (D1, C1), GAR-dPEG4-lys(DCC)dPEG8TS conjugate (F1, E1), and unmodified GAR pAb (G1, H1) recognition are shown.

[0110] [Figure 15] Figure 15 shows BLI ELISA assay profiles demonstrating GAR-Rb recognition (2400–4200 s) and subsequent dissociation (4200–6000 s) for GAR-lys(DCC)dPEG8TS conjugates (C1, C6; D1, D6), GAR-dPEG4-lys(DCC)dPEG8TS conjugates (F1, F6; E1, E6), and unmodified GAR pAb (H1, H6; G1, G6).

[0111] [Figure 16]Figure 16 shows BLI ELISA assay profiles shown for MS anti-TS mAb recognition of TS hapten-labeled (6000-7800 s) and subsequent Ab dissociation (7800-8800 s) GAR-lys(DCC)dPEG8TS conjugates (C1, C8; D1, D8), GAR-dPEG4-lys(DCC)dPEG8TS conjugates (E1, E8; F1, F8), and unmodified GAR pAb (G1, G8; H1, H8).

[0112] [Figure 17] Figure 17 shows the BLI Elisa assay profiles performed with Rb mAb immobilization (600-1800 seconds), GAR-Rb recognition (2400-4200 seconds), and anti-BF label recognition (6000-7800 seconds). GAR-dPEG8BF conjugates (G1, B1) and GAR-CLBF conjugates (yellow, teal) are shown. A negative control sensor without Rb mAb immobilization is shown (E1, A1).

[0113] [Figure 18] Figure 18 shows BLI Elisa assay profiles demonstrating GAR-Rb recognition (2400-4200 s) and subsequent dissociation (4200-6000 s) for GAR-dPEG8BF conjugates (B1, B6; G1, G6) and GAR-CLBF conjugates (A1, A8; E1, E8).

[0114] [Figure 19] Figure 19 shows BLI Elisa assay profiles shown for MS anti-BF mAb recognition of BF hapten-labeled (6000-7800 s) and subsequent Ab dissociation (7800-8800 s) GAR-dPEG8BF conjugates (B1, B8; G1, G8) and GAR-CLBF conjugates (A1, A8; E1, E8).

[0115] [Figure 20]FIG. 20 shows examples of labeling reagents for biological proteins, including, but not limited to, maleimides, NHS esters, hydrazides, and ethylcarbodiamides.

[0116] [Figure 21] FIG. 21 shows the absorbance spectra of coumarin-based conjugates of the present disclosure.

[0117] [Figure 22] FIG. 22 shows the absorbance spectra of coumarin-based conjugates of the present disclosure.

[0118] [Figure 23] FIG. 23 shows a table providing the ratio of the absorbance spectra at wavelengths of 280 nm and 346 nm.

[0119] [Figure 24] FIG. 24 shows size exclusion chromatography spectra of the three coumarin-based conjugates of FIG. 23 according to the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0120] It is also to be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0121] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprising" is defined inclusively, such that "including A or B" means including A, B, or A and B.

[0122] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., the inclusion of at least one of a number or list of elements, but including a plurality, and, where appropriate, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0123] Terms such as "comprises," "includes," and "having" are used interchangeably and have the same meaning. Similarly, terms such as "comprises," "includes," and "having" are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprises," and therefore is to be interpreted as an open term meaning "at least the following," and is not intended to exclude additional features, limitations, embodiments, etc. Thus, for example, "an apparatus having components a, b, and c" means that the apparatus comprises at least components a, b, and c. Similarly, the phrase "a method comprising steps a, b, and c" means that the method includes at least steps a, b, and c. Furthermore, although steps and processes may be outlined in a particular order herein, those skilled in the art will recognize that the ordering of steps and processes may vary.

[0124] As used in the specification and claims herein, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more elements of the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related to those elements specifically identified or not, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally including more than one A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); etc.

[0125] "Analog" or "derivative" is used according to its plain and ordinary meaning in chemistry and biology to refer to a compound that is structurally similar to another compound (i.e., a so-called "reference" compound) but differs in composition, for example, by the replacement of an atom with an atom of a different element, or by the presence of a particular functional group, or by the replacement of one functional group with another, or by the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, an analog is a compound that is similar or equivalent in function and appearance but not similar in structure or origin to the reference compound.

[0126] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, alkyl, alkyl group, alkyl radical, alkyl radicals, alkyl alkoxy ... 10 "Alkyl" refers to straight-chain (i.e., unbranched) or branched-chain, or combinations thereof, which can include di- and polyvalent radicals having 1 to 10 carbons (where "-" means 1 to 10 carbons). "Alkyl" is not cyclized. Examples of alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are those containing one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (—O—).

[0127] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from alkyl, exemplified, but not limited to, -CHCHCHCH-. Typically, an alkyl (or alkylene) group has from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms.

[0128] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable linear or branched chain, or combination thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyls are not cyclized. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive, e.g., -CH2-NH-OCH3.

[0129] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl," respectively. Cycloalkyls and heterocycloalkyls are not aromatic.

[0130] Each of the above terms (eg, "alkyl," "heteroalkyl," "cycloalkyl," etc.) includes both substituted and unsubstituted forms of the indicated radical.

[0131] Substituents for alkyl and heteroalkyl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to, -OR', -O, -NR', -N-OR', -NR'R-SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -C and m′ can be one or more of a variety of groups selected from: —OR′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)R′, —NR—C(NR′R″R′″)—NR″″, —NR—C(NR′R″)—NR′″, —S(O)R′, —S(O)R′, —S(O)NR′R″, —NRSO R′, —CN, and —NO, where m′ is the total number of carbon atoms in such radical. R′, R″, R′″, and R′″ each preferably independently represent hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 to 3 halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxy group, or arylalkyl group. When a compound of the invention includes multiple R groups, for example, each of the R groups is independently selected, as are each R', R", R'", and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those of skill in the art will understand that the term "alkyl" is meant to include groups that include carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.).

[0132] As used herein, the term "antibody" refers to an immunoglobulin or immunoglobulin-like molecule, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response of any vertebrate (e.g., in mammals such as humans, goats, rabbits, and mice), as well as antibody fragments that specifically bind to a molecule of interest (or a group of closely related molecules of interest) to the substantial exclusion of binding to other molecules. Antibody also refers to a polypeptide ligand comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. An antibody may be composed of heavy and light chains, each of which has a variable region referred to as the variable heavy (VH) region and the variable light (VL) region. Collectively, the VH and VL regions are responsible for binding the antigen recognized by the antibody. The term antibody also includes intact immunoglobulins, as well as variants and portions thereof that are well known in the art.

[0133] As used herein, the terms "biological sample," "tissue sample," "specimen," and the like refer to any sample containing biomolecules (such as proteins, peptides, nucleic acids, lipids, carbohydrates, or combinations thereof) obtained from any organism, including viruses. Other examples of organisms include mammals (such as humans, veterinary animals such as cats, dogs, horses, cows, and pigs, and laboratory animals such as mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections or needle biopsies of tissue), cell samples (such as cytological smears, such as Pap smears or blood smears, or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (obtained by lysing cells and separating their components, such as by centrifugation). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucous membranes, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical or needle biopsy), nipple aspirate, earwax, milk, vaginal fluid, saliva, swabs (such as cheek swabs), or any material containing biomolecules derived from an initial biological sample. In certain embodiments, the term "biological sample," as used herein, refers to a sample prepared from a tumor or portion thereof obtained from a subject (such as a homogenized or liquefied sample).

[0134] As used herein, the term "conjugate" refers to two or more molecules or moieties (including polymeric or supramolecular molecules) that are covalently linked into a larger construct. In some embodiments, a conjugate comprises one or more biomolecules (such as peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently linked to one or more other molecular moieties.

[0135] The term "coumarin" as used herein refers to a fluorescent derivatizing agent. As used herein, coumarin includes, for example, halo-substituted coumarins (e.g., chlorocoumarins, fluorocoumarins, bromocoumarins and their derivatives), hydroxycoumarins and their derivatives (including umbelliferone and its derivatives), cyanocoumarins and their derivatives, methylcoumarins and their derivatives, ethoxycoumarins and their derivatives, benzocoumarins and their derivatives, phenylcoumarins and their derivatives, acetylcoumarins and their derivatives), and their carboxylated derivatives and derivatives such as succinimidyl esters. The aforementioned derivatives are non-limiting and are provided merely as examples.

[0136] As used herein, the term "bond" or "bonded" refers to the joining, bonding (e.g., covalent bonding), or linking of one molecule or atom to another molecule or atom.

[0137] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0138] As used herein, a "hapten" is a small molecule that can specifically bind to an antibody but is typically substantially incapable of being immunogenic except in combination with a carrier molecule.

[0139] The term "heteroatom," as used herein, is meant to include boron (B), oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

[0140] As used herein, a "lower substituent" or "lower substituent group" means a group selected from all of the substituents described above for "substituent", wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5- to 7-membered heterocycloalkyl.

[0141] As used herein, the term "primary antibody" refers to an antibody that specifically binds to a target protein antigen in a tissue sample. A primary antibody is typically the first antibody used in immunohistochemistry.

[0142] As used herein, the term "reactive group" or "reactive functional group" means a functional group that can chemically react with a functional group of a different moiety to form a covalent bond.

[0143] As used herein, the term "secondary antibody" herein refers to an antibody that specifically binds to a primary antibody, thereby forming a bridge, if any, between the primary antibody and subsequent reagents (e.g., labels, enzymes, etc.). A secondary antibody is a second antibody typically used in immunohistochemistry.

[0144] As used herein, the term "specific binding entity" refers to a member of a specific binding pair. A specific binding pair is a pair of molecules characterized by binding to each other to the substantial exclusion of binding to other molecules (e.g., a specific binding pair can have a binding constant at least 10^3 M-1 greater, 10^4 M-1 greater, or 10^5 M-1 greater than the binding constant of either of the two members of the binding pair to other molecules in a biological sample). Specific examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidins such as streptavidin, and protein A). A specific binding moiety can also include a molecule (or portion thereof) that is specifically bound by such a specific binding protein.

[0145] As used herein, the terms "staining," "staining," and the like generally refer to any treatment of a biological specimen to detect and / or differentiate the presence, location, and / or amount (e.g., concentration) of a specific molecule (e.g., lipid, protein, or nucleic acid) or a specific structure (e.g., normal or malignant cells, cytosol, nucleus, Golgi apparatus, or cytoskeleton) in the biological specimen. For example, staining can provide contrast between a specific molecule or specific cellular structure and the surrounding area of ​​the biological specimen, and the intensity of the staining can provide a measure of the amount of a specific molecule in the specimen. Staining can be used to aid in the observation of molecules, cellular structures, and organisms using not only brightfield microscopes but also other observation tools such as phase-contrast microscopes, electron microscopes, and fluorescence microscopes. Some staining performed by the system can be used to visualize cell contours. Other staining performed by the system may depend on the specific cellular component (e.g., molecule or structure) being stained, with no or relatively little staining of other cellular components. Examples of types of staining methods performed by the system include, but are not limited to, histochemical methods, immunohistochemical methods, and other methods based on reactions between molecules (including non-covalent interactions), such as hybridization reactions between nucleic acid molecules. Specific staining methods include, but are not limited to, primary staining methods (e.g., H&E staining, Pap staining, etc.), enzyme-linked immunohistochemical methods, and in situ RNA and DNA hybridization methods, such as fluorescent in situ hybridization (FISH).

[0146] As used herein, the term "target" refers to any molecule whose presence, location, and / or concentration is or can be determined. Examples of target molecules include proteins, nucleic acid sequences, and haptens, such as haptens covalently bound to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label.

[0147] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0100] The headings provided herein are for convenience only and do not interpret the scope or meaning of the disclosed embodiments.

[0148] Coumarin-based reagents

[0149] As described in further detail herein, the present disclosure provides novel coumarin-based reagents, e.g., linkers, and conjugates comprising one or more of the disclosed coumarin-based reagents. In some embodiments, the presence of a coumarin moiety within a coumarin-based reagent allows for the detection of labels that are typically difficult to detect, such as specific haptens. In some embodiments, difficult-to-detect haptens include benzofuran haptens and thiazole sulfonamide haptens. In some embodiments, difficult-to-detect haptens include 5-nitro-3-pyrazolecarbamide (NP), 2-acetamido-4-methyl-5-thiazolesulfonamide (TS), 7-(diethylamino)-2-oxo-2H-chromene-3-carboxyylic acid (DCC), and 2,1,3-benzoxadiazole-5-carbamide (BF). Antibody denaturation and / or aggregation are generally believed to cause background UV / VIS absorption in the analyzed range for these difficult-to-detect labels. As a result, conjugates with difficult to detect hapten labels are generally overestimated in hapten loading, leading to an underestimation of conjugate concentration. The use of coumarin-based reagents disclosed herein is believed to alleviate this problem.

[0150] In some embodiments, the coumarin-based reagents of the present disclosure have an extinction coefficient of at least 10,000 M-1 cm-1. In other embodiments, the coumarin-based reagents of the present disclosure have an extinction coefficient of at least 15,000 M-1 cm-1. In still other embodiments, the coumarin-based reagents of the present disclosure have an extinction coefficient of at least 20,000 M-1 cm-1. In further embodiments, the coumarin-based reagents of the present disclosure have an extinction coefficient of at least 25,000 M-1 cm-1. In some embodiments, the coumarin-based compounds of the present disclosure have an absorbance wavelength of about 340 nm to about 500 nm.

[0151] In some embodiments, the coumarin-based reagents of the present disclosure have formula (IA) or (IB): TIFF0007743561000014.tif44170

[0152] (In the formula,

[0153] A and B are independently a reactive functional group, a detectable label, or an enzyme-reactive moiety;

[0154] L 1 and L 2 is the linker,

[0155] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin;

[0156] Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0157] m and n are independently integers ranging from 1 to 4. It has the following structure.

[0158] In some embodiments, m and n are independently 1 or 2. In some embodiments, at least one of m or n is 1. In some embodiments, both m and n are 1. In some embodiments, one of m or n is 1 and the other of m or n is 2.

[0159] In some embodiments, both A and B are reactive functional groups. An example of such a compound is provided herein as compound (42). Such compounds can be useful, for example, as cross-linking agents, e.g., to cross-link two antibodies or proteins to one another.

[0160] Groups A, B, L 1 , L 2 , W, m, and n are each defined in more detail herein.

[0161] In some embodiments, the coumarin-based reagents of the present disclosure have formula (IIA) or (IIB): TIFF0007743561000015.tif43170

[0162] (In the formula,

[0163] A 1 is a reactive functional group,

[0164] B 1 is a detectable label,

[0165] L 1 and L 2 is the linker,

[0166] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin;

[0167] Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0168] m and n are independently integers ranging from 1 to 4. It has the following structure.

[0169] In some embodiments, the detectable moiety is a hapten. In other embodiments, the detectable moiety is an enzyme. In other embodiments, the detectable moiety is a chromogen. In further embodiments, the detectable moiety is a fluorophore. Specific examples of each of these types of detectable labels are disclosed herein.

[0170] In some embodiments, the compounds of formula (IIA) and (IIB) can be conjugated to another macromolecule, drug, or biomolecule. For example, the reactive functional group may be capable of reacting with an amine group on a protein or antibody to form a conjugate of the protein or antibody with a coumarin-based reagent. Other conjugation methods are contemplated and disclosed herein. In some embodiments, such conjugates can be utilized as tissue labeling or staining reagents.

[0171] In some embodiments, the coumarin-based reagents of the present disclosure have formula (IIC) or (IID): TIFF0007743561000016.tif36170

[0172] (In the formula,

[0173] A 1 is a reactive functional group,

[0174] B 2 is a hapten,

[0175] L 1 and L 2 is the linker,

[0176] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin;

[0177] Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0178] m and n are independently integers ranging from 1 to 4. It has the following structure.

[0179] In some embodiments, the hapten is oxazole, pyrazole, thiazole, benzofurazan, triterpene, urea, thiourea other than rhodamine thiourea, nitroaryl other than dinitrophenyl or trinitrophenyl, rotenoid, cyclolignan, heterobiaryl, azoaryl, or benzodiazepine. In other embodiments, the hapten is benzofurazan or thiazole sulfonamide. In some embodiments, the hapten is 5-nitro-3-pyrazole carbamide, 2-acetamido-4-methyl-5-thiazole sulfonamide, 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid, and 2,1,3-benzoxadiazole-5-carbamide. Other suitable haptens are disclosed herein. In some embodiments, A 1 comprises a maleimide moiety or an NHS-ester group. 1 or L 2 At least one of L 1 or L 2 At least one of the groups comprises at least one dPEG group.

[0180] Non-limiting examples of compounds having formula (IIC) or (IID) are presented herein as compounds (16), (24), (34), (54), and (66).

[0181] In some embodiments, the compounds of formula (IIC) and (IID) can be conjugated to another macromolecule, drug, or biomolecule. For example, the reactive functional group may react with the amine group of a protein or antibody to form a conjugate of the protein or antibody with a coumarin-based reagent. Such a conjugate can be used as a tissue labeling or staining reagent.

[0182] In some embodiments, the coumarin-based reagents of the present disclosure have the formula (IIIA) or (IIIB): TIFF0007743561000017.tif43170

[0183] (In the formula,

[0184] A 2 is the enzyme-reactive moiety,

[0185] B 1 is a detectable label,

[0186] L 1 and L 2 is the linker,

[0187] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin;

[0188] Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0189] m and n are independently integers ranging from 1 to 4. It has the following structure.

[0190] an enzyme-reactive moiety (including a tyramide moiety, a moiety that is a derivative of a tyramide moiety, and a quinone methide precursor moiety), a detectable label, L 1 , L 2 , W, Z, m, and n are each described in further detail herein.

[0191] In some embodiments, A 2 is a moiety comprising a tyramide moiety or a tyramide derivative described herein. 2 is a quinone methide precursor moiety as described herein. In some embodiments, B 1 is a hapten. In some embodiments, the hapten is an oxazole, pyrazole, thiazole, benzofurazan, triterpene, urea, thiourea other than rhodamine thiourea, nitroaryl other than dinitrophenyl or trinitrophenyl, rotenoid, cyclolignan, heterobiaryl, azoaryl, or benzodiazepine. In other embodiments, B 1 is a chromogen. In yet another embodiment, B 1 is a fluorophore. In some embodiments, L 1 or L 2 At least one of L 1 or L 2 At least one of the groups comprises at least one dPEG group.

[0192] In some embodiments, the compounds of Formula (IIIA) and (IIIB) are suitable for use in detecting targets in biological samples that are labeled with an enzyme, such as a phosphatase or peroxidase. Such detection processes that can be adapted for use with the presently disclosed reagents are described in U.S. Patent Application Publication No. 2017 / 0089911, the disclosure of which is incorporated herein by reference in its entirety.

[0193] In some embodiments, the coumarin-based reagents of the present disclosure have formula (IIIC) or (IIID): TIFF0007743561000018.tif42170

[0194] (In the formula,

[0195] A 2 is the enzyme-reactive moiety,

[0196] B 2 is a hapten,

[0197] L 1 and L 2 is the linker,

[0198] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin;

[0199] Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0200] m and n are independently integers ranging from 1 to 4. It has the following structure.

[0201] As described above, the enzyme-reactive moiety (including tyramide moieties, moieties that are derivatives of tyramide moieties, and quinone methide precursor moieties), detectable label, L 1 , L 2 Each of W, Z, m, and n is described in further detail herein. In some embodiments, the hapten is oxazole, pyrazole, thiazole, benzofurazan, triterpene, urea, thiourea other than rhodamine thiourea, nitroaryl other than dinitrophenyl or trinitrophenyl, rotenoid, cyclolignan, heterobiaryl, azoaryl, or benzodiazepine. In other embodiments, the hapten is benzofurazan or thiazole sulfonamide. In some embodiments, the hapten is 5-nitro-3-pyrazolecarbamide, 2-acetamido-4-methyl-5-thiazolesulfonamide, 7-(diethylamino)-2-oxo-2H-chromene-3-carboxyylic acid, and 2,1,3-benzoxadiazole-5-carbamide. In some embodiments, L 1 or L 2 At least one of L 1 or L2 At least one of the groups comprises at least one dPEG group.

[0202] Non-limiting examples of compounds having formula (IIIC) or (IIID) are presented herein as compounds (17), (25), and (35).

[0203] In some embodiments, the compounds of formula (IIIC) and (IIID) are suitable for use in detecting targets in biological samples that are labeled with an enzyme, such as a phosphatase or peroxidase, as described herein.

[0204] In some embodiments, the coumarin-based reagents of the present disclosure have formula (IIIE) or (IIIF): TIFF0007743561000019.tif43170

[0205] (In the formula,

[0206] A 2 is the enzyme-reactive moiety,

[0207] B 3 is a reactive group capable of participating in a click chemistry reaction,

[0208] L 1 and L 2 is the linker,

[0209] W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin;

[0210] Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group;

[0211] m and n are independently integers ranging from 1 to 4. It has the following structure.

[0212] In some embodiments, B 3 is a DBCO group. In another embodiment, B 3 is an azide group. Still other suitable functional groups capable of participating in click chemistry reactions are disclosed herein. In some embodiments, L 1 or L 2 At least one of L 1 or L 2 At least one of the groups comprises at least one dPEG group.

[0213] In some embodiments, compounds of formula (IIIE) and (IIIF) are suitable for use in detecting targets in biological samples labeled with an enzyme, e.g., a phosphatase or peroxidase, including embodiments utilizing click conjugates attached to a reporter moiety, such as those described in PCT Publication No. WO / 2018 / 002016, the disclosure of which is incorporated herein by reference in its entirety.

[0214] A substituted or unsubstituted coumarin moiety, or a moiety that is a substituted or unsubstituted derivative or analogue of coumarin.

[0215] In some embodiments, W has formula (IVA) or (IVB): TIFF0007743561000020.tif15170

[0216] (wherein Q is a bond or a substituted or unsubstituted straight or branched chain C1-C 16 Alkyl group, -[(CH2CH2) j -O] k -CH2-, -[(CH2) j -O] k -CH2- (j is an integer ranging from 1 to 4, and k is an integer ranging from 1 to 16); -NR x -;-C(O)-NR x -; or -NR x -C(O)- and R xis H or a C1-C4 alkyl group,

[0217] Y is a substituted or unsubstituted coumarin moiety or a moiety that is a substituted or unsubstituted derivative or analog of coumarin. The structure is either of the following:

[0218] In some embodiments, (i) a substituted or unsubstituted coumarin moiety, or (ii) a moiety that is a substituted or unsubstituted derivative or analog of coumarin, is selected from the group consisting of a substituted or unsubstituted straight or branched chain C1-C6 alkyl group, a substituted or unsubstituted straight or branched chain C1-C6 alkoxy group, a substituted or unsubstituted straight or branched chain C1-C6 heteroalkyl group, a trifluoromethyl group, a hydroxyl group, a sulfate group, a cyano group, a halogen, a phosphate group, a sugar, a carboxylic acid group, a nitro group, or —C(O)NR x R y ;-SR x ;-SO2;-SO2Cl;-SO3H;-SO4H;-SO2NR x R y ;-N(H)-NR x R y ;-NR x R y and wherein R x and R y are independently H or a C1-C4 alkyl group.

[0219] In some embodiments, W has formula (VA) or (VB): TIFF0007743561000021.tif75170

[0220] (Wherein, the portion of formula (VA) is 0, 1, 2, 3, or 4 R t groups, and the compounds of formula (VB) may contain 0, 1, 2, 3, 4, or 5 R t groups, and each R t is a substituted or unsubstituted straight or branched chain C1-C 12 Alkyl groups; substituted or unsubstituted straight or branched chain C1-C12 Alkoxy groups, substituted or unsubstituted, straight or branched chain C1-C 12 Heteroalkyl groups; Trifluoromethyl groups; Hydroxyl groups; Sulfate groups; Cyano groups; Halogens; Phosphate groups; Sugars; Carboxylic acid groups; Nitro groups; -C(O)NR x R y ;-SR x ;-SO2;-SO2Cl;-SO3H;-SO4H;-SO2NR x R y ;-N(H)-NR x R y ;-NR x R y and wherein R x and R y are independently H or a C1-C6 alkyl group,

[0221] The formula (VA) consists of one -R 1 -X- group and one -R 5 -X- group and the moiety of formula (VB) is substituted with one -R 5 substituted with an -X- group,

[0222] R 1 is C1~C 10 substituted or unsubstituted branched or unbranched alkyl groups, -O-, -O-CH2-, -N(R x )-, or -S-,

[0223] R 5 is C1~C 10 a substituted or unsubstituted branched or unbranched alkyl group, —C(O)—O—, —C(O)—N(H)—;

[0224] Each X is independently a bond or a substituted or unsubstituted straight or branched chain C1-C 16 Alkyl group; -[(CH2) j -O] k -CH2- (j is an integer ranging from 1 to 4, and k is an integer ranging from 1 to 16); -NR x -;-C(O)-NR x -; or -NR x-C(O)-(R x is H or a C1-C4 alkyl group. The structure is either of the following:

[0225] In some embodiments, compounds of formula (VA) and (VB) have one R t In some embodiments, the compounds of formula (VA) and (VB) contain two R t Contains a group.

[0226] In some embodiments, R 1 is a C1-C6 branched or unbranched alkyl group. 5 is a C1 to C6 branched or unbranched alkyl group.

[0227] In some embodiments, W has formula (VC) or (VD): TIFF0007743561000022.tif94170

[0228] (In the formula,

[0229] R 2 , R 3 , R 4 , R 6 , and R 7 is a substituted or unsubstituted straight-chain or branched C1-C6 alkyl group; a substituted or unsubstituted straight-chain or branched C1-C6 alkoxy group; a substituted or unsubstituted straight-chain or branched C1-C6 heteroalkyl group; a trifluoromethyl group; a hydroxyl group; a sulfate group; a cyano group; a halogen; a phosphate group; a sugar; a carboxylic acid group; a nitro group; -C(O)NR x R y ;-SR x ;-SO2;-SO2Cl;-SO3H;-SO4H;-SO2NR x R y ;-N(H)-NR x R y ;-NR x R y and wherein Rx and R y are independently H or a C1-C4 alkyl group,

[0230] R 5 is C1~C 10 a substituted or unsubstituted branched or unbranched alkyl group, —C(O)—O—, —C(O)—N(H)—;

[0231] R 1 is C1~C 10 substituted or unsubstituted branched or unbranched alkyl groups, -O-, -O-CH2-, -N(R x )-, or -S-,

[0232] Each X is independently a bond or a substituted or unsubstituted straight or branched chain C1-C 16 Alkyl group; -[(CH2) j -O] k -CH2- (j is an integer ranging from 1 to 4, and k is an integer ranging from 1 to 16); -NR x -;-C(O)-NR x -; or -NR x -C(O)-(R x is H or a C1-C4 alkyl group. The structure is either of the following:

[0233] In some embodiments, R 1 is a C1-C6 branched or unbranched alkyl group. 5 is a C1 to C6 branched or unbranched alkyl group.

[0234] Linker

[0235] In some embodiments, the group L 1 and L 2 and each independently have a molecular weight in the range of about 20 g / mol to about 3000 g / mol. 1 and L 2and each independently have a molecular weight in the range of about 40 g / mol to about 2000 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 50 g / mol to about 1500 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 50 g / mol to about 1250 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 75 g / mol to about 1000 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 100 g / mol to about 800 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 100 g / mol to about 600 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 100 g / mol to about 400 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 100 g / mol to about 200 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 250 g / mol to about 500 g / mol. 1 and L 2 each independently has a molecular weight in the range of about 20 g / mol to about 150 g / mol.

[0236] In some embodiments, the group L 1 and L 2 and each independently have a length in the range of about 0.5 nm to about 150 nm. 1 and L 2 each independently have a length in the range of about 0.5 nm to about 100 nm. 1and L 2 each independently have a length in the range of about 0.5 nm to about 50 nm. In some embodiments, the group L 1 and L 2 and each independently have a length in the range of about 0.5 nm to about 30 nm. 1 and L 2 and each independently have a length of less than about 20 nm. 1 and L 2 each independently have a length of less than about 15 nm.

[0237] In some embodiments, L 1 and L 2 In some embodiments, the groups L 1 and L 2 contains one or more charged groups. For example, in some embodiments, the group L 1 and L 2 may contain one or more quaternary amine groups, pyridinium groups, cyclic urea, or sulfate groups.

[0238] In other embodiments, L 1 and L 2 may comprise a single amino acid residue (e.g., lysine), a peptide, or a polymer, which may be charged or uncharged. In some embodiments, L 1 and L 2 can independently comprise peptides having 2 to 20 amino acids, peptides having 2 to 10 amino acids, peptides having 2 to 8 amino acids, or peptides having 1 to 4 amino acids. By way of example, peptides can comprise lysine-[U] q-lysine, where U represents an amino acid, and q is 0 or an integer ranging from 1 to 18. When q is 1 or greater, U can represent a homogeneous or heterogeneous peptide sequence, i.e., one containing the same or different amino acids. In some embodiments, U is selected from lysine, alanine, arginine, guanine, glutamic acid, or any combination thereof. Suitable charged polymers are described in U.S. Patent Application Publication Nos. 2002 / 0052335, 2004 / 0058446, 2004 / 0197318, and 2004 / 0162235, the disclosures of each of which are incorporated herein by reference in their entirety.

[0239] In some embodiments, L 1 and L groups independently comprise one or more solubilizing groups. In some embodiments, the solubilizing groups comprise polyethylene glycol (PEG) groups (or individual PEGS (dPEG) available from Quanta Biodesign). In some embodiments, L 1 and L2 groups independently comprise from about 2 to about 24 PEG groups. In some embodiments, L 1 and L2 groups independently comprise from about 2 to about 18 PEG groups. In some embodiments, L 1 and L2 groups independently comprise from about 2 to about 12 PEG groups. In some embodiments, L 1 and L2 groups independently comprise from about 2 to about 6 PEG groups. 1 and L groups independently comprise four PEG groups. 1 and L groups independently comprise 8 PEG groups. 1 and L groups independently comprise 12 PEG groups. 1 and L2 groups independently comprise 16 PEG groups. 1 and L2 groups independently contain 24 PEG groups. The incorporation of such alkylene oxide linkers is believed to increase the hydrophilicity of the coumarin-based reagents.

[0240] In some embodiments, each L 1 and L 2 are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 2 to 200 carbon atoms and optionally one or more heteroatoms selected from O, N, or S. In some embodiments, each L 1 and L 2 are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 2 to 120 carbon atoms and optionally one or more heteroatoms selected from O, N, or S. In some embodiments, each L 1 and L 2 are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 2 to 80 carbon atoms and optionally one or more heteroatoms selected from O, N, or S. In some embodiments, each L 1 and L 2 are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 4 to 40 carbon atoms and optionally one or more heteroatoms selected from O, N, or S. In some embodiments, each L 1 and L 2 are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 6 to 20 carbon atoms and optionally one or more heteroatoms selected from O, N, or S.

[0241] In some embodiments, each L 1 and / or L 2 The groups are independently of the formula (VIA): TIFF0007743561000023.tif41170

[0242] (In the formula,

[0243] f is 0 or an integer ranging from 1 to 24;

[0244] j is an integer ranging from 1 to 24,

[0245] R 8 is a bond or O, S, -N(R c )(R d ), or -N + (R c )(R d )(R e ) and

[0246] R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or -N(R c )(R d ) and

[0247] R c , R d , and R e are independently selected from H or a C1-C4 alkyl group;

[0248] R 9 and R 10 are independently a bond or a group having up to 6 carbon atoms and including a carbonyl, amide, imide, ester, ether, amine, thione, or thiol. It has.

[0249] In some embodiments, R a or R b At least one of R is H. In some embodiments, a or R b At least one of is H and f is 1 or 2. In some embodiments, R a or R b is H, f is 1, and s is at least 2. In some embodiments, R a and R b is H and R 8is a bond. In some embodiments, the linker of formula (VIA) contains 2 to 40 carbon atoms. In some embodiments, the linker of formula (VIA) contains 4 to 20 carbon atoms. In some embodiments, the group L 1 and L 2 and each independently have a molecular weight in the range of about 100 g / mol to about 600 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 100 g / mol to about 400 g / mol. 1 and L 2 each independently has a molecular weight in the range of about 100 g / mol to about 200 g / mol.

[0250] In some embodiments, each L 1 and / or L 2 The groups are independently of formula (VIB): TIFF0007743561000024.tif39170

[0251] (wherein f is an integer ranging from 1 to 12;

[0252] R 8 is a bond or O, S, -N(R c )(R d ), or -N + (R c )(R d )(R e ) and

[0253] R c , R d , and R e are independently CH3 or H.

[0254] R 9 and R 10 is independently a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, or thiol;

[0255] j is an integer ranging from 1 to 24. It has.

[0256] In some embodiments, f is 1 and s is at least 2. In some embodiments, R 8 is a bond, f is 1, and j is 2 to 16. In another embodiment, R 8 is a bond, f is 1, and j is 2 to 12. In another embodiment, R 8 is a bond, f is 1, and j is 2 to 8.

[0257] In some embodiments, the linker of formula (VIB) contains 2 to 40 carbon atoms. In some embodiments, the linker of formula (VIB) contains 4 to 20 carbon atoms. In some embodiments, the group L 1 and L 2 and each independently have a molecular weight in the range of about 100 g / mol to about 600 g / mol. 1 and L 2 and each independently have a molecular weight in the range of about 100 g / mol to about 400 g / mol. 1 and L 2 each independently has a molecular weight in the range of about 100 g / mol to about 200 g / mol.

[0258] In some embodiments, each L 1 and / or L 2 The groups are independently of the formula (VIC): TIFF0007743561000025.tif39170

[0259] (wherein f is 0 or an integer ranging from 1 to 12;

[0260] j is an integer ranging from 1 to 24,

[0261] R 9 and R 10are independently a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, or thiol. It has.

[0262] In some embodiments, f is 1 and j is at least 2. In some embodiments, f is 1 and j is 2. In some embodiments, f is 1 and j is 3. In some embodiments, f is 1 and j is 4. In some embodiments, f is 2 and j is an integer ranging from 2 to 16. In some embodiments, f is 2 and j is an integer ranging from 2 to 12. In some embodiments, f is 2 and j is an integer ranging from 2 to 8. In some embodiments, f is 2 and j is at least 2. In some embodiments, f is 2 and j is at least 3. In some embodiments, f is 2 and j is at least 4.

[0263] Those skilled in the art will appreciate that when m and n in formula (IA) and / or (IIB) are greater than 1, multiple L 1 or L 2 groups may be bonded, and each L 1 or L 2 It will be understood that the groups may be different. For example, when m is 2, the group -[L 1 ]-[L 1 ]-Each L 1 may be the same or different. For example, the groups -[L 1 ]-[L 1 ]-One of the L 1 contains repeating alkylene oxide groups, and the group -[L 1 ]-[L 1 ]- the other L 1 unsubstituted C1 to C 10 As another example, the group -[L 1 ]-[L 1 ]-One of the L 1 contains at least one PEG or dPEG group, and 1 ]-[L 1 ]- the other L 1can include a lysine residue. 1 ]-[L 1 ]-One of the L 1 contains at least one PEG or dPEG group, and 1 ]-[L 1 ]- the other L 1 may contain lysine residues, and L 2 The group may include at least one PEG or dPEG group.

[0264] reactive functional groups

[0265] In general, a reactive functional group can be any group that facilitates the bonding of two molecules together to form an adduct. In some embodiments, the reactive functional group is one that can participate in nucleophilic substitution (e.g., reactions of amines and alcohols with acyl halides, activated esters), electrophilic substitution (e.g., enamine reactions), and addition to multiple carbon-carbon and carbon-heteroatom bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are reviewed, for example, in March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney, et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982.

[0266] In some embodiments, the reactive functional group is a carboxylic acid, an activated ester of a carboxylic acid, a carbodiimide, a sulfonyl halide, an acyl halide, a silyl halide, an acyl azide, an acyl nitrile, an acrylamide, an amine, an aldehyde, an alkyl halide (the halide may be subsequently displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, a thiol anion, a carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom), an aryl halide, an alkyl sulfonate, or an alkyl hydroxyl group. Examples of reactive functional groups include aryls, sulfonates, anhydrides, azides, aziridines, diazoalkanes, haloacetamides, halotriazines, hydrazines, hydroxylamines, isocyanates, isothiocyanates, maleimides, phosphoramidates, thiols (which in some embodiments can be converted to disulfides, reacted with acyl halides, or bonded to metals), hydroxyls (which can be converted to esters, ethers, aldehydes, etc.), hydrazines, and alkynes (which can undergo, for example, cycloaddition, acylation, or Michael addition). In some embodiments, the reactive functional group is a carboxyl group or various derivatives thereof, including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters. In some embodiments, the reactive functional group is a dienophile group capable of participating in a Diels-Alder reaction, such as a maleimide group. In some embodiments, the reactive functional group is an aldehyde or ketone group that is capable of subsequent derivatization, for example, via formation of a carbonyl derivative such as an imine, hydrazone, semicarbazone, or oxime, or via mechanisms such as Grignard addition or alkyllithium addition.

[0267] In other embodiments, the reactive functional group is selected from carboxylic acids, activated esters of carboxylic acids, sulfonyl halides, acyl halides, amines, alkyl or aryl halides, anhydrides, azides, haloacetamides, halotriazines, hydrazines, isocyanates, isothiocyanates, maleimides, phosphoramidates, thiols, hydroxyls, and alkynes. In still other embodiments, the reactive functional group is selected from carboxylic acids, activated esters of carboxylic acids, amines, azides, haloacetamides, hydrazines, isocyanates, maleimides, and alkynes.

[0268] In some embodiments, reactive functional groups can be selected so that they do not contribute to or interfere with the chemical stability of the conjugates described herein, or alternatively, reactive functional groups can be protected from participating in crosslinking reactions by the presence of protecting groups.

[0269] In some embodiments, the reactive functional group is a moiety capable of undergoing a "click chemistry" reaction. "Click chemistry" is a chemical philosophy originally defined by the Sharpless and Meldal groups that describes chemistry tailored to rapidly and reliably generate substances by linking small units. "Click chemistry" has been applied to a collection of reliable and autonomous organic reactions (Kolb, H.C. Finn, M.G.; Sharpless, K.B. Angew). Chem. Int. Ed. 2001, 40, 2004-2021). For example, the identification of copper-catalyzed azide-alkyne [3+2] cycloaddition as a reliable molecular coupling in water (Rostovtsev, VV; et al. Angew. Chem. Int. Ed. 2002, 41, 2596-2599) has been used to augment the investigation of several types of biomolecular interactions (Wang, Q.; et al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Speers, AE; et al. J. Am. Chem. Soc. 2003, 125, 4686-4687; Link, AJ; Tirrell, DAJA Am. Chem. Soc. 2003, 125, 11164-11165; Deiters, A.; et al.J.Am.Chem.Soc.2003,125,11782-11783).Furthermore, they have been used in organic synthesis (Lee, LV; et al. J. Am. Chem. Soc. 2003, 125, 9588-9589), drug discovery (Kolb, HC; Sharpless, KB Drug Disc. Today 2003, 8, 1128-1137; Lewis, WG; et al. Angew. Chem. Int. Ed. 2002, 41, 1053-1057), and surface functionalization (Meng, J.-C.; et al. Angew. Chem. Int. Ed. 2004, 43, 1255-1260; Fazio, F.; et al. J. Am. Chem. Soc. 2002, 124, 14397-14402; Collman, JP; et al. Langmuir 2004, ASAP, in Applications of click chemistry to new applications (see press; Lummerstorfer, T.; Hoffmann, H.J. Phys. Chem. B 2004, in press) are also emerging. In general, click chemistry promotes reactions that are broad in scope, have high chemical yields, produce harmless by-products, are chemically specific, require simple reaction conditions, use readily available starting materials and reagents, are solvent-free or use mild solvents (e.g., water) to facilitate easy product isolation, have a large thermodynamic driving force favoring reactions with a single reaction product, and / or have a high atom economy, and have modular applications. While certain general criteria may be subjective in nature, it is not necessary to meet all criteria.

[0270] Those skilled in the art will understand that any of the compounds of formula (IA), (IB), (IIIE), (IIIF) can be terminated with a suitable reactive group (e.g., a DBCO group) that can form a click adduct with another appropriately functionalized compound (e.g., a compound bearing an azide group) (including those described in PCT Publication WO / 2018 / 002015, the disclosure of which is incorporated herein by reference in its entirety). Pairs of reactive groups that can react in a click chemistry reaction are shown in the following table. TIFF0007743561000026.tif86170

[0271] Detectable Label

[0272] Generally, detectable labels include chromogenic, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance to another to provide a detectable difference (such as by converting a colorless substance to a colored substance or vice versa, or by producing a precipitate or increasing the turbidity of the sample), additional detectable-label antibody conjugates, and haptens that can be detected by antibody-hapten binding interactions using paramagnetic and magnetic molecules or materials. Of course, the reporter moiety itself can also be indirectly detected; for example, if the detectable label is a hapten, an additional antibody specific for the detectable label can be utilized to detect the detectable label, as known to those skilled in the art.

[0273] In some embodiments, suitable haptens include, but are not limited to, pyrazoles (e.g., nitropyrazoles), nitrophenyl compounds, benzofurazans, triterpenes, ureas (e.g., phenylureas), thioureas (e.g., phenylthioureas), rotenone and rotenone derivatives, oxazoles (e.g., oxazole sulfonamides), thiazoles (e.g., thiazole sulfonamides), coumarin and coumarin derivatives, and cyclolignans. Additional non-limiting examples of haptens include thiazoles, nitroaryls, benzofurans, triperpenes, and cyclolignans. Specific examples of haptens include dinitrophenyl, biotin, digoxigenin, and fluorescein, as well as any derivatives or analogs thereof. Other suitable haptens are described in U.S. Patent Nos. 8,846,320, 8,618,265, 7,695,929, 8,481,270, and 9,017,954, the disclosures of which are incorporated herein by reference in their entireties. Other suitable haptens are described in U.S. Patent Application Publication Nos. 2013 / 0109019 and 2010 / 0184087, the disclosures of which are incorporated herein by reference in their entireties.

[0274] In some embodiments, suitable fluorophores belong to several general chemical classes, including coumarins, fluoresceins (or fluorescein derivatives and analogs), rhodamines, resorufins, luminophores, and cyanines. Additional examples of fluorescent molecules can be found in "Molecular Probes Handbook - A Guide to Fluorescent Probes and Labeling Technologies," Molecular Probes, Eugene, OR, TheroFisher Scientific, 11th Edition. In other embodiments, the fluorophore is selected from xanthene derivatives, cyanine derivatives, squaraine derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, and tetrapyrrole derivatives.In other embodiments, the fluorescent moiety is selected from the group consisting of CF dyes (available from Biotium), DRAQ and CyTRAK probes (available from BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (e.g., DyLight 649) (available from Thermo Scientific, Pierce), Atto and Tracy (available from Sigma-Aldrich), FluoProbes (available from Interchim), Abberior Dyes (available from Abberior), DY and MegaStokes Dyes (available from Dyomics), Sulfo Cy dyes (available from Cyandye), HiLyte Fluor (available from AnaSpec), Seta, SeTau and Square Dyes (available from SETA BioMedicals), Quasar and Cal Fluor dyes (available from Biosearch Technologies), SureLight Dyes (available from APC, RPEPerCP, Phycobilisomes) (Columbia Biosciences), as well as APC, APCXL, RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen).

[0275] In some embodiments, suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, or β-lactamase. In other embodiments, the enzyme includes an oxidoreductase or peroxidase (e.g., HRP, AP). In these embodiments, the enzyme conjugated to the anti-label antibody catalyzes the conversion of a chromogenic substrate into a reactive moiety that covalently binds to the sample proximal to or directly from the target.

[0276] Specific non-limiting examples of chromogenic compounds / substrates include diaminobenzidine (DAB), 4-nitrophenyl phosphate (pNPP), Fast Red, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), BCIP / NBT, Fast Red, AP Orange, AP Blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulfonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal). These include methylumbelliferyl-β-D-galactopyranoside (MU-Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue, tetrazolium violet, N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCO purple), and rhodamine 110 (rhodamine). DAB, which is oxidized in the presence of peroxidase and hydrogen peroxide, results in the deposition of a brown, alcohol-insoluble precipitate at the site of enzymatic activity. In some embodiments, the chromogenic substrate is a signaling conjugate that includes a latent reactive moiety and a chromogenic moiety.

[0277] In some embodiments, the latent reactive moiety of the signaling conjugate is configured to undergo catalytic activation to form a reactive species that can covalently bond with a sample or other detection component. Catalytic activation is driven by one or more enzymes (e.g., oxidoreductase enzymes and peroxidase enzymes such as horseradish peroxidase) and results in the formation of reactive species. These reactive species can react with a chromogenic moiety proximal to their generation, i.e., near the enzyme. Specific examples of signaling conjugates are described in U.S. Patent Application Publication No. 2013 / 0260379, the disclosure of which is incorporated herein by reference in its entirety.

[0278] Other suitable detectable labels are described in U.S. Patent Application Publication No. 2018 / 002015, the disclosure of which is incorporated herein by reference in its entirety. For example, suitable labels include multi-dye conjugates having at least two chromophores directly or indirectly attached to each other.

[0279] Further examples of detectable labels include, but are not limited to, DAB, AEC; CN; BCIP / NBT; Fast Red; Fast Blue; Fuchsin; NBT; ALK GOLD; Cascade Blue acetyl azide; Dapoxyl sulfonic acid / carboxylic acid succinimidyl ester; DY-405; Alexa Fluor 405 succinimidyl ester; Cascade Yellow succinimidyl ester; Pyridyloxazole succinimidyl ester (PyMPO); Pacific Blue succinimidyl ester; DY-415; 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester; DYQ-425; 6-FAM phosphoramidite; Lucifer Yellow; iodoacetamide; Alexa Fluor 430 succinimidyl ester; Dabcyl succinimidyl ester; NBD chloride / fluoride; QSY 35 succinimidyl ester; DY-485XL; Cy2 succinimidyl ester; DY-490; Oregon Green 488 carboxylic acid succinimidyl ester; Alexa Fluor 488 succinimidyl ester; BODIPY 493 / 503 C3 succinimidyl ester; DY-480XL; BODIPY FL C3 succinimidyl ester; BODIPY FL C5 succinimidyl ester; BODIPY FL-X succinimidyl ester; DYQ-505; Oregon Green 514 carboxylic acid succinimidyl ester; DY-510XL; DY-481XL; 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester (JOE); DY-520XL; DY-521XL; BODIPY R6G C3 succinimidyl ester; erythrosine isothiocyanate; 5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl ester; Alexa Fluor 532 succinimidyl ester; 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein succinimidyl ester (HEX); BODIPY 530 / 550 C3 succinimidyl ester; DY-530; BODIPY TMR-X succinimidyl ester; DY-555; DYQ-1; DY-556; Cy3 succinimidyl ester;DY-547; DY-549; DY-550; Alexa Fluor 555 succinimidyl ester; Alexa Fluor 546 succinimidyl ester; DY-548; BODIPY 558 / 568 C3 succinimidyl ester; Rhodamine Red-X succinimidyl ester; QSY 7 succinimidyl ester; BODIPY 564 / 570 C3 succinimidyl ester; BODIPY 576 / 589 C3 succinimidyl ester; Carboxy-X-rhodamine (ROX); succinimidyl ester; Alexa Fluor 568 succinimidyl ester; DY-590; BODIPY 581 / 591 C3 succinimidyl ester; DY-591; BODIPY TR-X succinimidyl ester; Alexa Fluor 594 succinimidyl ester; DY-594; Carboxynaphthofluorescein succinimidyl ester; DY-605; DY-610; Alexa Fluor 610 succinimidyl ester; DY-615; BODIPY 630 / 650-X succinimidyl ester; Erioglaucine; Alexa Fluor 633 succinimidyl ester; Alexa Fluor 635 succinimidyl ester; DY-634; DY-630; DY-631; DY-632; DY-633; DYQ-2; DY-636; BODIPY 650 / 665-X succinimidyl ester; DY-635; Cy5 succinimidyl ester; Alexa Fluor 647 succinimidyl ester; DY-647; DY-648; DY-650; DY-654; DY-652; DY-649; DY-651; DYQ-660; DYQ-661; Alexa Fluor 660 succinimidyl ester; Cy5.5 succinimidyl ester; DY-677; DY-675; DY-676; DY-678; Alexa Fluor 680 succinimidyl ester; DY-679; DY-680; DY-682; DY-681; DYQ-3; DYQ-700; Alexa Fluor 700 succinimidyl ester; DY-703; DY-701; DY-704; DY-700; DY-730; DY-731; DY-732; DY-734; DY-750; Cy7 succinimidyl ester; DY-749;DYQ-4; and Cy7.5 succinimidyl ester.

[0280] The reporter moiety, such as that attached to the secondary antibody, can be selected from any of the detectable labels described above.

[0281] Enzyme-reactive moiety

[0282] In some embodiments, enzyme-reactive moieties include tyramides, tyramide derivatives, and quinone methide precursors and their derivatives.

[0283] In some embodiments, the enzyme-reactive moiety is tyramide or its derivative or analogue.Suitable tyramide moieties and tyramide derivatives include those described in US Patent Application Publication No. 2012 / 0171668, the disclosure of which is incorporated herein by reference in its entirety.In some embodiments, the tyramide moiety or tyramide derivative is a substrate for an enzyme, for example, a peroxidase enzyme, such as horseradish peroxidase enzyme.

[0284] In some embodiments, the enzyme-reactive moiety has formula (VIIA): TIFF0007743561000027.tif42170 (in the formula, each R 11 groups are independently selected from H or lower alkyl groups having 1 to 4 carbon atoms, and wherein R x is H or a C1-C4 alkyl group) The structure is given by:

[0285] In some embodiments, the enzyme-reactive moiety has formula (VIIB): It has the structure provided by TIFF0007743561000028.tif29170.

[0286] In some embodiments, the enzyme-reactive moiety is a quinone methide precursor or a derivative thereof. Suitable quinone methide precursors or derivatives are described in U.S. Patent Application Publication No. 2017 / 0089911, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the quinone methide precursor or derivative thereof is a substrate for an enzyme, such as a phosphatase or alkaline phosphatase.

[0287] In some embodiments, the enzyme-reactive moiety has formula (VIIIA): TIFF0007743561000029.tif36170 (in the formula, R 2 is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or sugar; R 13 is a halide, R 15 , R 16 , R 17 , and R 18 are independently selected from hydrogen or an aliphatic group having 1 to 4 carbon atoms; R 14 is -(CH2) w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2) w NH-, -C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -, -N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(o)N(H)(CH2CH2O) w -, -(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-, -C(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) wCH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) w NH- or -N(H)(CH2) w NH-, and w is an integer ranging from 1 to 12. The structure is given by:

[0288] In some embodiments, the enzyme-reactive moiety has formula (VIIIB): It has the structure provided by TIFF0007743561000030.tif28170.

[0289] In some embodiments, the enzyme-reactive moiety has formula (VIIIC): TIFF0007743561000031.tif40170

[0290] (In the formula, R 13 -(CH2) w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2) w NH-, C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -, -N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(o)N(H)(CH2CH2O) w -, -(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-, -C(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) wNH- or -N(H)(CH2) w NH-, where w is independently an integer ranging from 1 to 12. In some embodiments, R 7 is C(O)N(H)(CH2) w NH and w is as defined above. In other embodiments, R 7 is C(O)N(H)(CH2) w NH, and w is in the range of 2 to 6.

[0291] In some embodiments, the enzyme-reactive moiety has formula (VIIID): TIFF0007743561000032.tif49170, where w ranges from 1 to 12. In some embodiments, w ranges from 1 to 8. In other embodiments, w ranges from 2 to 8. In still other embodiments, w ranges from 2 to 6. In a further embodiment, w is 6.

[0292] Further Coumarin-Based Reagents

[0293] In some embodiments, the coumarin-based reagent is: TIFF0007743561000033.tif180170 (in the formula, R v is an enzyme-reactive moiety or reactive functional group) It has the following structure.

[0294] In some embodiments, the coumarin-based reagent is: TIFF0007743561000034.tif211170 (in the formula, R v is an enzyme-reactive moiety or reactive functional group) It has the following structure.

[0295] In some embodiments, the coumarin-based reagent is:

[0296] TIFF0007743561000035.tif115170 (in the formula, R vis an enzyme-reactive moiety or reactive functional group) It has the following structure.

[0297] In some embodiments, the coumarin-based reagent is: TIFF0007743561000036.tif148170 (in the formula, R v is an enzyme-reactive moiety or reactive functional group) It has the following structure.

[0298] In some embodiments, the coumarin-based reagent is: TIFF0007743561000037.tif198170 (in the formula, R v is an enzyme-reactive moiety or reactive functional group) It has the following structure.

[0299] In some embodiments, the coumarin-based reagent is: TIFF0007743561000038.tif168170 (in the formula, R v is an enzyme-reactive moiety or reactive functional group) It has the following structure.

[0300] Conjugates containing coumarin-based reagents

[0301] The present disclosure also provides conjugates of coumarin-based reagents with another substituent, such as a polymer, a drug, or a biomolecule. In some embodiments, a coumarin-based reagent of formula (IA), (IB), (IIA), (IIB), (IIC), or (IID) can be reacted with a polymer, a drug, or a biomolecule. In some embodiments, the conjugate is T-[coumarin-based reagent] o where T is a specific binding entity, amino acid, peptide, protein, saccharide, nucleoside, nucleotide, oligonucleotide, drug, lipid, or nanoparticle, and o is an integer ranging from 1 to 10.

[0302] There are various methods that can be used to conjugate coumarin-based reagents to macromolecules, drugs, or biomolecules. For example, to facilitate this conjugation, coumarin-based reagents can be attached to biomolecule reactive groups, such as active ester groups, amino groups, sulfhydryl groups, carbohydrate groups, azide groups, or carboxy groups. There are various methodologies for reacting biomolecule reactive groups with macromolecules or macromolecular fragments. Examples of such methodologies are photocrosslinking and glutaraldehyde crosslinking. Still other methods for achieving such conjugation will be found by those skilled in the art. For examples of such methods, see Hermanson, GT, Bioconjugate Techniques, Elsevier Science, London, 2008.

[0303] The active ester groups of the present invention must be selected so as not to impair the binding of the extended linking group to a protein or polymer. Those skilled in the art will understand that active esters such as N-hydroxysuccinimide or N-hydroxysulfosuccinimide can be used in the present invention. Alternatively, the primary amino group of the extended linking group can be coupled to the primary amino group of the protein with glutaraldehyde. The amino group of the protein can be coupled to the carboxyl group of the extended linking group. Furthermore, the extended linking group can be modified with nitrophenyl azide so that coupling to the protein occurs upon irradiation with visible light. Other methods for achieving such binding will be apparent to those skilled in the art.

[0304] In some embodiments, the conjugate of the present disclosure has formula (IXA) or (IXB): TIFF0007743561000039.tif66170, wherein T is a substituent selected from a specific binding entity, an amino acid, a peptide, a protein, a saccharide, a nucleoside, a nucleotide, an oligonucleotide, a drug, a lipid, or a nanoparticle; R z is a detectable label as defined above, o is an integer ranging from 1 to 10; L 1 and L 2 is the linker, W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin; Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group; m and n are independently integers ranging from 1 to 4. The structure is either of the following:

[0305] In some embodiments, the specific binding entity is an antibody, an antibody fragment, a drug / antibody conjugate, or a nucleic acid. In some embodiments, the antibody is a primary antibody. In other embodiments, the antibody is a secondary antibody.

[0306] In some embodiments, R z is a hapten. In some embodiments, R z is an oxazole, pyrazole, thiazole, benzofurazan, triterpene, urea, thiourea other than rhodamine thiourea, nitroaryl other than dinitrophenyl or trinitrophenyl, rotenoid, cyclolignan, heterobiaryl, azoaryl, benzodiazepine. z is a benzofurazan or a thiazole sulfonamide.

[0307] In other embodiments, R z is an enzyme, e.g., a peroxidase or a phosphatase. z is the fluorophore.

[0308] In some embodiments, the conjugate of the present disclosure has formula (IXC) or (IXD): TIFF0007743561000040.tif66170Ab is an antibody, R z is a detectable label as defined above, o is an integer ranging from 1 to 10; L 1 and L 2 is the linker, W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin; Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group; m and n are independently integers ranging from 1 to 4. The structure is either of the following:

[0309] In some embodiments, the conjugate of the present disclosure has formula (IXE) or (IXF): TIFF0007743561000041.tif65170 (wherein NA is a nucleic acid; R z is a detectable label as defined above, o is an integer ranging from 1 to 10; L 1 and L 2 is the linker, W comprises a moiety that is a substituted or unsubstituted coumarin moiety or a substituted or unsubstituted derivative or analog of coumarin; Z is a bond, a -CH- group, a -CH-CH- group, or a -CH-CH- group; m and n are independently integers ranging from 1 to 4. The structure is either of the following:

[0310] In some embodiments, R z is a hapten. In some embodiments, R z is an oxazole, pyrazole, thiazole, benzofurazan, triterpene, urea, thiourea other than rhodamine thiourea, nitroaryl other than dinitrophenyl or trinitrophenyl, rotenoid, cyclolignan, heterobiaryl, azoaryl, benzodiazepine. z is a benzofurazan or a thiazole sulfonamide.

[0311] In some embodiments, a coumarin-based reagent (e.g., one having formula (IIA) or (IIB)) can be attached to any portion of an antibody. Three functional groups on an antibody are sites of covalent modification: amines (-NH2), thiol groups (-SH), and carbohydrate residues (Shrestha D, et al, 2012). Thus, any of the coumarin-based reagents having at least one reactive functional group disclosed herein can be attached to an amine residue, a thiol residue, a carbohydrate residue, or any combination thereof. In some embodiments, a coumarin-based reagent is attached to the Fc portion of an antibody. In other embodiments, a coumarin-based reagent is attached to the hinge region of an antibody. In some embodiments, a coumarin-based reagent is attached to one or more Fc regions of an antibody and one or more hinge regions of an antibody. Indeed, any combination is contemplated by the present disclosure.

[0312] Amino groups are generally preferred, primarily due to the abundance of these moieties on antibodies. However, the random nature of amino groups poses the risk of inactivating the antibody. (Adamczyk M, et al, 1999, Bioconjug Chem; Jeanson A, et al, 1988, J Immunol Methods; Vira S, et al, 2010, Anal Biochem; Pearson JE et al, 1998, J Immunol Methods). In some embodiments, one or more coumarin-based reagents are coupled to amino groups of the antibody.

[0313] On the other hand, under appropriate reaction conditions, sulfhydryl labeling provides highly specific targeting of the disulfide bond between the two heavy chains of an antibody in the hinge region. Because the hinge region is far from the antigen-binding site, this modification is believed to better preserve the binding affinity of the antibody. In some embodiments, one or more coumarin-based reagents are bound to the thiol groups of the antibody.

[0314] Conjugation to carbohydrate moieties present in the Fc portion of an antibody is similar to conjugation of thiol groups, with modification occurring at -CHO groups away from the antigen-binding site. Again, carbohydrate conjugation is believed to have less of a negative effect on the binding affinity of the antibody. The degree of labeling varies depending on the glycosylation state of a particular antibody. However, loss of antibody affinity has still been reported (Jeanson A, et al., 1988, J Immunol Methods). In some embodiments, one or more coumarin-based reagents are conjugated to carbohydrate groups on the antibody.

[0315] Detection of coumarin-based conjugates

[0316] In some embodiments, the conjugate of any of Formulas (IXA), (IXB), (IXC), and (IXD) comprises a detectable label that facilitates direct detection of the conjugate. For example, when the label of a coumarin-based conjugate comprises a fluorophore or chromophore, the fluorophore or chromophore can be directly detected according to methods known to those skilled in the art.

[0317] In other embodiments, specific reagents are utilized to allow detection of any conjugate of formula (IXA), (IXB), (IXC), (IXD), (IXE), and (IXF), and thus the target in a tissue sample. In some embodiments, a detection reagent specific to the particular detectable label of the conjugate is utilized. In some embodiments, the detection reagent comprises a secondary antibody specific to the label of the conjugate, e.g., the hapten moiety of the conjugate. For example, the secondary antibody can be an anti-label antibody or an anti-hapten antibody that is itself conjugated to a reporter moiety.

[0318] In some embodiments, the secondary antibody or anti-hapten antibody can be conjugated to a "reporter moiety" to achieve detection of conjugates of formula (IXA), (IXB), (IXC), (IXE), and (IXF). Any of the detectable labels described above are suitable for this purpose, i.e., can function as a reporter moiety. In some embodiments, the reporter moiety of the secondary antibody includes chromogenic, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance to another to provide a detectable difference (such as by converting a colorless substance to a colored substance, or vice versa, or by producing a precipitate or increasing the turbidity of the sample), additional detectably labeled antibody conjugates, and haptens that can be detected by antibody-hapten binding interactions using paramagnetic and magnetic molecules or materials. Of course, the detectable label itself can also be indirectly detected; for example, if the detectable label is a hapten, an additional antibody specific for that hapten (e.g., an anti-hapten antibody), as described herein and known to those skilled in the art, can be utilized to detect the detectable label.

[0319] Methods for detecting targets using coumarin-based conjugates of any of formulas (IXA), (IXB), (IXC), and (IXD) and detection reagents

[0320] The present disclosure also provides methods for detecting one or more targets in a biological sample using any of the conjugates of formulae (IXA), (IXB), (IXC), (IXD), (IXE), and (IXF) described herein. In some embodiments, a conjugate of any of formulae (IXA), (IXB), (IXC), (IXD), (IXE), and (IXF) can be used in a simplex assay to directly or indirectly detect a specific target (e.g., a biomarker) in a biological sample (e.g., PD-L1, ER, PR, HER2, CD68, Ki67, CD20, etc.). In some embodiments, the biomarker is a nucleic acid (e.g., DNA, RNA, mRNA, etc.). For example, FIG. 1 illustrates staining with a coumarin-based reagent conjugated to a nucleic acid probe (such as when using conjugates of formulae (IXE) and (IXF)) (the coumarin-based reagent is bound to DIG, which can be detected by application of an appropriate detection reagent known in the art).

[0321] In some embodiments, the conjugate of any of Formulas (IXA), (IXB), (IXC), and (IXD) comprises a primary antibody (e.g., an antibody specific for PD-L1, ER, PR, HER2, CD68, Ki67, CD20, etc.). In these embodiments, the conjugate comprising the primary antibody can be used to directly "label" the target with the conjugate. In other embodiments, the conjugate of any of Formulas (IXA), (IXB), (IXC), and (IXD) comprises a secondary antibody. In these embodiments, and as discussed in more detail herein, the target (e.g., a protein target or a nucleic acid target) can be labeled with a primary antibody (in the case of IHC) or a nucleic acid conjugate (e.g., a nucleic acid sequence bound to a hapten in the case of ISH), and then the primary antibody or nucleic acid conjugate can be "labeled" with a conjugate of any of Formulas (IXA), (IXB), (IXC), and (IXD) comprising a secondary antibody (which can then be detected). These and other embodiments are further described herein.

[0322] As an example, Figures 2 and 3 show a secondary antibody-coumarin-based reagent conjugate used to label a primary antibody deposited on tissue. In these figures, the secondary antibody is an anti-DIG antibody, and the conjugate is conjugated to DIG. DIG can be detected or visualized using an anti-DIG antibody conjugated to an enzyme, followed by the introduction of a chromogen, such as DAB.

[0323] In some embodiments, the coumarin-based conjugate comprises a primary antibody, the coumarin-based primary antibody conjugate being specific to a target of interest, and application of the coumarin-based primary antibody conjugate to a tissue sample results in the formation of a target-coumarin-based primary antibody conjugate complex. Following application of the coumarin-based primary antibody conjugate, a detection reagent (e.g., an anti-label antibody or an anti-hapten antibody) can be subsequently applied so that the target-coumarin-based primary antibody conjugate complex can be detected. In some embodiments, the detection reagent comprises an anti-hapten antibody specific to a particular hapten-detectable label of the coumarin-based primary antibody conjugate, the anti-hapten antibody comprising a reporter moiety. The single target can then be visualized or otherwise detected.

[0324] In other embodiments, a tissue sample is first contacted with a primary antibody or nucleic acid probe to form either a target-primary antibody complex or a target-nucleic acid probe complex. Subsequently, a coumarin-based conjugate containing a secondary antibody is introduced into the tissue sample, where the secondary antibody portion of the coumarin-based conjugate is specific for either (i) the primary antibody, (ii) a label conjugated to the primary antibody, or (iii) a label conjugated to the nucleic acid probe. Application of the coumarin-based secondary antibody conjugate allows the formation of a secondary complex, thereby "labeling" the target. Following application of the coumarin-based secondary antibody conjugate and formation of the secondary complex, a detection reagent (e.g., an anti-label antibody, an anti-hapten antibody) can be applied so that the secondary complex can be detected. In some embodiments, the detection reagent includes an anti-hapten antibody specific for a particular label of the coumarin-based secondary antibody conjugate, where the anti-hapten antibody includes a reporter moiety. The target can then be visualized or otherwise detected via the bound reporter moiety.

[0325] In some embodiments of the present disclosure, a method for multiplex detection, including automated multiplex detection, is provided. Figure 4 provides a flow chart showing one method for multiplex detection of targets, in which a tissue sample is simultaneously contacted with multiple coumarin-based conjugates (step 100), each coumarin-based conjugate being specific for a particular target, and each coumarin-based conjugate containing a different detectable label. Figure 4 shows the application of coumarin-based conjugates, and those skilled in the art will understand that the coumarin-based conjugates can include coumarin-based nucleic acid conjugates, coumarin-based primary antibody conjugates, and coumarin-based secondary antibody conjugates, depending on the target in the sample (e.g., nucleic acid sequence, protein target recognized by primary antibody coumarin-based conjugate, or pre-deposited primary antibody recognized by secondary antibody coumarin-based conjugate).

[0326] In some embodiments, the sample can be contacted with two coumarin-based conjugates, each specific for a particular target and each containing a different detectable label. In other embodiments, the sample can be contacted with three coumarin-based conjugates, each specific for a particular target and each containing a different detectable label.

[0327] Coumarin-based conjugates (such as those of formula (IXA) or (IXB)) can be applied to tissue samples as a "pool" or "cocktail" containing each coumarin-based conjugate required for a particular assay. Pooling of coumarin-based conjugates is believed possible because the coumarin-based conjugates are not believed to cross-reactive with each other, at least to the extent that cross-reactivity would interfere with staining performance. Each coumarin-based conjugate binds to its respective target, forming a detectable target-coumarin-based conjugate complex. In some embodiments, and following application of the coumarin-based conjugates, a blocking step is performed.

[0328] Following simultaneous application of the coumarin-based conjugates (step 100), multiple detection reagents are simultaneously applied to the tissue sample (step 110), each detection reagent facilitating detection of one of the initially applied coumarin-based conjugates (step 100), and each detection reagent contains a different detectable label. In other embodiments, the detection reagent is a secondary antibody specific for the detectable label of the coumarin-based conjugate (e.g., an anti-label antibody specific for the label of the coumarin-based conjugate). In embodiments in which anti-hapten antibodies are used, the anti-hapten antibodies can be provided to the tissue sample as a pool or cocktail containing each of the anti-hapten antibodies necessary for detection of the target-coumarin-based antibody conjugate complex. Following application of the detection reagents, in some embodiments, the tissue sample can be stained with a counterstain. The signals from each of the reporter moieties can be visualized or otherwise detected (e.g., can be simultaneously visualized or detected).

[0329] An example of a multiplex assay utilizing coumarin-based conjugates is as follows: A first coumarin-based antibody conjugate containing a first detectable label and specific for a first target (e.g., specific for one of PD-L1, ER, PR, HER2, CD68, Ki67, CD20, etc.) is introduced into a tissue sample. In some embodiments, the first coumarin-based antibody conjugate forms a detectable first target-coumarin-based antibody conjugate complex. Simultaneously, a second coumarin-based antibody conjugate containing a detectable label and specific for a second target (e.g., another one of PD-L1, ER, PR, HER2, CD68, Ki67, CD20, etc.) is introduced into the sample to form a second target-coumarin-based antibody conjugate complex. Third, fourth, and subsequent coumarin-based antibody conjugates specific for other targets (forming "n" target detection probe complexes) and bearing different detectable labels are then introduced into the sample. 第 n additional coumarin-based conjugates are further introduced simultaneously with the first and second coumarin-based conjugates.

[0330] After the coumarin-based antibody conjugates are deposited, they can be detected directly or indirectly, depending, of course, on their composition. In some embodiments, anti-label antibodies are introduced to allow detection of each of the target-coumarin-based antibody conjugate complexes. In some embodiments, the anti-label antibodies are specific for different reporter moieties of the coumarin-based conjugate, and each anti-label antibody is conjugated to a different detectable label. In some embodiments, the detectable reagents are each anti-label antibodies conjugated to a fluorophore. In some embodiments, the first, second, and 第 n anti-labeled antibodies are introduced simultaneously, and the first, second, and 第 Each of the n detection reagents is specific for a different coumarin-based conjugate, and each of the anti-label antibodies is conjugated to a fluorophore. 第 n anti-labeled antibodies are introduced sequentially, the first, second, and 第 Each of the n detection reagents is specific for a different coumarin-based conjugate, and each of the anti-label antibodies is conjugated to an enzyme.

[0331] As a further example of a multiplex assay according to the present disclosure, a first coumarin-based antibody conjugate specific for a first target (e.g., PD-L1, ER, PR, HER2) is introduced into a tissue sample, where the first coumarin-based antibody bears a first detectable label. In some embodiments, the first coumarin-based antibody conjugate forms a detectable first target-coumarin-based antibody conjugate complex. Simultaneously or subsequently, a second coumarin-based antibody conjugate specific for a second target (e.g., PD-L1, ER, PR, HER2) is introduced into the sample to form a second target-coumarin-based antibody conjugate complex, where the second coumarin-based antibody conjugate bears a second detectable label. Third, fourth, and 第The n additional coumarin-based antibody conjugates, each specific for a different target (forming "n" target-coumarin-based antibody conjugate complexes), may be further introduced sequentially or simultaneously with the first and / or second coumarin-based antibody conjugate, and may be introduced sequentially with the third, fourth, and 第 Each of the n coumarin-based antibody conjugates further comprises a different detectable label. The coumarin-based antibody conjugates can be detected after deposition. In some embodiments, an additional detection reagent is introduced to enable detection of the target, including those described herein (e.g., chromogenic detection reagents). In some embodiments, the first, second, and 第 n detection reagents are introduced sequentially, first, second, and 第 Each of the n detection reagents comprises (i) a secondary antibody, i.e., an anti-label antibody, specific for each of the detectable labels of the coumarin-based antibody conjugates, and, if the secondary antibody is conjugated to an enzyme, (ii) a chromogenic substrate, 第 Each of the n chromogenic substrates is different.

[0332] In yet another embodiment, the multiplex detection method includes the steps of: (i) contacting the biological sample with a first coumarin-based antibody conjugate to form a first target-coumarin-based antibody conjugate complex; (ii) contacting the biological sample with a first labeled conjugate, the first labeled conjugate comprising a first enzyme (the first labeled conjugate is an anti-label antibody that specifically binds to the first coumarin-based antibody conjugate and is configured to label the target with the enzyme); (iii) contacting the biological sample with a first signaling conjugate comprising a first latently reactive moiety and a first chromogenic moiety (see, e.g., U.S. Patent Application No. 13 / 849,160, the disclosure of which is incorporated herein by reference for its description of signaling conjugates and their components); and (iv) inactivating the first enzyme, such as by contacting the sample with a first enzyme inactivation composition to substantially inactivate or completely inactivate the first enzyme contained in the biological sample.

[0333] After the first enzyme is inactivated (optionally), the multiplex method includes the steps of: (v) contacting the biological sample with a second coumarin-based antibody conjugate to form a second target coumarin-based antibody conjugate; (vi) contacting the biological sample with a second labeled conjugate, the second labeled conjugate comprising a second enzyme (the second labeled conjugate is an anti-label antibody that specifically binds to the second coumarin-based antibody conjugate and is configured to enzymatically label the target); (vii) contacting the biological sample with a second signaling conjugate comprising a second latently reactive moiety and a second chromogenic moiety; and (viii) contacting the sample with a first enzyme inactivating composition to inactivate the second enzyme, such as by substantially inactivating or completely inactivating the first enzyme contained in the biological sample.

[0334] After the second enzyme is inactivated, the method can be repeated so that additional coumarin-based antibody conjugates can be introduced along with additional detection reagents to achieve detection of other targets. Following the introduction of all coumarin-based antibody conjugates (and other detection probes) and their respective detection reagents or kits, the method can include steps of counterstaining the sample and / or detecting the first, second, and third coumarin-based antibody conjugates. 第 detecting (manually or via automated methods) a signal from the n chromogenic moieties, n Each of the chromogenic moieties is different. Alternatively, each of the coumarin-based antibody conjugates can be added simultaneously or sequentially, but before adding the labeled conjugate. As another example, three coumarin-based antibody conjugates can be applied sequentially first before introducing any detection reagent, and then each detection reagent can be added sequentially.

[0335] In the context of a multiplex assay in which multiple targets are detected sequentially and detection employs the use of enzymes, it is desirable to inactivate any reagents or endogenous enzymes between successive detection steps. As a result, it is believed that the enzymes present in any one detection step do not interfere with the enzymes of subsequent detection steps. This, in turn, is believed to improve visualization and detection of the different detectable moieties used in the multiplex assay. Any enzyme inactivation composition known in the art can be used for this purpose. In some embodiments, an enzyme inactivation composition is applied to inactivate reagents or endogenous enzymes after each detection step. Exemplary enzyme inactivation compositions are disclosed in U.S. Patent Application Publication No. 2018 / 0120202, the disclosure of which is incorporated herein by reference in its entirety.

[0336] In some embodiments, the denaturing step prevents the enzyme used in the first set of detection reagents from acting on the second substrate. In some embodiments, the denaturant is a substance that denatures the enzyme in the first set of detection reagents. In some embodiments, the denaturant is, for example, formamide, alkyl-substituted amide, urea or urea-based denaturants, thiourea, guanidine hydrochloride, or a derivative thereof. Examples of alkyl-substituted amides include, but are not limited to, N-propylformamide, N-butylformamide, N-isobutylformamide, and N,N-dipropylformamide. In some embodiments, the denaturant is provided in a buffer. For example, formamide is prepared in a solution of 20 mM dextran sulfate (50-57% formamide (UltraPure formamide stock solution), 2x SSC (20x SSC stock solution containing 0.3 M citrate and 3 M NaCl), 2.5 mM EDTA (0.5 M EDTA stock solution), 5 mM Tris, pH 7.4 (1 mM Tris, pH 7.4 (stock solution), 0.05% Brij-35 (10% stock solution containing polyoxyethylene (23) lauryl ether), pH 7.4. In some embodiments, the sample is treated with a denaturing agent for a period of time under conditions sufficient to denature the first target probe detection enzyme, e.g., alkaline phosphatase. In some embodiments, the sample is treated with the denaturing agent at about 37°C for about 15 to about 30 minutes, preferably about 20 to 24 minutes. In some embodiments, the sample is treated with the denaturing agent for a period of time and under conditions sufficient to denature the target enzyme while maintaining hybridization of the second nucleic acid probe to the target.

[0337] In these embodiments using an anti-labeled antibody conjugated to an enzyme, suitable conditions are used to introduce the signaling conjugate or chromogenic substrate with the biological sample, typically including providing a reaction buffer or solution containing a peroxide (e.g., hydrogen peroxide) and having a suitable salt concentration and pH to allow or promote the enzyme to perform its desired function. Generally, this step of the method is performed at a temperature ranging from about 35°C to about 40°C, although one of skill in the art can select an appropriate temperature range appropriate for the selected enzyme and signaling conjugate. For example, it is believed that these conditions allow the enzyme and peroxide to react and promote radical formation at the latent reactive moiety of the signaling conjugate. The latent reactive moiety, and therefore the signaling conjugate as a whole, is covalently deposited in the biological sample, particularly at one or more of the proximal tyrosine residues of the immobilized enzyme conjugate, the tyrosine residues of the enzyme portion of the enzyme conjugate, and / or the tyrosine residues of the antibody portion of the enzyme conjugate. The biological sample can then be illuminated with light, and the target can be detected by the absorbance of light produced by the chromogenic moiety of the signaling conjugate.

[0338] Methods for detecting conjugates of any of formulae (IXA), (IXB), (IXC), (IXD), (IXE), and (IXF) in combination with other specific binding entities

[0339] In some embodiments of the present disclosure, conjugates of any of formulae (IXA), (IXB), (IXC), and (IXD) are used in combination with other specific binding entities to provide multiplexed detection of targets in tissue samples. Those skilled in the art will appreciate that any of the above-identified methods and procedures can be appropriately adapted to any assay using both conjugates of any of formulae (IXA), (IXB), (IXC), and (IXD) and other specific binding entities. In some embodiments, the other specific binding entities include nucleic acids for in situ hybridization and unmodified antibodies for IHC. As used herein, the terms "unmodified antibody" or "unmodified antibody" refer to antibodies that do not include the coumarin-based reagents disclosed herein, but include antibodies conjugated to a hapten or another label. Essentially, "unmodified antibodies" are native antibodies traditionally used in IHC assays, specific for a particular target (e.g., anti-CD3 antibodies), and can be detected with an anti-species secondary antibody, or, if they contain a label, an anti-labeled antibody, etc. For example, a rabbit anti-CD3 antibody can be detected with a goat anti-rabbit antibody. Similarly, a rabbit anti-CD3 antibody bound to a hapten can be detected with an anti-hapten antibody. For example, an unmodified antibody specific for ER can be introduced into a sample and subsequently detected, while a coumarin-based conjugate of the present disclosure specific for PR can be introduced into the same sample simultaneously or sequentially and then detected.

[0340] Methods for detecting a target in a sample using coumarin-based reagents of formula (IIIA), (IIIB), (IIIC), and (IIID)

[0341] The present disclosure also provides methods for detecting one or more targets in a tissue sample using coumarin-based reagents, such as those compounds of formula (IIIA), (IIIB), (IIIC), and (IIID).

[0342] In some embodiments, compounds of Formulae (IIIA), (IIIB), (IIIC), and (IIID) having an enzyme-reactive moiety (e.g., when "A" is a tyramide derivative or a quinone methide derivative) and a detectable label (e.g., when "B" is a hapten) are contacted with a target-binding enzyme (e.g., an antibody-enzyme conjugate or a nucleic acid-enzyme conjugate already deposited in a biological sample) to generate a reactive intermediate. For example, an antibody-peroxidase conjugate may be deposited on a target, and the peroxidase enzyme may react with the enzyme-reactive moiety of any compound of Formulae (IIIA), (IIIB), (IIIC), and (IIID) upon introduction into a sample. As another example, a nucleic acid-phosphatase conjugate may hybridize to a target in a sample, and the conjugated phosphatase enzyme may react with the enzyme-reactive moiety of any compound of Formulae (IIIA), (IIIB), (IIIC), and (IIID) upon introduction into a sample.

[0343] In some embodiments, the reactive intermediate forms a covalent bond to a nucleophile on or within the biological sample to provide an immobilized tissue-coumarin-based compound complex. The detectable label of the compound of formula (IIIA), (IIIB), (IIIC), and (IIID) allows visualization of the tissue-coumarin-based compound complex. For example, if the detectable label is a hapten, an anti-hapten antibody (e.g., a chromogen or fluorophore) conjugated to a reporter can be introduced to detect the tissue-coumarin-based compound complex. Detection and visualization methods using compounds with enzyme-reactive moieties are described in U.S. Patent Application Publication Nos. 2017 / 0089911 and 2012 / 0171668, the disclosures of which are incorporated herein by reference in their entireties.

[0344] Figure 5 shows the use of a coumarin-based moiety of any of formulas (IIIA), (IIIB), (IIIC), and (IIID) conjugated to a tyramide moiety and a hapten, such as DIG, and compares the staining performance with a tyramide moiety conjugated to DIG.

[0345] Methods for detecting a target in a sample using click conjugates of formula (IIIE) and (IIIF)

[0346] Reagents of formula (IIIE) and (IIIF) can be used to detect targets in samples using specific click conjugates. In some embodiments, a reagent having formula (IIIE) or (IIIF), which includes an enzyme-reactive moiety and further includes a functional group capable of participating in a click chemistry reaction, is contacted with a target-binding enzyme (e.g., the target-binding enzyme can be an antibody-enzyme conjugate already deposited in a biological sample) to generate a reactive intermediate. In some embodiments, the reactive intermediate forms a covalent bond to a nucleophile on or within the biological sample to provide an immobilized tissue-coumarin-based compound complex. The immobilized tissue click conjugate complex can then react with a click conjugate that includes a reporter moiety and a functional group capable of participating in a click chemistry reaction, provided that the click conjugate and the immobilized tissue click conjugate complex have reactive functional groups capable of reacting with each other to form a covalent bond. The reaction product of the immobilized tissue click conjugate complex and the click conjugate generates an immobilized tissue click adduct complex. The tissue click adduct complex can be detected by a signal transmitted from the linked reporter moiety. Useful click conjugates for reacting with immobilized tissue click conjugate complexes include those described in PCT Publication No. WO / 2018 / 002016, the disclosure of which is incorporated herein by reference in its entirety. Further details of detection methods are also disclosed in PCT Publication No. WO / 2018 / 002016, the disclosures of which are each incorporated herein by reference in their entirety.

[0347] Synthesis of Coumarin-Based Reagents

[0348] Methods for synthesizing various types of coumarin-based reagents, such as reagents of either formula (IA) or (IB), are described below.

[0349] Synthesis of 2,5-dioxopyrrolidin-1-yl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate

[0350] 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic acid (Sigma-Aldrich, 500 mg, 2.27 mmol) was dissolved in 10 mL of dry DMF, and DSC (641 mg, 2.50 mmol) and DMAP (416 mg, 3.40 mmol) were added to the solution. The solution was stirred at room temperature (rt) for 1 h and then diluted with 50 mL of DCM. The solution was washed three times with 50 mL of water and once with 50 mL of brine, then concentrated in vacuo and purified by silica gel chromatography (2% isopropanol / DCM to 10% isopropanol / DCM) to give 632 mg (86% yield) of 2,5-dioxopyrrolidin-1-yl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (Compound 10). TIFF0007743561000042.tif27170

[0351] Synthesis of tert-butyl (1-(7-hydroxy-2-oxo-2H-chromen-3-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamate

[0352] In this example, a linker group is introduced into the coumarin moiety.

[0353] 2,5-Dioxopyrrolidin-1-yl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (500 mg, 1.65 mmol) was dissolved in 10 mL of DCM. To the solution was added tert-butyl (3-(2-(3-aminopropoxy)ethoxy)propyl)carbamate (547 mg, 1.98 mmol) and triethylamine (334 mg, 3.3 mmol). The reaction was stirred at room temperature for 16 hours. Upon completion, the reaction was concentrated under reduced pressure and purified by silica gel chromatography (2% methanol / DCM to 10% methanol / DCM) to afford 688 mg (82% yield) of tert-butyl (1-(7-hydroxy-2-oxo-2H-chromen-3-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamate (Compound 11) as a yellow oil. TIFF0007743561000043.tif24170

[0354] Synthesis of tert-butyl 2-((3-((2,2-dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetate

[0355] tert-Butyl (1-(7-hydroxy-2-oxo-2H-chromen-3-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamate (407 mg, 0.80 mmol) was dissolved in 10 mL of dry DMF, and to the solution was added tert-butyl 2-bromoacetate (172 mg, 0.884 mmol) and potassium carbonate (330 mg, 2.41 mmol), and the reaction was stirred in an oil bath at 60° C. After 3 hours, the reaction was diluted with 70 mL of DCM, and the solution was washed three times with 100 mL of water and once with 100 mL of brine. The solution was concentrated in vacuo and then purified by silica gel chromatography (2% methanol / DCM to 12% methanol / DCM) to give 398 mg (80% yield) of tert-butyl 2-((3-((2,2-dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetate (compound 12). TIFF0007743561000044.tif23170

[0356] Synthesis of 2-((3-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid

[0357] tert-Butyl 2-((3-((2,2-dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetate (398 mg, 0.64 mmol) was dissolved in 20 mL of 35% TFA / DCM and stirred at room temperature until the reaction was complete by HPLC (approximately 4 hours). The reaction was then concentrated in vacuo, azeotroped three times with toluene, and dried overnight under high vacuum to give 372 mg of 2,2,2-trifluoroacetic acid compound along with 2-((3-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (compound 13) (1:1), which was used without further purification. TIFF0007743561000045.tif25170

[0358] Synthesis of 2-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid

[0359] In this example, a hapten moiety is introduced.

[0360] 2,5-Dioxopyrrolidin-1-yl 7-(2-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)acetamido)heptanoate (90 mg, 0.1 mL) in 3 mL of dry DMF To a solution of 2-((3-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid 2,2,2-trifluoroacetate (1:1) (120 mg, 0.205 mmol) and Hunig's base (82 μL, 0.480 mmol) was added and the reaction was stirred at room temperature overnight. The crude reaction was filtered through a 0.2 micron filter and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 116 mg (83% yield) of 2((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (Compound 14) as an off-white powder. TIFF0007743561000046.tif36170

[0361] Synthesis of 1-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oic acid

[0362] In this example, a second linker is introduced into the coumarin moiety.

[0363] To a solution of 2-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (130 mg, 0.129 mmol) in 10 ml of dry DCM was added N-hydroxysuccinimide (19 mg, 0.167 mmol) and 1.0 M HCl in DCM. DCC (167 μL, 0.167 mmol) was added, and the reaction was stirred at room temperature until formation of the active ester was complete (approximately 4 h) as determined by HPLC. The urea by-product was removed by filtration, and 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (44 mg, 0.167 mmol) and Hunig's base (66 μL, 0.167 mmol) were added to the solution, and the reaction was stirred at room temperature overnight. The reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 125 mg (76% yield) of 1-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oic acid (compound 15) as an off-white powder. TIFF0007743561000047.tif39170

[0364] Synthesis of 2,5-dioxopyrrolidin-1-yl 1-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate

[0365] In this example, the starting material is derivatized to allow the resulting reagent to be conjugated to a macromolecule such as a protein or antibody.

[0366] 1-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trimethyl-1H-cyclopenta[a]phenanthren-3-yl)oxy) in 10 ml of dry DCM To a solution of (oxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (25 mg, 0.02 mmol), N-hydroxysuccinimide (2.3 mg, 0.03 mmol) and 1.0 M DCC (30 μL, 0.03 mmol) in DCM were added, and the reaction was stirred at room temperature until formation of the active ester was complete (approximately 4 h) as determined by HPLC. The urea byproduct was removed by filtration, and the reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 22 mg (80% yield) of 2,5-dioxopyrrolidin-1-yl 1-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate (Compound 16) as an off-white powder. TIFF0007743561000048.tif25170

[0367] Synthesis of 4-hydroxyphenylethyl 1-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate

[0368] In this example, an enzyme-reactive moiety, specifically a tyramide moiety, is introduced.

[0369] 2,5-Dioxopyrrolidin-1-yl 1-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa- To a solution of (3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oate (20 mg, 0.015 mmol) was added tyramine (4.1 mg, 0.030 mmol) and triethylamine (4.2 μL, 0.030 mmol), and the reaction was stirred at room temperature overnight. The crude reaction was filtered through a 0.2 micron filter and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 17 mg (81% yield) of 4-hydroxyphenylethyl 1-((3-((1-(((3S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2,10-dioxo-15,18,21-trioxa-3,11-diazatetracosan-24-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate (compound 17) as an off-white powder. TIFF0007743561000049.tif25170

[0370] Synthesis of 2-((3-((2,2-dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid

[0371] To a solution of the 2,2,2-trifluoroacetate salt of 2-((3-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (1:1) (8.03 g, 13.83 mmol) in 50 ml of dioxane / water (1:1) was added (Boc)O (3.02 g, 13.83 mmol) and triethylamine (4.38 ml, 31.46 mmol) and the reaction was stirred at room temperature overnight. The reaction was diluted with 100 ml of DCM and washed twice with 50 ml of water and once with brine. The organic phase was dried by filtering through a plug of MgSO4 and then concentrated before purification by silica gel chromatography (4% methanol / DCM to 15% methanol / DCM) to give 6.48 g (83% yield) of 2-((3-((2,2-dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (compound 20). TIFF0007743561000050.tif28170

[0372] Synthesis of 1-((3-((2,2-dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid

[0373] To a solution of 2-((3-((2,2-dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (6.48 g, 11.4 mmol) in 40 ml of dry DCM was added 1.0 M DCC in DCM (17.16 ml, 17.16 mmol) and NHS (1.97 g, 17.16 mmol), and the reaction was stirred at room temperature until ester formation was complete (5 hours). The urea by-product was removed by filtration, and to the solution was added 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (4.55 g, 17.16 mmol) and Hunig's base (2.22 g, 17.16 mmol), and the reaction was stirred at room temperature overnight. The reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 7.42 g (80% yield) of 1-((3-((2,2-dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18)-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (Compound 21). TIFF0007743561000051.tif26170

[0374] Synthesis of 1-((3-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)-propyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oic acid (1:1)

[0375] 1-((3-((2,2-Dimethyl-4-oxo-3,9,12,15-tetraoxa-5-azaoctadecan-18-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (7.42 g, 9.12 mmol) was dissolved in 50 ml of 35% TFA / DCM and stirred at room temperature until the reaction was complete by HPLC (approximately 4 hours). The reaction was then concentrated in vacuo, azeotroped three times with toluene, and dried under high vacuum overnight to give 7.92 g of the 2,2,2-trifluoroacetate salt of 1-((3-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (compound 22) (1:1) as a pale yellow oil. TIFF0007743561000052.tif37170

[0376] Synthesis of 1-((3-((1-(benzo[c][1,2,5]oxadiazol-5-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oic acid

[0377] To a solution of benzo[c][1,2,5]oxadiazole-5-carboxylic acid (13 mg, 0.079 mmol) in 5 mL of dry DCM was added 1.0 M DCC in DCM (100 μl, 0.10 mmol) and N-hydroxysuccinimide (12 mg, 0.10 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 4 hours). The urea by-product was removed by filtration, and the solution was

[0378] The 2,2,2-trifluoroacetic acid salt of 1-((3-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (1:1) (82.8 mg, 0.10 mmol) and triethylamine (30 mg, 0.30 mmol) were added and the reaction was stirred at room temperature overnight. The reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 42 mg (62% yield) of 1-((3-((1-(benzo[c][1,2,5]oxadiazol-5-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oic acid (compound 23). TIFF0007743561000053.tif25170

[0379] Synthesis of 2,5-dioxopyrrolidin-1-yl 1-((3-((1-(benzo[c][1,2,5]oxadiazol-5-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate

[0380] To a solution of 1-((3-((1-(benzo[c][1,2,5]oxadiazol-5-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (42 mg, 0.049 mmol) in 10 mL of dry DCM was added 1.0 M DCC in DCM (60 μL, 0.06 mmol) and N-hydroxysuccinimide (12 mg, 0.06 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 4 h). The urea by-product was removed by filtration, and the reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 38 mg (81% yield) of 2,5-dioxopyrrolidin-1-yl 1-((3-((1-(benzo[c][1,2,5]oxadiazol-5-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate (compound 24). TIFF0007743561000054.tif24170

[0381] Synthesis of N-(1-(7-((21-(4-hydroxyphenyl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazahenicosyl)oxy)-2-oxo-2H-chromen-3-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)benzo[c][1,2,5]oxadiazole-5-carboxamide

[0382] To a solution of benzo[c][1,2,5]oxadiazole-5-carboxylic acid (13 mg, 0.079 mmol) in 5 ml of dry DCM was added 1.0 M DCC in DCM (100 μl, 0.10 mmol) and N-hydroxysuccinimide (12 mg, 0.10 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 4 hours). The urea by-product was removed by filtration, and to the solution was added N-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-7-((21-(4-hydroxyphenyl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazahenicosyl)oxy)-2-oxo-2H-chromene-3-carboxamide 2,2,2-trifluoroacetate (94.7 mg, 0.10 mmol) and triethylamine (30 mg, 0.30 mmol), and the reaction was stirred at room temperature overnight. The reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 40 mg (52% yield) of N-(1-(7-((21-(4-hydroxyphenyl)-2,18-dioxo-6,9,12,15-tetraoxa)-3,19-diazahenicosyl)oxy)-2-oxo-2H-chromen-3-yl)-1-oxo-6,9,12-trioxa-2-azapentadecan-15-yl)benzo[c][1,2,5]oxadiazole-5-carboxamide (compound 25). TIFF0007743561000055.tif25170

[0383] Synthesis of tert-butyl 3-(2-acetamido-5-methylthiazole-4-sulfonamido)propanoate

[0384] To a solution of 2-acetamido-5-methylthiazole-4-sulfonyl chloride (1.01 g, 3.97 mmol) in 30 mL of dry DCM was added tert-butyl 3-aminopropanoate (1.44 g, 7.94 mmol) and triethylamine (2.21 mL, 15.88 mmol), and the reaction was stirred at room temperature for 1 hour. The reaction was concentrated to approximately 5 mL and then purified by silica gel chromatography (1% methanol / DCM to 15% methanol / DCM) to afford 1.33 g (92% yield) of tert-butyl 3-(2-acetamido-5-methylthiazole)-4-sulfonamido)propanoate (Compound 30). TIFF0007743561000056.tif26170

[0385] Synthesis of 3-(2-acetamido-5-methylthiazole-4-sulfonamido)propanoic acid

[0386] tert-Butyl 3-(2-acetamido-5-methylthiazole-4-sulfonamido)propanoate (1.33 g, 3.65 mmol) was taken up in 30 ml of 30% TFA / DCM and the reaction was stirred at room temperature until the reaction was complete by HPLC (approximately 3 h). The reaction was then concentrated in vacuo, azeotroped three times with toluene, and dried under high vacuum overnight to give 3-(2-acetamido-5-methylthiazole-4-sulfonamido)propanoic acid (compound 31), which was used without further purification. TIFF0007743561000057.tif32170

[0387] Synthesis of 2-((3-((17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl-2-oxo-2H-chromen-7-yl)oxy)acetic acid

[0388] To a solution of 3-(2-acetamido-5-methylthiazole-4-sulfonamido)propanoic acid) (325 mg, 1.06 mmol) in 20 mL of dry DCM was added 1.0 M DCC in DCM (1.27 mL, 1.27 mmol) and N-hydroxysuccinimide (146 mg, 1.27 mmol). The solution was stirred at room temperature until the reaction was complete by HPLC (approximately 4 h). The urea by-product was removed by filtration, and 2-((3-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamoyl-2-oxo-2H-chromen-7-yl)oxy)acetic acid 2,2,2-trifluoroacetate (1:1)) (850 mg, 1.46 mmol) and triethylamine (442 μL, 3.17 mmol) were added to the solution, and the reaction was stirred at room temperature. The crude reaction mixture was eluted with 0.05% HCl. The residue was filtered through a 0.2 micron filter and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 627 mg (79% yield) of 2-((3-((17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10)-trioxa-14-azaheptadecyl)carbamoyl-2-oxo-2H-chromen-7-yl)oxy)acetic acid (Compound 32). TIFF0007743561000058.tif32170

[0389] Synthesis of 1-((3-((17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oic acid

[0390] To a solution of 2-((3-((17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl-2-oxo-2H-chromen-7-yl)oxy)acetic acid (142 mg, 0.188 mmol) in 10 ml of dry DCM was added 1.0 M DCC (0.226 μL, 0.226 mmol) and N-hydroxysuccinimide (26 mg, 0.226 mmol) were added, and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 3.5 hours). The urea by-product was removed by filtration, and 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (60 mg, 0.226 mmol) and triethylamine (79 μL, 0.564 mmol) were added to the solution, and the reaction was stirred at room temperature overnight. The reaction was concentrated. The crude product was taken up in minimal methanol and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 158 mg (84% yield) of 1-((3-((17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (compound 33). TIFF0007743561000059.tif25170

[0391] Synthesis of 2,5-dioxopyrrolidin-1-yl 1-((3-((17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl))carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate

[0392] To a solution of 1-((3-((17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (250 mg, 0.216 mmol) in 10 mL of dry DCM was added 1.0 M DCC (275 μL, 0.275 mmol) and N-hydroxysuccinimide (32 mg, 0.275 mmol) in DCM, and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 3 h). The urea byproduct was removed by filtration, and the reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 193 mg (83% yield) of 2,5-dioxopyrrolidin-1-yl 1-((3-((17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate (Compound 34). TIFF0007743561000060.tif25170

[0393] Synthesis of N-(17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)-7-((21-(4-hydroxyphenyl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazahenicosyl)oxy)-2-oxo-2H-chromene-3-carboxamide

[0394] To a solution of 2,5-dioxopyrrolidin-1-yl 1-((3-((17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate (50 mg, 0.045 mmol) in 5 ml of dry DCM was added tyramine (8 mg, 0.059 mmol) and triethylamine (19 μL, 0.135 mmol) and the reaction was stirred at room temperature overnight. The crude reaction was filtered through a 0.2 micron filter and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 43 mg (86% yield) of N-(17-(2-acetamido-5-methylthiazole-4-sulfonamido)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)-7-((21-(4-hydroxyphenyl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazahenicosyl)oxy)-2-oxo-2H-chromene-3-carboxamide (compound 35). TIFF0007743561000061.tif24170

[0395] Synthesis of 2-((3-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid

[0396] To a solution of 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (500 mg, 2.96 mmol) in 20 ml of dry DCM was added 1.0 M DCC in DCM (3.55 ml, 3.55 mmol) and N-hydroxysuccinimide (408 mg, 3.55 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 1 h). The urea by-product was removed by filtration, and to the solution was added 2-((3-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid 2,2,2-trifluoroacetate (1:1) (1.20 g, 2.00 mmol) and triethylamine (700 μL, 5.00 mmol), and the reaction was stirred at room temperature overnight. The crude reaction was filtered through a 0.2 micron filter and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 889 mg (72% yield) of 2-((3-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15)-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (compound 40). TIFF0007743561000062.tif27170

[0397] Synthesis of 1-((3-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oic acid

[0398] To a solution of 2-((3-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (573 mg, 0.928 mmol) in 20 ml of dry DCM was added 1.0 M DCC in DCM (1.20 ml, 0.120 mmol) and N-hydroxysuccinimide (139 mg, 0.126 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 3 hours). The urea by-product was removed by filtration, and 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (369 mg, 1.80 mmol) and triethylamine (388 μL, 0.564 mmol) were added to the solution, and the reaction was stirred at room temperature overnight. The reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 586 mg (73% yield) of 1-((3-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15)-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oic acid (compound 41). TIFF0007743561000063.tif21170

[0399] To a solution of 2-((3-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (573 mg, 0.928 mmol) in 20 ml of dry DCM was added 1.0 M DCC in DCM (1.20 ml, 0.120 mmol) and N-hydroxysuccinimide (139 mg, 0.126 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 3 hours). The urea by-product was removed by filtration, and 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (369 mg, 1.80 mmol) and triethylamine (388 μL, 0.564 mmol) were added to the solution, and the reaction was stirred at room temperature overnight. The reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 586 mg (73% yield) of 1-((3-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15)-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oic acid (compound 41).

[0400] Synthesis of 2,5-dioxopyrrolidin-1-yl 1-((3-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate

[0401] To a solution of 1-((3-((17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (367 mg, 0.424 mmol) in 5 ml of dry DMF was added DSC (bis(2,5-dioxopyrrolidin-1-yl)carbonate) (120 mg, 0.467) and DMAP (78 mg, 0.636 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 30 min). The crude reaction was filtered through a 0.2 micron filter and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 351 mg (86% yield) of 2,5-dioxopyrrolidin-1-yl 1-((3-((17-(2,5-dioxo-2,5-dihydro-1H)-pyrrol-1-yl)-15-oxo-4,7,10-trioxa-14-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate (compound 42). TIFF0007743561000064.tif24170

[0402] Synthesis of N-(1-(7-amino-4-methyl-2-oxo-2H-chromen-3-yl)-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)benzo[c][1,2,5]oxadiazole-5-carboxamide

[0403] To a solution of 2-(7-amino-4-methyl-2-oxo-2H-chromen-3-yl)acetic acid (152 mg, 0.652 mmol) in 5 mL of dry DMF was added DSC (176 mg, 0.685) and DMAP (120 mg, 0.978 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 30 min). Next, N-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)benzo[c][1,2,5]oxadiazole-5-carboxamide 2,2,2-trifluoroacetate (360 mg, 0.749 mmol) and triethylamine (273 μL, 1.956 mmol) were added, and the reaction was stirred at room temperature overnight. The reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 269 mg (71% yield) of N-(1-(7-amino-4-methyl-2-oxo-2H-chromen-3-yl)-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)benzo[c][1,2,5]oxadiazole-5-carboxamide (compound 50). TIFF0007743561000065.tif29170

[0404] Synthesis of tert-butyl 2-((3-(1-(benzo[c][1,2,5]oxadiazol-5-yl)-1,17-dioxo-6,9,12-trioxa-2,16-diazaoctadecan-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)acetate

[0405] To a solution of N-(1-(7-amino-4-methyl-2-oxo-2H-chromen-3-yl)-2-oxo-7,10,13-trioxa-3-azahexadecan-16-yl)benzo[c][1,2,5]oxadiazole-5-carboxamide (200 mg, 0.344 mmol) in 10 mL of dry DMF was added tert-butyl 2-bromoacetate (67 mg, 0.344 mmol) and triethylamine (144 μL, 1.03 mmol), and the reaction was stirred overnight at 60° C. The reaction was diluted with 50 mL of DCM and washed twice with 50 mL of saturated sodium bicarbonate, followed by two 50 mL washes with brine. The organic phase was concentrated to approximately 5 mL and then purified by silica gel chromatography (2% methanol / DCM to 16% methanol / DCM) to give 218 mg (91% yield) of tert-butyl 2-((3-(1-(benzo[c][1,2,5]oxadiazol-5-yl)-1,17-dioxo-6,9,12-trioxa-2,16-diazaoctadecan-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)acetate (51). TIFF0007743561000066.tif30170

[0406] Synthesis of 2-((3-(1-(benzo[c][1,2,5]oxadiazol-5-yl)-1,17-dioxo-6,9,12-trioxa-2,16-diazaoctadecan-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)acetic acid

[0407] tert-Butyl 2-((3-(1-(benzo[c][1,2,5]oxadiazol-5-yl)-1,17-dioxo-6,9,12-trioxa-2,16-diazaoctadecan-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)acetate (218 mg, 0.313 mmol) was taken up in 20 ml of 30% TFA / DCM and the reaction was stirred at room temperature until the reaction was complete by HPLC (approximately 4.5 hours). The reaction was then concentrated in vacuo, azeotroped three times with toluene, and dried under high vacuum overnight to give 220 mg (quantitative yield) of 2-((3-(1-(benzo[c][1,2,5]oxadiazol-5))-yl)-1,17-dioxo-6,9,12-trioxa-2,16-diazaoctadecan-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)acetic acid, which was used without further purification (52). TIFF0007743561000067.tif33170

[0408] Synthesis of 1-((3-(1-(benzo[c][1,2,5]oxadiazol-5-yl)-1,17-dioxo-6,9,12-trioxa-2,16-diazaoctadecane-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid

[0409] To a solution of 2-((3-(1-(benzo[c][1,2,5]oxadiazol-5-yl)-1,17-dioxo-6,9,12-trioxa-2,16-diazaoctadecan-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)acetic acid (220 mg, 0.345 mmol) in 10 ml of dry DCM was added 1.0 M DCC in DCM (413 μL, 0.413 mmol) and N-hydroxysuccinimide (47 mg, 0.413 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 4 hours). The urea by-product was removed by filtration, and 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (182 mg, 0.686 mmol) and triethylamine (200 μL, 1.44 mmol) were added to the solution, and the reaction was stirred at room temperature overnight. The reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 517 mg (85% yield) of 1-((3-(1-(benzo[c][1,2,5]oxadiazol-5-yl)-1,17-dioxo)-6,9,12-trioxa-2,16-diazaoctadecan-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (compound 53). TIFF0007743561000068.tif24170

[0410] Synthesis of 2,5-dioxopyrrolidin-1-yl 1-((3-(1-(benzo[c][1,2,5]oxadiazol-5-yl)-1,17-dioxo-6,9,12)-trioxa-2,16-diazaoctadecane-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate

[0411] To a solution of 1-((3-(1-(benzo[c][1,2,5]oxadiazol-5-yl)-1,17-dioxo-6,9,12-trioxa-2,16-diazaoctadecane-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (120 mg, 0.135 mmol) in 10 mL of dry DCM was added 1.0 M DCC (156 μL, 0.156 mmol) and N-hydroxysuccinimide (18 mg, 0.156 mmol) in DCM, and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 5 h). The urea byproduct was removed by filtration, and the reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 109 mg (82% yield) of 2,5-dioxopyrrolidin-1-yl 1-((3-(1-(benzo[c][1,2,5]oxadiazol-5)-yl)-1,17-dioxo-6,9,12-trioxa-2,16-diazaoctadecan-18-yl)-4-methyl-2-oxo-2H-chromen-7-yl)amino)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-oate (Compound 54). TIFF0007743561000069.tif23170

[0412] Synthesis of 2-((tert-butoxycarbonyl)amino)-6-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)hexanoic acid

[0413] To a solution of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (1.57 g, 6.02 mmol) in 25 mL of dry DCM was added 1.0 M DCC (7.22 mL, 7.22 mmol) in DCM and N-hydroxysuccinimide (830 mg, 7.22 mmol). The solution was stirred at room temperature until the reaction was complete by HPLC (approximately 4 h). The urea by-product was removed by filtration, and 6-amino-2-((tert-butoxycarbonyl)amino)hexanoic acid (1.77 g, 0.686 mmol) and triethylamine (2.52 mL, 18.06 mmol) were added to the solution. The reaction was stirred overnight at room temperature. The reaction was diluted with 50 mL of DCM and washed twice with 50 mL of saturated sodium bicarbonate, followed by two 50 mL washes with brine. The organic phase was concentrated to approximately 5 ml and then purified by silica gel chromatography (3% methanol / DCM to 16% methanol / DCM) to give 2.62 g (89% yield) of 2-((tert-butoxycarbonyl)amino)-6-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)hexanoic acid (compound 60). TIFF0007743561000070.tif40170

[0414] Synthesis of 2-amino-6-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)hexanoic acid

[0415] 2-((tert-Butoxycarbonyl)amino)-6-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)hexanoic acid (2.62 g, 5.36 mmol) was dissolved in 50 mL of 35% TFA / DCM and stirred at room temperature until the reaction was complete by HPLC (approximately 3 h). The reaction was then concentrated in vacuo, azeotroped three times with toluene, and dried under high vacuum overnight to give the 2,2,2-trifluoroacetic acid salt (1:1) of 2-amino-6-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)hexanoic acid (Compound 61), which was used without further purification. TIFF0007743561000071.tif33170

[0416] Synthesis of 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid

[0417] To a solution of 2-acetamido-5-methylthiazole-4-sulfonyl chloride (246 mg, 0.967 mmol) in 15 mL of dry DCM was added 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid (507 mg, 0.967 mmol) and triethylamine (350 μL, 2.50 mmol), and the reaction was stirred at room temperature overnight. The reaction was diluted with 20 mL of DCM and washed twice with 30 mL of saturated sodium bicarbonate, followed by two washes with 30 mL of brine. The organic phase was concentrated to approximately 5 ml and then purified by silica gel chromatography (4% methanol / DCM to 15% methanol / DCM) to give 530 mg (83% yield) of 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid (compound 62). TIFF0007743561000072.tif22170

[0418] Synthesis of 2,5-dioxopyrrolidin-1-yl 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oate

[0419] To a solution of 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid (530 mg, 0.803 mmol) in 20 mL of dry DCM was added 1.0 M DCC (964 μL, 0.964 mmol) and N-hydroxysuccinimide (111 mg, 0.964 mmol). The solution was stirred at room temperature until the reaction was complete by HPLC (approximately 3 h). The urea byproduct was removed by filtration, and the reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 510 mg (84% yield) of 2,5-dioxopyrrolidin-1-yl 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oate (compound 63). TIFF0007743561000073.tif26170

[0420] Synthesis of [1-(2-acetamido-5-methylthiazole-4-sulfonamido)-29-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontan-30-oic acid

[0421] To a solution of 2,5-dioxopyrrolidin-1-yl 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oate (608 mg, 0.803 mmol) in 25 mL of dry DCM was added the 2,2,2-trifluoroacetate salt of 2-amino-6-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)hexanoic acid (1:1) (485, 0.9636 mmol) and triethylamine (336 μL, 2.409 mmol). The solution was stirred at room temperature until the reaction was complete by HPLC (approximately 6 h). The reaction was diluted with 30 mL of DCM and washed twice with 40 mL of saturated sodium bicarbonate, followed by two 40 mL washes with 40 mL of brine. The organic phase was concentrated, taken up in minimal methanol, and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 637 mg (77% yield) of 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-29-(4-(7-(diethylamino)-2-oxo-2H)-chromene-3-carboxamido)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontan-30-oic acid (Compound 64). TIFF0007743561000074.tif29170

[0422] Synthesis of 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-29-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)butyl)-27,30-dioxo-3,6,9,12,15,18,21,24,34,37,40,43-dodecaoxa-28,31-diazahexatetracontan-46-oic acid

[0423] To a solution of 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-29-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontan-30-oic acid (310 mg, 0.301 mmol) in 20 mL of dry DCM was added 1.0 M DCC in DCM (450 μL, 0.450 mmol) and N-hydroxysuccinimide (52 mg, 0.450 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 3.5 hours). The urea by-product was removed by filtration, and 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (96 mg, 0.361 mmol) and triethylamine (126 μL, 0.903 mmol) were added to the reaction, which was stirred overnight at room temperature. The reaction was diluted with 30 mL of DCM and washed twice with 50 mL of saturated sodium bicarbonate, followed by two 50 mL washes with 50 mL of brine. The organic phase was concentrated, taken up in minimal methanol, and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 327 mg (97% yield) of 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-29-(4-(7-(diethylamino)-2-oxo-2H)-chromene-3-carboxamido)butyl)-27,30-dioxo-3,6,9,12,15,18,21,24,34,37,40,43-dodecaoxa-28,31-diazahexatetracontan-46-oic acid (compound 65). TIFF0007743561000075.tif41170

[0424] Synthesis of 2,5-dioxopyrrolidin-1-yl 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-29-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido))butyl)-27,30-dioxo-3,6,9,12,15,18,21,24,34,37,40,43-dodecaoxa-28,31-diazahexatetracontan-46-oate

[0425] To a solution of 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-29-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)butyl)-27,30-dioxo-3,6,9,12,15,18,21,24,34,37,40,43-dodecaoxa-28,31-diazahexatetracontan-46-oic acid (178 mg, 0.139 mmol) in 15 ml dry DCM was added 1.0 M DCC in DCM (167 μL, 0.167 mmol) and N-hydroxysuccinimide (19 mg, 0.167 mmol), and the solution was stirred at room temperature until the reaction was complete by HPLC (approximately 4.5 h). The urea by-product was removed by filtration, and the reaction was concentrated, taken up in minimal methanol, and purified by preparative HPLC. Pure fractions were combined and lyophilized to give 168 mg (88% yield) of 2,5-dioxopyrrolidin-1-yl 1-(2-acetamido-5-methylthiazole-4-sulfonamido)-29-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carboxamido)butyl)-27,30-dioxo-3,6,9,12,15,18,21,24,34,37,40,43-dodecaoxa-28,31-diazahexatetracontan-46-oate (Compound 66). TIFF0007743561000076.tif49170

[0426] automation

[0427] Multiplex assays and methods can be automated and can be combined with a specimen processing device. The specimen processing device can be an automated device such as the BENCHMARK XT instrument and SYMPHONY instrument sold by Ventana Medical Systems, Inc., which is the assignee of several U.S. patents disclosing systems and methods for performing automated analyses, including U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Patent Application Publication Nos. 2003 / 0211630 and 2004 / 0052685, each of which is incorporated herein by reference in its entirety. Alternatively, specimens can be processed manually.

[0428] The specimen processor can apply fixatives to the specimen, including crosslinkers (e.g., aldehydes such as formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde crosslinkers), oxidizing agents (e.g., metal ions and complexes such as osmium tetroxide and chromate), protein denaturants (e.g., acetic acid, methanol, ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), compounding reagents (e.g., Carnoy's fixative, methacarn, Bouin's solution, B5 fixative), Rossmann's solution, Gendre's solution, microwave, and other fixatives (e.g., excluding volume fixation and vapor fixation).

[0429] If the specimen is a paraffin-embedded sample, the specimen can be deparaffinized using an appropriate deparaffinizing solution using a specimen processor. After the waste removal agent removes the deparaffinizing solution(s), any number of substances can be applied sequentially to the specimen. The substances can be for pretreatment (e.g., protein cross-linking, exposing nucleic acids, etc.), denaturation, hybridization, washing (e.g., stringent washing), detection (e.g., linking visual or marker molecules to probes), amplification (e.g., amplification of proteins, genes, etc.), counterstaining, coverslipping, etc.

[0430] The specimen processing device can apply a wide range of substances to the specimen. Substances include, but are not limited to, stains, probes, reagents, rinses, and / or conditioners. Substances can be fluids (e.g., gases, liquids, or gas / liquid mixtures). Liquids can be solvents (e.g., polar solvents, nonpolar solvents, etc.), solutions (e.g., aqueous or other types of solutions), etc. Reagents can include, but are not limited to, stains, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen retrieval solutions (e.g., aqueous or non-aqueous-based antigen retrieval solutions, antigen retrieval buffers, etc.). Probes can be isolated nucleic acids or isolated synthetic oligonucleotides attached to a detectable label or reporter molecule. Labels can include radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.

[0431] After the specimen has been processed, the user can transport the slide containing the specimen to an imaging device. The imaging device used here is a brightfield imager slide scanner. One brightfield imager is the iScan Coreo™ brightfield scanner sold by Ventana Medical Systems, Inc. In an automated embodiment, the imaging device is a digital pathology device such as those disclosed in International Patent Application No. PCT / US2010 / 002772 (Publication No. WO 2011 / 049608) entitled "IMAGING SYSTEM AND TECHNIQUES" or U.S. Patent Application Publication No. 2014 / 0178169, filed September 9, 2011, entitled "IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME."

[0432] Counterstaining

[0433] Counterstaining is a method of staining a sample with a chemical agent and then post-processing the sample to detect one or more targets so that target structures can be more easily visualized under a microscope. For example, counterstaining is sometimes used before mounting to enhance the clarity of immunohistochemical staining. Counterstains differ in color from the primary stain. Many well-known counterstains include hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, and nuclear fast red. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that can be used.

[0434] In some instances, multiple stains can be mixed together to create a counterstain. This provides flexibility and the ability to select stains. For example, a first stain can be selected for a mixture that has a particular attribute but not another desired attribute. A second stain can be added to the mixture that displays the missing desired attribute. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed to form a counterstain.

[0435] Imaging

[0436] Certain aspects, or all, of the disclosed embodiments can be automated and facilitated by computer analysis and / or image analysis systems. In some applications, precise color or fluorescence ratios are measured. In some embodiments, optical microscopy is utilized for image analysis. Certain disclosed embodiments include digital image acquisition, which can be achieved by linking a digital camera to a microscope. Digital images obtained from stained samples are analyzed using image analysis software. Color or fluorescence can be measured in several different ways. For example, color can be measured by red, blue, and green values; hue, saturation, and intensity values; and / or by measuring specific wavelengths or wavelength ranges using a spectral imaging camera. Samples can also be evaluated qualitatively and semi-quantitatively. Qualitative evaluations include assessing staining intensity, identifying positively stained cells and subcellular compartments involved in the staining, and assessing the quality of the entire sample or slide. Individual evaluations are performed on test samples, and this analysis can include comparison to known averages to determine whether the sample represents an abnormal condition.

[0437] Samples and targets

[0438] A sample contains biological components, generally suspected of containing one or more target molecules of interest. The target molecules may be on the surface of cells, or the cells may be in suspension or in tissue sections. The target molecules may also be intracellular and can be detected upon cell lysis or permeabilization of the cells with a probe. Those skilled in the art will understand that methods for detecting target molecules in a sample will vary depending on the type of sample and probe used. Methods for collecting and preparing samples are known in the art.

[0439] Samples used in embodiments of the methods using the compositions disclosed herein, such as tissue or other biological samples, can be prepared by a skilled artisan using any method known in the art. Samples can be obtained from subjects for routine screening or from subjects suspected of having a disorder, such as a genetic abnormality, infection, or neoplasm. The described embodiments of the disclosed methods can also be applied to samples that do not have a genetic abnormality, disease, disorder, etc., referred to as "normal" samples. Such normal samples are particularly useful as controls for comparison with other samples. Samples can be analyzed for various purposes. For example, samples can be used in scientific research, for the diagnosis of suspected illnesses, or as prognostic indicators of treatment success, survival, etc.

[0440] The sample may contain multiple targets that can be specifically bound by probes or reporter molecules. The targets may be nucleic acid sequences or proteins. In some examples, the targets are proteins or nucleic acid molecules derived from pathogens such as viruses, bacteria, or intracellular parasites such as viral genomes. For example, the target proteins may be produced from target nucleic acid sequences that are related to (e.g., correlated with, causally related to, etc.) a disease.

[0441] Those skilled in the art will appreciate that coumarin-based conjugates specific for any of the following targets can be developed:

[0442] In certain non-limiting examples, the target protein is produced by a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) associated with neoplasia (e.g., cancer).A large number of chromosomal abnormalities (including translocations and other rearrangements, amplifications or deletions) have been identified in tumor cells, particularly cancer cells such as B-cell and T-cell leukemia, lymphoma, breast cancer, colon cancer, and neural cancer.Therefore, in some examples, at least a portion of the target molecule is produced by a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) that is amplified or deleted in at least a subset of cells in the sample.

[0443] In another example, the target protein is produced from a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) that is a tumor suppressor gene that is deleted (lost) in malignant cells. For example, the p16 region on chromosome 9p21 (including D9S1749, D9S1747, p16(INK4A), p14(ARF), D9S1748, p15(INK4B), and D9S1752) is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the short arm of chromosome 1 (e.g., encompassing SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322) and the pericentromeric region of chromosome 19 (e.g., 19p13-19q13) (e.g., encompassing MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1) are characteristic molecular features of certain types of solid tumors of the central nervous system.

[0444] Many other cytogenetic abnormalities correlated with neoplastic transformation and / or proliferation are known to those skilled in the art. Target proteins produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences) that are correlated with neoplastic transformation and are useful in the disclosed methods also include the EGFR gene (7p12; e.g., GENBANK™ Accession No. NC-000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK™ Accession No. NC-000008, nucleotides 128817498-128822856), the D5S271 ( 5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK™ Accession No. NC-000008, nucleotides 19841058-19869049), RB1 (13q14; e.g., GENBANK™ Accession No. NC-000013, nucleotides 47775912-47954023), p53 (17p13.1; e.g., GENBANK™ Accession No. NC-000017, complement, nucleotides 7512464-7531642), )), N-MYC (2p24; e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides 151835231-151854620), CHOP (12q13; e.g., GENBANK™ Accession No. NC-000012, complement, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK™ Accession No. NC-000016, nucleotides 31098954-31110601), FKHR (13p1 4; e.g., GENBANK™ Accession No. NC-000013, complement, nucleotides 40027817-40138734), as well as, e.g., ALK (2p23; e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides 29269144-29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK™ Accession No. NC-000011, nucleotides 69165054-69178423), BCL2 (18q21.3; e.g., GENBANK™ Accession No. NC-000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK™ Accession No. NC-000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-p31; e.g., GENBANK™ Accession No. NC-000001, complement, nucleotides 59019051-59022373), TOP2A (17q21-q22; e.g., G ENBANK™ Accession No. NC-000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No. NC-000021, complement, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK™ Accession No. NC-000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3; e.g., GENBANK™ Accession No. GENBANK™ Accession No. NC-000007, complement, nucleotides 13897379-13995289), EWS (22q12.2; e.g., GENBANK™ Accession No. NC-000022, nucleotides 27994271-28026505); FLI1 (11q24.1-q24.3; e.g., GENBANK™ Accession No. NC-000011, nucleotides 128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides 13897379-13995289), complement, nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK™ Accession No. NC-000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK™ Accession No. NC-000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g., GENBANK™ Accession No. NC-000019, complement, nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK™ Accession No. NC-000001, complement, nucleotides 40133685-40140274), REL (2p13-p12; e.g., GENBANK™ Accession No. NC-000002, nucleotides 60962256-61003682), and CSF1R (5q33-q35; e.g., GENBANK™ Accession No. NC-000005, complement, nucleotides 149413051-149473128).

[0445] [Example]

[0446] Synthesis and purification of bioconjugates

[0447] BSA-hapten conjugates

[0448] The BSA protein solid (fraction V, 60 mg) was dissolved in 3 mL of PBS (100 mM phosphate, 150 mM NaCl, pH 7.5) and rotated at room temperature for 1 h. The resulting solution was filtered through a 0.2 μm Pall GHP syringe filter and analyzed for concentration by UV-VIS to yield an 18.1 mg / mL solution. The BSA stock solution (1 mL) was treated with 20 equivalents of the NHS ester of the appropriate labeling reagent in anhydrous DMF. The amount of DMF was maintained below approximately 15% (v / v). The resulting mixture was rotated overnight at room temperature. The resulting mixture was filtered through a 0.2 μm Pall GHP syringe filter and purified by size exclusion chromatography. The resulting conjugate fractions were combined and analyzed for concentration and label / protein ratio by UV-VIS. The sample was stored at 2–6 °C until use.

[0449] GAR-hapten conjugates

[0450] Goat anti-rabbit pAb (1 mg / mL in PBS) was treated with 30 equivalents of NHS ester of the appropriate labeling reagent in anhydrous DMF. The amount of DMF was kept below approximately 15% (v / v). The resulting mixture was rotated overnight at room temperature. The resulting mixture was filtered through a 0.2 μPall GHP syringe filter and purified by size exclusion chromatography. The resulting conjugate fractions were combined and analyzed for concentration and label / protein ratio by UV-VIS. The samples were stored at 2–6 °C until use.

[0451] Biolayer Interference (BLI) Analysis

[0452] Individual protein conjugates (antibody conjugates or BSA immunogens) were labeled with the novel coumarin-based reagents disclosed herein, as well as tyramide-dPEG8-hapten and NHS-dPEG8-hapten labeling reagents (see Figure 20) (and PCT Application No. PCT / US2011 / 042849, the disclosure of which is incorporated herein by reference in its entirety). Biolayer interferometry (BLI) was used to characterize the effect of protein labeling on protein interactions on a Pall-ForteBio Octet Red instrument equipped with an amine-reactive second-generation (AR2G) biosensor (Figure 6). The AR2G biosensor provides a high density of carboxylic acids on the surface, reducing the propensity for nonspecific interactions. Protein immobilization was achieved by standard EDC-catalyzed amide bond formation to create a covalent bond between the reactive amines on the protein and the carboxy-terminal biosensor surface. Carboxylic acids were activated by reaction with EDC (1-ethyl-3-[3-dimethyl-aminopropyl]carbodiimide hydrochloride) and s-NHS (N-hydroxysulfosuccinimide) to generate highly reactive NHS esters. The esters reacted rapidly with primary amines of proteins to form highly stable amide bonds. Covalent immobilization anchored proteins to biosensor surfaces, enabling analysis and kinetic characterization of binding events.

[0453] Representative BLI assay

[0454] Octet RED BLI analysis assays were performed using Pall-ForteBio AR2G biosensor chips. Assay conditions were performed as suggested in Pall-ForteBio Dip and Read™ Amine Reactive 2nd Generation Technical Note 26, with minor modifications. Activation and quenching of the AR2G biosensor were performed as suggested in Technical Note 26. Individual reagent concentrations were modified as needed for each assay to maximize signal-to-noise ratio while minimizing background binding events. All BLI assay steps were performed at 37°C. A representative assay for each is described below.

[0455] Coumarin-based reagent antibody cross-reactivity studies

[0456] This assay was performed to assess any potential cross-reactivity with the coumarin-based reagents described herein. The BSA-CLBF immunogen was immobilized on an AR2G biosensor at a concentration of 1.25 μg / mL in 100 mM NaOAc (pH=5). Assessment of anti-hapten cross-reactivity was performed with 725 ng / mL of anti-hapten mAb in PBS (100 mM phosphate, 150 mM NaCl, pH=7.5) using Pall-ForteBio Kinetics Additive. The following table shows the time steps: TIFF0007743561000077.tif44170

[0457] Effect of coumarin-based reagents on antigen and anti-label recognition [Rb immobilization - labeled GAR recognition - anti-label recognition]

[0458] This assay was performed to better understand how the coumarin-based reagent structure affects the antigen detection ability of labeled antibodies. Additionally, a secondary anti-labeled antibody was used to better understand how the linker structure affects label detection. Rabbit pAb was immobilized on the AR2G biosensor at a concentration of 25 μg / mL in 100 mM NaOAc (pH = 5). GAR recognition of Rb mAb was performed at 3 μg / mL in PBS (100 mM phosphate, 150 mM NaCl, pH = 7.5) using Pall-ForteBio Kinetics Additive. Anti-label recognition was performed at 3 μg / mL in PBS (100 mM phosphate, 150 mM NaCl, pH = 7.5) using Pall-ForteBio Kinetics Additive. The time steps are shown in the following table. TIFF0007743561000078.tif55170

[0459] Results and Discussion

[0460] Antibody cross-reactivity studies of coumarin-based reagents

[0461] The use of coumarin-based reagents in this disclosure enables the detection of difficult-to-detect hapten labels. Hapten labels are commonly used in multiplex staining assays, which sets the stage for understanding whether interactions with anti-hapten antibody libraries are observed. Biolayer interference studies were performed where the BSA-CLBF immunogen was immobilized on the tip. The anti-hapten antibody library was then screened for potential cross-reactivity. Figure 7 shows an example of the expected interaction of MS anti-BF mAb (H1, H6) with the modified chip. MS anti-BF mAb binding to the sensor chip causes a net increase in layer growth on the chip, resulting in a positive association signal for 4800 seconds. Net dissociation is displayed in the last frame after 4800 seconds. MS anti-PPT mAb (C1, C6) and MS anti-DABSYL mAb (D1, D6) antibodies show no significant interaction with the coumarin linker-modified biosensor chip.

[0462] The remaining Ms anti-hapten library was screened against a similar BSA-CLBF-modified biosensor chip in a separate BLI experiment. No significant interactions were observed with the coumarin-based reagent-modified biosensor chip and the Ms anti-NP mAb (B1, B6; C1, C6) and Ms anti-TS mAb (H1, H6; G1, G6) antibodies (Figure 8). The Ms anti-NCA mAb (B1, B6; C1, C6) and Ms anti-HQ mAb (H1, H6; G1, G6) antibodies also showed no significant interactions with the coumarin-based reagent-modified biosensor chip (Figure 8). No significant interactions were observed with the coumarin-based reagent-modified biosensor chip and the Ms anti-DCC mAb (B1, B6; D1, D6) and Ms anti-ROT mAb (F1, F6; G1, G6) antibodies (Figure 10). The BLI results in Figure 11 show that no interaction was observed with the Ms anti-DIG mAb (red, light blue), whereas the Ms anti-DNP mAb (B1, B6; C1, C6) antibodies exhibited positive interaction with the coumarin-based reagent. The observed Ms anti-DNP mAb BLI binding response (80 pm) was approximately 15% of the Ms anti-BF mAb BLI binding response (520 pm, Figure 7). Furthermore, nonspecific binding of the Ms anti-DNP mAb antibodies was not easily dissociated from the biosensor tip, suggesting that background staining of the tissue was observed. Ms anti-DNP mAb (F1, F6; G1, G6) coumarin linker recognition was confirmed in another BLI experiment shown in Figure 11. Again, the Ms anti-DNP mAb antibodies were not easily dissociated from the biosensor tip. The BLI results in Figure 12 also showed that no interaction was observed between the Ms anti-BD mAbs (C1, C6; D1, D6) and the biosensor chip.

[0463] Effect of linker on antigen and anti-label recognition [Rb immobilization - labeled GAR recognition - anti-label recognition]

[0464] GAR-lys(DCC)dPEG8TS vs. GAR-dPEG4-lys(DCC)dPEG8TS conjugates:

[0465] The GAR-lys(DCC)dPEG8TS and GAR-dPEG4-lys(DCC)dPEG8TS conjugates utilized a lysine amino acid core as a branched ligand. The easily detectable DCC coumarin hapten was used as a potential chromophore and directly attached to the primary amine of lysine. A second dPEGTS hapten group was attached to the secondary amine of the lysine core (see Figure 13, Compound A) due to the difficulty of label quantification. This group was directly conjugated to proteins by attaching an NHS group directly to the lysine branch. A second crosslinked lysine linker core was generated with a dPEG4 handle added to provide separation from the lysine bridge (see Figure 13, Compound B). The extra dPEG4 spacer group was expected to reduce steric interactions, provide more flexibility for the labeling group, and facilitate both protein loading and assay detection performance (primary antigen and anti-label recognition).

[0466] GAR pAb (1 mg, 1 mg / mL) was conjugated with 30 equivalents of both linker constructs. The GAR-lys(DCC)dPEG8TS conjugate (GAR + Compound A) gave a linker / GAR ratio of 3.3 (913 mg, 89% yield). The GAR-dPEG4-lys(DCC)dPEG8TS conjugate (GAR + Compound B) gave a linker / GAR ratio of 5.4 (913 mg, 84% yield). The extra dPEG4 spacer between the lysine bridge and the NHS ester worked as expected, providing higher antibody label loading. Comparable yields were obtained with both conjugates.

[0467] The BLI assay was designed to mimic a tissue staining assay. Rabbit mAb was bound to a biosensor chip and recognized by the above conjugates. Additionally, an unmodified GAR standard was used to compare the effect of the label on rabbit recognition. Tertiary recognition of the TS hapten label was performed using Ms anti-TS mAb (see Figure 14). The GAR antibody conjugate recognized the rabbit antibody-loaded biosensor chip similarly to the unmodified GAR antibody. A slightly thicker ELISA layer was observed for the GAR-dPEG4-lys(DCC)dPEG8TS conjugate than for the GAR-lys(DCC)dPEG8TS conjugate (see Figure 15). No difference was observed in the dissociation rate of the modified GAR conjugates. The additional dPEG4 spacer between the lysine bridge and the NHS ester in GAR-dPEG4-lys(DCC)dPEG8TS also provided more Ms anti-TS mAb recognition (see Figure 16).

[0468] GAR-CLBF conjugate

[0469] GAR pA (2 mg, 1 mg / mL) was labeled with 30 equivalents of BFdPEG8NHS and BFCLNHS reagents. UV / VIS analysis of the GAR-CLBF conjugate showed a BF / GAR ratio of 5.85 (1.6 mg, 80% yield). A detectable moiety of CL coumarin provided the conjugate with a label / Ab ratio and yield in the expected range. UV / VIS analysis of the GAR-dPEG8BF conjugate overestimated the expected BF / GAR ratio of 38.1 (860 mg, 43% yield). The GAR-dPEG8BF conjugate data is imprecise and should be used as a relative approximation.

[0470] The BLI assay was again designed to mimic the tissue staining assay. Rabbit mAb was bound to a biosensor chip and recognized by the above conjugate. Tertiary recognition of the BF hapten label was performed using Ms anti-BF mAb (see Figure 17). The GAR antibody conjugate also recognized a biosensor chip loaded with rabbit antibody (see Figure 18). A slightly thicker ELISA layer was observed for the GAR-CLBF conjugate than for the GAR-dPEG8BF conjugate. No significant difference was observed in the dissociation rate of the modified GAR conjugates. The -dPEG4-coumarin-dPEG4-BF linker is believed to be more rigid than the -dPEG-BF label. The GAR-CLBF conjugate provided a thicker ELISA layer on the biosensor tip when recognized by Ms anti-BF mAb (see Figure 19). The Ms anti-BF mAb dissociated slightly faster from the GAR-CLBF conjugate than from the GAR-dPEG8BF conjugate.

[0471] This increased dissociation of the Ms-anti-BF mAb from the GAR-CLBF conjugate may be the result of loosening of the bond (faster koff) caused by increased affinity and avidity interactions between the Ms-anti-BF mAb and the coumarin linker. As previously mentioned, the CL coumarin linker is expected to be more rigid. The added flexibility of the -dPEG-BF label may allow the unbound BF label to bend and bind to the other complementary domain of the Ms-anti-BF mAb via affinity interactions, resulting in stronger binding. Avidity interactions (2-1) always result in less dissociation than affinity interactions (1-1), which may compete with the coumarin linker. A similar dissociation phenomenon was observed in cross-reactivity studies (see Figure 7 above).

[0472] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to use concepts from the various patents, applications, and publications to provide further embodiments.

[0473] While the present disclosure has been described with reference to certain exemplary embodiments, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that would fall within the spirit and scope of the principles of the present disclosure. More particularly, reasonable variations and modifications are possible in the components and / or arrangements of the combined configuration of the subject matter within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the present disclosure. In addition to variations and modifications of the components and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

1. Formula (IXA) or (IXB): (In the formula, T is an antibody, R ｚ is a detectable label, o is an integer ranging from 1 to 10; L １ and L ２ is a linker, L 1 and L 2 independently contain 2 to 12 PEG groups; W is a group represented by the formula (VC) (In the formula, R 2 , R 3 , R 4 , and R 6 are independently selected from a substituted or unsubstituted, linear or branched C 1 -C 6 alkyl group; a substituted or unsubstituted, linear or branched C 1 -C 6 alkoxy group; a substituted or unsubstituted, linear or branched C 1 -C 6 heteroalkyl group; a trifluoromethyl group; a hydroxyl group; a sulfate group; a cyano group; a halogen; a phosphate group; a sugar; a carboxylic acid group; a nitro group; —C(O)NR x R y ; —S—R x ; —SO 2 ; —SO 2 Cl; —SO 3 H; —SO 4 H; —SO 2 NR x R y ; —N(H)—NR x R y ; —NR x R y ; R 5 is a C 1 to C 10 substituted or unsubstituted branched or unbranched alkyl group, —C(O)—O—, or —C(O)—N(H)—; R 1 is a C 1 to C 10 substituted or unsubstituted branched or unbranched alkyl group, —O—, —O—CH 2 —, —N(R x )—, or —S—; each X is independently a bond or a substituted or unsubstituted straight or branched chain C 1 -C 16 alkyl group; —[(CH 2 ) j —O] k —CH 2 — (j is an integer ranging from 1 to 4 and k is an integer ranging from 1 to 16); —N—R x —; —C(O)—N—R x —; or —N—R x —C(O)—; wherein each R x and R y is independently H or a C 1 -C 4 alkyl group. having the structure Z is a -CH- group, -CH-CH ２ - group, or -CH ２ is a —CH— group, m and n are independently integers ranging from 1 to 4. A compound defined by one of the structures:

2. The compound of claim 1 , wherein the antibody is a primary antibody.

3. The compound of claim 1 , wherein the antibody is a secondary antibody.

4. R ｚ The compound according to any one of claims 1 to 3, wherein is a hapten.

5. R ｚ The compound according to any one of claims 1 to 3, wherein is an enzyme.

6. 6. The compound of claim 5, wherein the enzyme is selected from the group consisting of peroxidases and phosphatases.

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