Benzothienopyrrole cyanine dyes

Abstract

The present disclosure provides novel benzothienopyrrole (Bt)-cyanine (Cy) fluorescent compounds, water soluble BtCy fluorescent dyes, water soluble BtCy binding partner conjugates, and tandem dyes thereof. Yellow, orange, red, NIR or IR wavelengths can be used to excite the BtCy fluorescent compounds, conjugates, and tandem dyes. The fluorescent dyes can be conjugated to a binding partner for detecting a target analyte in a biological sample, and are suitable for flow cytometry assays. These dyes have the general formula wherein Ar1 is selected from the group consisting of substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl, polycyclic aryl, monocyclic heteroaryl and polycyclic heteroaryl.

Description

[0001] This application was filed on March 15, 2024 as a PCT international patent application and claims the benefit and priority of U.S. Provisional Application Serial No. 63 / 490,932, filed March 17, 2023, and U.S. Provisional Application Serial No. 63 / 510,018, filed June 23, 2023, each of which is incorporated herein by reference in its entirety. Background Technology

[0002] The demand for a variety of fluorescent dyes and dye conjugates for use in flow cytometry and spectroscopic flow cytometry instruments is constantly increasing. Water-soluble fluorescent compounds and their conjugates can be used for a variety of biological applications by generating signals that can be monitored in real time, and provide a simple and rapid method for detecting biological targets and events, such as in diagnostic kits, in microscopy, or in drug screening.

[0003] Molecular recognition involves the specific binding of two molecules. Molecules that bind specifically to target biomolecules can be used in a variety of research and diagnostic applications, such as analyte labeling and separation, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation, and chromatography. Target biomolecules can be detected by labeling them with fluorescent dyes.

[0004] Several fluorescent dyes and their antibody conjugates that can be excited by 355 nm and 405 nm lasers have been previously developed. For example, U.S. Patent No. 11,208,527 describes a fluorescent polymer dye based on water-soluble dihydrophenanthrene (DHP), which exhibits an excitation maxima between 395 and 415 nm, with emission light between approximately 415 and 475 nm. U.S. Patent Application Publication US2020 / 0190253 describes a purple excitable polymer and tandem dye based on water-soluble DHP.

[0005] There is an increasing demand for multicolor panels for both conventional and spectroscopic flow cytometry, requiring additional fluorescent dyes that can be excited by other lasers (e.g., 488 nm, 563 nm, 638 nm, and 808 nm). Parameters that users consider when selecting fluorescent dyes may include the maximum excitation wavelength, the maximum emission wavelength, the dye's brightness, and its fluorescence lifetime. The dye's brightness is a combination of the extinction coefficient (ε, a measure of the amount of light absorbed at a given wavelength) and the fluorescence quantum yield (Φ, a measure of the light emitted in radiative form from its singlet excited states).

[0006] Water-soluble fluorescent dyes that can be excited by blue, green, yellow, orange, red, near infrared (NIR), and infrared (IR) wavelengths are desirable. Summary of the Invention

[0007] This disclosure provides benzothienopyrrole-cyanine (BtCy) compounds, which can be water-soluble fluorescent BtCy dyes. It also provides BtCy tandem dyes comprising a BtCy fluorescent compound as an acceptor or donor chromophore. Furthermore, it provides labeled specific binding pairs and complexes thereof, said labeled specific binding pairs comprising a BtCy compound or BtCy tandem dye according to the invention conjugated to the specific binding pair. The BtCy compounds, labeled specific binding pairs, and tandem dyes according to this disclosure can be used in biological applications, including for detecting target analytes and for use in diagnostic kits. For example, a labeled specific binding pair comprising a specific binding pair according to the invention and a BtCy compound or BtCy tandem dye can be used in methods for detecting analytes in a sample.

[0008] Novel fluorescent dyes based on the benzo[4,5]thieno[2,3b]pyrrole-cyanine structure have been provided. Water solubility has been improved by adding a water-soluble moiety. Synthetic routes have been designed for conjugation with antibodies and for the preparation of tandem dyes.

[0009] This disclosure provides fluorescent compounds comprising the structure according to formula (I):

[0010] in

[0011] Selected from substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl, polycyclic aryl, monocyclic heteroaryl and polycyclic heteroaryl;

[0012] Selected from substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl and polycyclic aryl;

[0013] Each T is independently for NR 4’ CR 1 CR 1 R 2 , O, S, Se or Te;

[0014] V is NR 11 CR 8 CR 8 R 9O, S, Se or Te, or each TV together can represent SO2, -CR 1 -O-,-O-CR 1 -,-CO-O-,-O-CO-,-CO-NR 1 1-, or -NR 4’ -CO- structural elements;

[0015] G is either C or N;

[0016] Each R 1 R 2 R 8 and R 9 Independently selected from water-soluble moieties, linked water-soluble moieties, linker moieties, E, linked E, reactive groups, linked reactive groups, conjugated tags, linked conjugated tags, conjugated mating bodies, linked conjugated mating bodies, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonic acid, sulfonate / ester, alkylsulfonate / ester, alkylsulfonate, alkoxysulfonate / ester, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, alkoxycarboxylate / ester, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylsulfonamide PEG, alkylamide.

[0017]

[0018]

[0019] Each R 4 R 4’ R 10 and R 11 Independently selected from the following: linker portion, chromophore, linked chromophore, reactive group, linked reactive group, conjugated tag, linked conjugated tag, water-soluble portion, linked water-soluble portion, conjugated mating body, linked conjugated mating body, E, linked E, H, halogenated alkyl, alkenyl, alkynyl, PEG group, linked PEG group, cycloalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, sulfonic acid, sulfonate / ester, alkyl sulfonate / ester, alkyl sulfonate, carboxylic acid, carboxylate / ester, alkyl carboxylate / ester, alkyl sulfonamide, alkyl sulfonamide PEG, alkylamide, alkylamide-PEG.

[0020]

[0021] Or its protected group; or R 4 and R 12 Together, R 14 and R 10 Together, R 4 and R 13 Together, R 13 and R 10 Together, R 13 and R 11 Together, R 12 and R 14 Together, R 4’ and R 13 Together, R 4’ and R 12 Together, R 13 and R 10 Together, R 4 R 12 R 14 and R 10 Together, R 4 R 13 and R 10 Together, R 4 R 13 and R 11 Together, R 4 R 12 and R 14 Together, R 4’ R 13 and R 11 Together, and R 12 R 14 and R 10 One, two, three, or four together form unsubstituted or substituted unsaturated or partially unsaturated C3-C atoms. 10 cycloalkyl; unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 10 Heterocyclic alkyl groups; unsubstituted or substituted unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polycyclic alkyl; or unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polyhedraloyl groups;

[0022] Each R 3Independently selected from H, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, alkyl sulfonate / ester, alkyl carboxylate / ester, water-soluble moiety, linked water-soluble moiety, chromophore, linked chromophore, functional moiety, linked functional moiety, conjugated tag, linked conjugated tag, conjugated partner, linked conjugated partner, PEG group and linked PEG group;

[0023] Each Q is independently a bond, O, NH, NR 4 C1-C 12 Alkylene, CHR 4 Or CH2;

[0024] Each Z is independently CH2, CHR 4 O, NR 4 Or NH;

[0025] Each W 1 It is a water-soluble component on its own.

[0026] L 1 L 2 and L 3 Each connector is selected independently.

[0027] Each E is independently selected from chromophores, functional moieties, substrates, reactive groups, conjugation tags, linked conjugation tags, and binding partners;

[0028] Each R 7 Independently selected from H, hydroxyl, C1-C 12 Alkyl, C1-C 12 Heteroalkyl, C2-C 12 Olefins, C2-C 12 Alkynes, C3-C 12 cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy group, C2-C 18 (Miscellaneous) arylamino, carboxylates / esters, carboxylic acids, C2-C 12 Alkyl carboxylic acids, C2-C 12 Alkyl carboxylates / esters, C2-C 12 Alkyl carboxylic acid esters, aryl carboxylic acids, aryl carboxylic acid esters, C1-C 12 Alkyl groups, water-soluble moiety, PEG moiety, protected or unprotected functional groups, chemoselective functional groups, conjugated labels, linked conjugated labels, connectors, sulfonic acids, sulfonates / esters, C1-C 12 Alkyl sulfonates / esters, sulfonamides, and combinations thereof;

[0029] Each R 12 R 13 and R 14 Independently selected from hydrogen, halogen, one or more heteroatoms, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C1-C6 olefin, substituted or unsubstituted C3-C 10 Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C3-C 10 Heterocyclic alkyl groups, CO2R 1 CONR 1 R 2 -O-CH2CH2-PEG-R 7 -S-CH2CH2-PEG-R 7 -N-CH2CH2-PEG-R 7 O-aryl, S-aryl, N-aryl, -O-alkyl, S-alkyl, N-alkyl, wherein each alkyl or aryl group may optionally be represented by one or more R groups. 7 PEG or PEG-R 7 Replace, optionally, each of R 12 R 13 and R 14 Independently replaced by one or more R 7 Group; or R 1 R 2 R 4 R 4’ R 8 R 9 R 10 R 11 R 12 R 13 , and R 14 At least two of them together, including but not limited to R 1 R 2 R 4 R 4' R 8 R 9 R 10 R 11 R 12 R 13 and R 14 Two, three, or four of them together, optionally including but not limited to R 1 R 2 R 4 R 4’ R 8 R 9 R 10 R 11R 12 R 13 , and R 14 One, two, three or four together, including but not limited to R 4 and R 12 Together, R 14 and R 10 Together, R 4 and R 13 Together, R 13 and R 10 Together, R 13 and R 11 Together, R 12 and R 14 Together, R 4' and R 13 Together, R 4’ and R 12 Together, R 13 and R 10 Together, R 4 R 12 R 14 and R 10 Together, R 4 R 13 and R 10 Together, R 4 R 13 and R 11 Together, R 4 R 12 and R 14 Together, R 4’ R 13 and R 11 Together, and R 12 R 14 and R 10 Formation of unsaturated or partially unsaturated C3-C, whether substituted or substituted. 10 Cycloalkyl, unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 10 Heterocyclic alkyl groups, unsubstituted or substituted, unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polycyclic alkyl groups, or substituted unsaturated or partially unsaturated C3-C groups, either unsubstituted or optionally O-substituted. 14 C3-C 10 Or C3-C8 polyhedraloyl groups;

[0030] Each K is independently a covalent bond, O, S, Se, P, NR 1 or CR 1 R 2 ;

[0031] Each f is an independent integer from 0 to 50, 1 to 30, or 2 to 20;

[0032] Each m and m' is independently 0, 1, 2, or 3;

[0033] Each n is an independent integer from 1 to 20; from 1 to 10; or 0, 1, 2, or 3.

[0034] Each p is independently 1, 2, 3 or 4;

[0035] Each s is independently 1 or 2;

[0036] Each t is independently 0, 1, 2, 3, or 4; and

[0037] X is a counter ion.

[0038] In some cases, m is 0 or 1.

[0039] In some cases, p is 1, 2, or 3. In some cases, p is 1. In some cases, p is 2. In some cases, p is 3. In some cases, p is 4.

[0040] In some cases, when m is 0, each T is independently CR. 1 or CR 1 R 2 In some cases, when m is 0, T is not NR. 4’ S or O. In some cases, when m is 1, each T is independently NR. 4’ O, S, Se, or Te. In some cases, when m is 1, T is not CR. 1 or CR 1 R 2 .

[0041] A BtCy tandem dye is provided, comprising: a fluorescent BtCy compound of the present disclosure; and a chromophore, donor dye, or acceptor dye covalently linked to the fluorescent BtCy compound or a labeled specific binding partner. The BtCy compound of the present invention can be a donor dye. The BtCy compound of the present invention can be an acceptor dye.

[0042] A labeled specific binding partner is provided, comprising: a fluorescent BtCy compound or a BtCy tandem dye according to the present disclosure; and a specific binding partner covalently linked to the fluorescent BtCy compound.

[0043] A method for detecting a target analyte in a sample is provided, the method comprising: providing a sample suspected of containing the analyte; and contacting the sample with a specific binding partner conjugated to a fluorescent BtCy compound or a BtCy tandem dye of the present disclosure, wherein the specific binding partner is capable of interacting with the target analyte.

[0044] A kit is provided comprising at least one fluorescent BtCy compound according to this disclosure, a labeled specific binding partner, or a tandem dye. The compound or tandem dye may contain a conjugation tag. Attached Figure Description

[0045] Figure 1 The chemical structures of prior art indole-cyanine dyes Cy3, C5, and Cy7, as well as the benzothiophene-pyrrole-cyanine dyes BtCy3, BtCy5, and BtCy7 of the present invention, are shown. Compared with the common indole-cyanine compounds Cy3, Cy5, and Cy7, the spectra of BtCy3, BtCy5, and BtCy7 compounds are red-shifted by approximately 200 nm.

[0046] Figure 2 Schemes 1 and 2 are shown, which respectively illustrate benzothiophene-pyrrole intermediates. 3 and 6 The representative synthetic route is shown in Scheme 1, which illustrates the intermediate compound starting from 2-iodobenzo[b]thiophene. 3 3-(2,3,3-trimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Synthesis of 1-(methyl)propane-1-sulfonate / ester. Scheme 2 illustrates the synthesis of intermediate compounds starting from ethyl 2-methyl-3-oxobutyrate. 6 3-(2,3-dimethyl-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Synthesis of 1-yl)propane-1-sulfonate / ester.

[0047] Figure 3 Schemes 3A, 3B, and 4 are shown, which respectively illustrate the benzothiophene-pyrrole intermediate. 7 and 8 and indole intermediates 10 A representative synthetic route. As shown in Scheme 3A, benzothiophene-pyrrole intermediate compounds 7 1-(2-Carboxyethyl)-2,3,3-trimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Iodides are composed of intermediate compounds 2 Prepared with 3-iodopropionic acid. As shown in Scheme 3B, benzothiophene-pyrrole intermediate compound 81-(2-Carboxyethyl)-2,3-dimethyl-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Iodides are composed of intermediate compounds 5 Prepared with 3-iodopropionic acid. As shown in Scheme 4, indole intermediate compound 10 5-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)oxy)-1-(2-carboxyethyl)-2,3,3-trimethyl-3H-indole-1- The iodide is prepared in two steps from 2,3,3-trimethyl-3H-indole-5-ol.

[0048] Figure 4 Schemes 5, 6, and 7 are shown, which respectively illustrate compounds derived from benzothiophene-pyrrole intermediates. 3 Initial symmetrical benzothiophene-pyrrole-cyanine dye compounds 11 (BtCy3) 12 (BtCy5) and 13 Representative synthetic routes for (BtCy7).

[0049] Figure 5 Scheme 8 shows the intermediate compound derived from benzothiophene-pyrrole. 6 Initial symmetrical benzothiophene-pyrrole-cyanine dye compounds 14 The representative synthetic route.

[0050] Figure 6 Schemes 9 and 10 are shown, which illustrate asymmetric benzothiophene-pyrrole-cyanine dye compounds. 15 and 17 Representative synthetic routes. Scheme 9 illustrates the synthesis of 1,2,3,3-tetramethyl-3H-indole-1- Iodides and intermediate compounds 3 The initial compound 15 : 4-((Z)-3,3-dimethyl-2-((2E,4E)-5-(1,3,3-trimethyl-3H-indole-1- -2-yl)pent-2,4-dien-1-ylidene)-2,3-dihydro-1H-benzo[4,5]thieno[2,3-b]pyrrolo-1-yl)butane-1-sulfonate / ester. Scheme 10 illustrates the preparation of 1-(2-carboxyethyl)-2,3,3-trimethyl-5-sulfono-3H-indole-1- Iodide intermediate compounds 16 and benzothiophene-pyrrole intermediate compounds 3 The initial compound 17: 1-(2-Carboxyethyl)-2-((1E,3E,5Z)-5-(3,3-dimethyl-1-(4-sulfonylbutyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-ylidene)pent-1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1- Preparation of sodium 5-sulfonate.

[0051] Figure 7 Schemes 11 and 12 are shown, which respectively illustrate asymmetric benzothiophene-pyrrole-cyanine dye compounds. 19 and 20 A representative synthetic route. Scheme 11 illustrates the synthesis from an indole intermediate compound. 18 and benzothiophene-pyrrole intermediate compounds 6 The initial compound 19 1-(2-Carboxyethyl)-3,3-dimethyl-2-((1E,3E,5E,7Z)-7-(3-methyl-1,3-bis(3-sulfopropyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-ylidene)hept-1,3,5-trien-1-yl)-3H-indole-1- Preparation of iodides. Scheme 12 shows the compound. 20 :3-(2-((1E,3E,5E)-7-((Z)-5-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)oxy)-1-(2-carboxyethyl)-3,3-dimethylindoline-2-yl)hept-1,3,5-trien-1-yl)-3-methyl-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- The preparation of 1-yl)propane-1-sulfonate / ester, which is derived from indole intermediate compounds 10 and benzothiophene-pyrrole intermediate compounds 6 Produced.

[0052] Figure 8 Scheme 13 is shown, which illustrates the use of N,N'-diphenylmantadin to extract from indole intermediate compounds 18 and benzothiophene-pyrrole intermediate compounds 6 Initial asymmetric benzothiophene-pyrrole-cyanine dye compounds 21 1-(2-Carboxyethyl)-3,3-dimethyl-2-((1E,3Z)-3-(3-methyl-1,3-bis(3-sulfopropyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-ylidene)prop-1-en-1-yl)-3H-indole-1- Representative synthetic pathways for iodides.

[0053] Figure 9 Schemes 14 and 15 are shown, which illustrate asymmetric benzothiophene-pyrrole-cyanine dye compounds. 24 Representative synthetic routes. Scheme 14 shows intermediate compounds starting from 2,3,3-trimethyl-3H-indole-5-ol. 23 1-(2-Carboxyethyl)-2,3,3-trimethyl-5-(3-sulfonylpropoxy)-3H-indole-1- Synthetic pathways for iodides. Scheme 15 illustrates the synthesis from indole intermediate compounds. 23 and benzothiophene-pyrrole intermediate compounds 6 The initial compound 24 The representative synthetic route.

[0054] Figure 10 Symmetrical benzothiophene-pyrrole-cyanine dye compounds are shown. 12 The chemical structure of (BtCy5) is shown. The absorption spectrum of BtCy5 is shown in the lower left, exhibiting an extinction coefficient ε of 88k at 859 nm in MeOH. The emission spectrum of BtCy5 is shown in the lower right, exhibiting a maximum λem of 878 nm in methanol (MeOH).

[0055] Figure 11 Asymmetric benzothiophene-pyrrole-cyanine compounds were shown. 15 The chemical structure of the compound. 15 The absorption spectrum in methanol (MeOH) is shown in the lower left, exhibiting a maximum λabs at 740 nm and an extinction coefficient of approximately 90 k at 740 nm. Compound 15 The emission spectrum in MeOH is shown in the lower right corner, with a maximum λem of 765 nm.

[0056] Figure 12 Symmetrical benzothiophene-pyrrole-cyanine dye compounds are shown. 13 The chemical structure of (BtCy7). The absorption spectrum of BtCy7 is shown in the lower left, with a maximum λabs of 973 nm. The emission spectra of BtCy7 in methanol after excitation at 972 nm (a) or 808 nm (b) are shown in the lower right. Symmetrical benzothiophene-pyrrole-cyanine dye compound. 13 It exhibits a maximum λem of 997nm.

[0057] Figure 13 This illustrates the relationship between symmetrical benzothiophene-pyrrole-cyanine dye compounds. 14 Symmetrical benzothiophene-pyrrole-cyanine dye compounds with overlapping absorption spectra in MeOH (maximum λabs 863 nm) or PBS 12Absorption spectra of these materials in MeOH (maximum λabs 860 nm), and their chemical structures.

[0058] Figure 14 An exemplary synthetic scheme for preparing and purifying BtCy-antibody conjugates is shown.

[0059] Figure 15 Scheme 17 is shown, which illustrates the relationship between indole intermediates and benzothiophene-pyrrole intermediates. 3 Initial asymmetric benzothiophene-pyrrole-cyanine dye compounds 25 The representative synthetic route.

[0060] Figure 16 Schemes 18 and 19 are shown, which illustrate benzothiophene-pyrrole intermediate compounds. 26 Asymmetric benzothiophene-pyrrole-cyanine dye compounds 27 The representative synthetic route.

[0061] Figure 17 Schemes 20 and 21 are shown, which illustrate indole intermediate compounds. 28 Asymmetric benzothiophene-pyrrole-cyanine dye compounds 29 The representative synthetic route.

[0062] Figure 18 Scheme 22 is shown, which illustrates an asymmetric benzothiophene-pyrrole-cyanine dye compound. 30 The representative synthetic route.

[0063] Figure 19 Schemes 23 and 24 are shown, which illustrate benzoindole intermediate compounds. 32 Asymmetric benzothiophene-pyrrole-cyanine dye compounds 33 The representative synthetic route. Detailed Implementation

[0064] I. Overview

[0065] This disclosure provides novel BtCy fluorescent compounds, and labeled specific binding pairs comprising BtCy compounds having covalently linked specific binding pairs. In some embodiments, the BtCy compound and the labeled specific binding pair have been designed to be water-soluble. This disclosure also provides tandem dyes comprising the BtCy compound and / or the labeled specific binding pair.

[0066] The BtCy compounds disclosed herein exhibit excitation maxima of about 500 to 1200 nm, 575 to 1200 nm, or 750 to 1200 nm, or 600 to 1000 nm.

[0067] In some embodiments, the BtCy compounds of this disclosure can be excited using blue, green, yellow, orange, red, near-infrared (NIR), or infrared (IR) wavelengths.

[0068] In some embodiments, the BtCy compounds of this disclosure can be excited using yellow, orange, red, or near-infrared (NIR) wavelengths.

[0069] A method is provided for detecting target analytes in a sample using fluorescent BtCy compounds or BtCy tandem dyes conjugated with binding couplers.

[0070] A kit is also provided, comprising at least one fluorescent BtCy compound according to this disclosure, a labeled specific binding partner, or a tandem dye. The fluorescent BtCy compound or tandem dye may contain a conjugation tag.

[0071] II. Definition

[0072] The abbreviations used in this article have their conventional meanings in the fields of chemistry and biology.

[0073] Reference will now be made in detail to certain embodiments of the disclosed subject matter, some examples of which are partially illustrated in the accompanying drawings. Although the disclosed subject matter will be described in conjunction with the recited claims, it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0074] Throughout this document, values ​​expressed in range format should be interpreted flexibly to include not only the explicitly stated numerical value as a limit to the range, but also all individual numerical values ​​or subranges covered within that range, as if each numerical value and subrange were explicitly stated. For example, the range “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. Unless otherwise stated, the expression “about X to Y” has the same meaning as “about X to about Y”. Similarly, unless otherwise stated, the expression “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z”.

[0075] In this document, unless the context clearly specifies otherwise, nouns without quantifiers are used to include one / more / a kind. Unless otherwise stated, the term "or" is used to mean non-exclusive "or". The expressions "at least one of A and B" or "at least one of A or B" have the same meaning as "A, B, or A and B". Furthermore, it should be understood that the wording or terms used herein, without further definition, are for descriptive purposes only and not for limitation. The use of any section headings is intended to aid reading the document and is not to be construed as restrictive; information relating to a section heading may appear within or outside that particular section. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if individually. In the event of any inconsistency between the usage in this document and those documents incorporated by reference, the usage in the incorporated references shall be considered supplementary to the usage in this document; in the case of irreconcilable inconsistencies, the usage in this document shall prevail.

[0076] In the methods described herein, actions may be performed in any order without departing from the principles of this disclosure, unless the timing or sequence of operations is explicitly specified. Furthermore, unless the explicit language of the claims states that the specified actions are performed separately, they may be performed simultaneously. For example, the claimed action of X and the claimed action of Y may be performed simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0077] The term “about” as used herein may allow for a degree of variability in the value or range, for example, within 10%, 5%, or 1% of the limit of the value or range, and includes the exact value or range. The term “substantially” as used herein means most or the majority, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or greater, or 100%. The term “substantially free” as used herein may mean that the substance is absent or present in trace amounts such that the amount of the substance present does not affect the material properties of the composition comprising the substance, such that about 0% by weight to about 5% by weight, or about 0% by weight to about 1% by weight, or about 5% by weight or less, or less than or equal to about 4.5% by weight, 4% by weight, 3.5% by weight, 3% by weight, 2.5% by weight, 2% by weight, 1.5% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0.7% by weight, 0.6% by weight, 0.5% by weight, 0.4% by weight, 0.3% by weight, 0.2% by weight, 0.1% by weight, 0.01% by weight, or about 0.001% by weight or less, or about 0% by weight, of the substance.

[0078] The term "ambient room temperature" refers to approximately 23 degrees Celsius.

[0079] Unless otherwise stated, the terms "percentage" or "%" refer to weight percentage.

[0080] The term "reactive group" refers to a functional group that, under certain conditions, can selectively react with another compatible functional group to form a covalent bond after one of the optionally activated functional groups. Chemically selective functional groups of interest include, but are not limited to, thiols, maleimides, halogenated maleimides, iodoacetamides, amines, alkyl carboxylates / esters, alkyl sulfonates / esters, carboxylic amines, carbamates, carboxylic esters, N-hydroxysuccinimidyl (NHS), imine esters, halogens, borate esters, boric acids, hydrazones, carboxylic acids or their reactive esters, and groups that can react with each other via click chemistry, such as azides and alkynes (e.g., cyclooctyne), tetrazides and olefins (e.g., cyclooctene), dienes and dienophiles, sulfur(VI) fluorine exchange chemistry (SuFEX), sulfonyl fluorides, and hydroxyl, hydrazide, hydrazine, aldehydes, ketones, azides, alkynes, phosphine, epoxides, etc., or their protected groups. Reactive groups can be conjugated tags. The chemically selective functional group can be protected or unprotected.

[0081] The term "amine reactive group" refers to any group that forms a chemical bond with a primary amine. Amine reactive groups of interest include, but are not limited to, isothiocyanates / esters, isocyanates / esters, acyl azides, NHS esters, imine esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, ethylene oxides, carbonates / esters, aryl halides, imine esters, carbodiimides, acid anhydrides, and fluorophenyl esters. The amine reactive group can be either an NHS ester or an imine ester.

[0082] In some cases, non-covalent linkages can involve specific binding between two target moieties (e.g., two affinity moieties, such as a hapten and antibody, or a biotin moiety and streptavidin). In other cases, non-covalent linkages can involve the uptake of a substrate.

[0083] When referring to compounds of this disclosure, the term "symmetric" means that each terminal heterocyclic ring system is identical, and the substituents may be the same or different. In some symmetric compounds, n = 2.

[0084] When referring to compounds of this disclosure, the term "asymmetric" means that each terminal heterocyclic ring system is different. In some asymmetric compounds, n = 3.

[0085] The term "counter ion" refers to an ion that is in charge balance with the fluorescent compound according to this disclosure. Counter ions can be cations. Counter ions can be anions. In some cases, counter ions can be halide ions, perchlorate ions, PF6 ions, etc. 6- Phosphate ions, sulfate ions, etc. Counter ions can be F... - Cl - , Br - I - ClO4 - CF3CO2 - CH3CO2 - PO4 3- SO4 2- BF4 - Etc. In some cases, the counter ion can be Na+. + K + Mg ++ Ca ++ wait.

[0086] For example, antibodies labeled with benzothiophene-pyrrole-cyanine dyes according to this disclosure can be used as reagents for displaying fluorescent signals in flow cytometry. Additionally, orthogonal "functional groups" may be included, which can be used for the bioconjugation or linkage of acceptor signal transduction chromophores in donor-acceptor benzothiophene-pyrrole tandem dyes.

[0087] As used herein, the term "organic group" refers to any carbon-containing functional part. Some examples may include: oxygen-containing groups, such as alkoxy, aryloxy, arylalkoxy, and oxo (carbonyl) groups; amino groups, including alkylamine esters and sulfonamide groups; carboxyl groups, including carboxylic acids, carboxyl salts / esters, and carboxylic esters; sulfur-containing groups, such as alkyl and aryl sulfide groups, thiols, thiol reactive groups, and sulfone groups; maleimide; iodoacetamide; azide groups; alkyne groups; and other heteroatom-containing groups. Some non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O)R, methylenedioxy, ethylenedioxy, N(R)2, N3, S(H)R, SOR, SO2R, SO2N(R)2, SOxR, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2) 0-2 N(R)C(O)R,(CH2) 0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N( R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (C1-C 100 R can be a hydrocarbon group, wherein R can be hydrogen (in instances containing other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

[0088] As used herein, the term "heteroatom" refers to any suitable non-carbon atom inserted between adjacent carbon atoms in an organic group, such as N, O, S, Se, P, B, Al, Si, and Ge. The organic group can be cyclic, aryl, or straight-chain or branched (e.g., alkyl or olefinic). More than one heteroatom may be inserted between adjacent carbon atoms (e.g., 1, 2, 3, 4, or 5 heteroatoms). Heteroatoms may be oxidized to form, for example, but not limited to, -S(O)- and S(O)2-, sulfinates / esters, and sulfonamide moieties.

[0089] The term “substituted” as used herein in conjunction with the molecule or organic group as defined herein refers to a state in which one or more hydrogen atoms contained herein are replaced by one or more non-hydrogen atoms (e.g., alkyl, aryl, or functional groups). A “substituted” group may contain one or more groups selected from halogen, hydroxy, amino, alkylamino, amide, acyl, nitro, cyano, and alkoxy.

[0090] As used herein, the terms “functional group,” “functional moiety,” or “substituent” refer to a group that can be substituted into or is substituted into a molecule or an organic group. Some examples of substituents or functional groups include, but are not limited to: halogens (e.g., F, Cl, Br, and I); alkenes; oxygen atoms in groups such as hydroxyl, alkoxy, aryloxy, arylalkoxy, oxo (carbonyl) groups, carboxyl groups (including carboxylic acids, carboxylates / esters, and carboxylic esters); sulfur atoms in groups such as thiols, alkyl and aryl sulfides, sulfoxides, sulfones, sulfonyl groups, and sulfonamides; nitrogen atoms in groups such as amines, hydroxylamines, nitriles, nitro groups, N-oxides, hydrazides, azides, tetrazides, imides (e.g., maleimides), and enamines; and other heteroatoms in a variety of other groups. Some non-limiting examples of substituents that can bond with substituted carbon (or other) atoms include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azides, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2) 0-2 N(R)C(O)R,(CH2) 0-2 N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C (O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R,

[0091] Where R can be hydrogen or a carbon-based fraction; for example, R can be hydrogen, (C1-C2)2, or (C1-C2)2. 100The R group may be a hydrocarbon group, alkyl group, acyl group, cycloalkyl group, aryl group, aralkyl group, heterocyclic group, heteroaryl group, or heteroarylalkyl group; or the two R groups bonded to a nitrogen atom or adjacent nitrogen atom may form a heterocyclic group together with one or more nitrogen atoms. The subject fluorescent compound may contain one or more "functional groups" provided for bioconjugation. In some cases, such functionality may be used to covalently link biomolecules or binder couplers, such as proteins, peptides, affinity ligands, antibodies, antibody fragments, polynucleotides, or aptamers. In some cases, the functional group may be selected from amines, carbamates, carboxylic acids, carboxylates / esters, maleimides, activated esters, N-hydroxysuccinimides, hydrazides, acylhydrazides, hydrazones, isothiocyanates, azides, alkynes, alkenes, tetrazides, aldehydes, thiols, and their protected groups for conjugation with substrates, acceptor dyes, functional moieties, or binder couplers. The functional group may be protected or unprotected. The functional group can be a reactive or chemoselective functional group that can react with another group via click chemistry, such as cycloalkenes (e.g., cyclooctene), alkynes, cycloalkynes (e.g., cyclooctynyl, such as bicyclo[6.1.0]nonyne (BCN)), dibenzocyclooctyne (DBCO)), cycloalkenes (e.g., cyclooctenyl, such as COtrans-cyclooctene (TCO)), azide groups, and tetrazinyl groups.

[0092] As used herein, the term “activated ester” or “activated ester” either on its own or as part of another substituent refers to a carboxyl-activating group in peptide chemistry that facilitates the condensation of a carboxyl group with a free amino group of an amino acid derivative. Descriptions of these carboxyl-activating groups can be found in common textbooks of peptide chemistry, such as K.D. Kopple, “Peptides and Amino Acids,” W.A. Benjamin, Inc., New York, 1966, pp. 50–51; and E. Schroder and K. Lubke, “The Peptides,” Vol. 1, Academic Press, New York, 1965, pp. 77–128, each of which is incorporated herein by reference in its entirety.

[0093] The term “ammonium” as used herein, either on its own or as part of another substituent, refers to ammonium having the formula NHR3. + The cation, wherein each R group is independently hydrogen or a substituted or unsubstituted alkyl, aryl, aralkyl, or alkoxy group. Preferably, each R group is hydrogen.

[0094] As used herein, the term "hydrocarbon" or "hydrocarbon group" refers to a molecule or functional group containing carbon and hydrogen atoms. The term may also refer to a molecule or functional group that typically contains both carbon and hydrogen atoms, but in which some or all of the hydrogen atoms are substituted by another functional group. The term "hydrocarbon group" refers to a functional group derived from a straight-chain, branched, or cyclic hydrocarbon and can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. A hydrocarbon group can be represented as (C... a -C b (C1-C4) hydrocarbon group, where a and b are integers and refer to a number of carbon atoms from a to b. For example, (C1-C4) hydrocarbon group means that the hydrocarbon group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0-C5) hydrocarbon group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4). b The term "hydrocarbon group" means that in some embodiments there is no hydrocarbon group. A hydrocarbon subgroup is a divalent hydrocarbon, for example, a hydrocarbon bonded at two positions.

[0095] As used herein, the term "alkyl" itself, or as part of another substituent, refers to a straight-chain or branched saturated aliphatic group having the indicated number of carbon atoms. Alkyl groups can be optionally substituted. For example, C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, etc. Alkyl groups can contain any number of carbons, for example, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, and 5 to 6 carbons. Alkyl groups can also refer to alkyl groups having up to 20 carbon atoms, such as, but not limited to, heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or unsubstituted. Unless otherwise stated, a “substituted alkyl” group may be substituted by one or more groups selected from halogens, hydroxyl groups, amino groups, alkylamino groups, amide groups, acyl groups, nitro groups, cyano groups, and alkoxy groups. Alkyl groups are usually monovalent, but can also be divalent, for example, when the alkyl group links two parts together.

[0096] As used herein, the term "alkylene" refers to an alkyl group as defined above (i.e., a divalent alkyl group) that is attached to at least two other groups. The two portions attached to the alkylene group may be attached to the same carbon atom or different carbon atoms of the alkylene group.

[0097] As used herein, the term "alkoxy" itself, or as part of another substituent, refers to an alkyl group as defined above, having an oxygen atom connecting the alkyl group to a linking point. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkoxy groups may be further substituted by a variety of substituents described herein. For example, alkoxy groups may be halogenated to form a "halo-alkoxy" group.

[0098] As used herein, the term "olefin" or "alkenyl" itself, or as part of another substituent, refers to a straight-chain, branched, or cyclic hydrocarbon having at least one double bond between two carbon atoms. Some examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hextrienyl. Alkenyl groups are typically monovalent, but can also be divalent, for example, when the alkenyl group links two parts together.

[0099] As used herein, the term "alkynyl" or "alkynyl group" refers, either on its own or as part of another substituent, to a straight-chain or branched hydrocarbon having at least one triple bond between two carbon atoms. Some examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butyrynyl, 1-pentynyl, 2-pentynyl, isopentenynyl, 1,3-pentyrynyl, 1,4-pentyrynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hextriynyl. Alynyl groups are typically monovalent, but can also be divalent, for example, when the alkynyl group links two parts together.

[0100] As used herein, the term "acyl" refers to a group containing a carbonyl moiety, wherein the group is bonded by a carbonyl carbon atom. The carbonyl carbon atom is bonded to hydrogen to form a "formyl" group, or to another carbon atom that may be part of an alkyl, aryl, aralkylcycloalkyl, cycloalkylalkyl, heterocyclic, heterocyclic alkyl, heteroaryl, heteroaryl, or heteroarylalkyl group. An acyl group may contain 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group may contain double or triple bonds as defined herein. An acyl group may optionally also contain heteroatoms as defined herein. Some examples of acyl groups include, but are not limited to, nicotinyl (pyridyl-3-carbonyl)acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl. When the group containing a carbon atom bonded to the carbonyl carbon atom contains a halogen, the group is called a "haloacyl" group. An example is trifluoroacetyl.

[0101] As used in this article, the term "aldehyde" itself, or as part of another substituent, refers to compounds having a -CHO group.

[0102] As used herein, the term "aryl" itself, or as part of another substituent, refers to a cyclic aromatic hydrocarbon group that does not contain heteroatoms in an aromatic ring assembly. An "aryl" group can be a monocyclic or fused bicyclic, tricyclic, tetracyclic, pentacyclic, or larger aromatic ring assembly containing 6 to 22, 14 to 22, 17 to 22, or 6 to 16 ring carbon atoms. For example, an aryl group can be, but is not limited to, phenyl, azulel, heptalenyl, biphenyl, indacenyl, fluorenel, phenanthrenyl, triphenylene, pyrene, and naphthacenyl. Aryl, biphenyl, anthracene, benzyl, or naphthyl, etc. In some cases, the aryl group can be phenyl.

[0103] The term "monocyclic aryl" refers to an unsubstituted or substituted aryl ring system comprising one aryl ring having or not having a fused cycloalkyl or cycloalkenyl ring.

[0104] The term "monocyclic heteroaryl" refers to an unsubstituted or substituted heteroaryl ring system comprising one aryl ring having or not having a fused cycloalkyl or cycloalkenyl ring, wherein the monocyclic ring system comprises one or more, two or more, three or more, or four or more heteroatoms. Some non-limiting examples of monocyclic heteroaryls are substituted or unsubstituted pyridyl, pyranyl, thiophenyl, furanyl, pyrroleyl, pyrazolyl, imidazolyl, thiazolyl, etc. Azolium group.

[0105] The term "polycyclic aryl" refers to an unsubstituted or substituted polycyclic system comprising 2 to 9, 2 to 8, or 2 to 6 aryl rings, with or without fused cycloalkyl or cycloalkenyl rings. Some examples of polycyclic aryl groups may include fluorene, 9H-fluorene, phenanthrene, dihydrophenanthrene, 9,10-dihydrophenanthrene, naphthalene, anthracene, tetraphenylene, pentaphenylene, etc.

[0106] The term "polycyclic heteroaryl" refers to an unsubstituted or substituted polycyclic ring system comprising 2 to 9, 2 to 8, or 2 to 6 aryl rings having or not having fused cycloalkyl or cycloalkenyl rings, wherein the polycyclic ring system contains one or more, two or more, three or more, or four or more heteroatoms. Some non-limiting examples of polycyclic heteroaryl systems may include quinoline, benzo[a], etc. azole, benzothiazole, benzimidazole, indole, benzoindole, pyridine Benzopyran Thiopyran 6,8-Dihydro-5H-naphtho[2,1-f]indole, 4,5-Dihydro-3H-naphtho[2,1-e]indole, 6,7-Dihydro-3H-naphtho[2,1-g]indole, 5,6-Dihydrophenanthrene[3,2-d]thiazole, 4,5-Dihydrophenanthrene[2,1-d]thiazole, 6,7-Dihydrophenanthrene[4,3-d]thiazole, 5,6-Dihydrophenanthrene[3,2-d] Azole, 4,5-dihydrophenanthrene[2,1-d] Azole, 6,7-dihydrophenanthrene[4,3-d] 5,6-Dihydronaphtho[2,1-g]quinoline, 7,8-Dihydronaphtho[2,1-h]quinoline, 5,6-Dihydronaphtho[2,1-f]quinoline, 5,6-Dihydro-8λ2-phenanthro[3,2-d]imidazole, 4,5-Dihydro-3λ2-phenanthro[2,1-d]imidazole, 6,7-Dihydro-3λ2-phenanthro[4,3-d]imidazole, 5,6-Dihydronaphtho[1,2-g]quinoxaline, 5,6-Dihydronaphtho[2,1- [f]quinoxaline, 7,8-dihydronaphtho[1,2-f]quinoxaline, 5,6-dihydropentanofenzo[3,2,1-cd:10,11,12-c'd']diindole, 3,8-dihydrophenanthrene[2,3-e:7,6-e']diindole, 3,5,6,8-tetrahydrophenanthrene[2,3-e:7,6-e']diindole, 1,2,3,5,6,8-hexahydrophenanthrene[2,3-e:7,6-e']diindole-1,10-di Salt, 3,5,6,8-tetrahydrophenanthrene[2,3-e:7,6-e']diindole-ethane(1 / 1), 5,6-dihydropentanfen[3,4-d:10,9-d']bis( 1,2,5,6,9,10-hexahydropentanol[3,4-d:10,9-d']bis(thiazolyl)-1,10-di 1,2,9,10-Tetrahydropentafenofenza[3,4-d:10,9-d']bis(thiazole), 1,2,9,10-tetrahydropentafenofenza[3,4-d:10,9-d']bis(thiazole)-1,10-di 6,7-Dihydrophenanthrene[2,3-f:7,6-f']diquinoline-1,12-di 6,7-Dihydrophenanthrene[2,3-f:7,6-f']diquinoline, 2,6,7,11-tetrahydrobenzo[1,2-g:4,3-g']dichromene, 2,11-dihydrobenzo[1,2-g:4,3-g']dichromene, 5,10-dihydro-6H-naphtho[2,1-g]chromene, 10H-naphtho[2,1-g]chromene, 2,6,7,11-tetrahydrobenzo[1,2-g:4,3-g']bis(thiochromene), 2,11-dihydrobenzo[1,2-g:4,3-g']bis(thiochromene), etc.

[0107] The term "arylene" refers to a divalent group derived from an aryl group. The aryl group can be selected from one, two, or three of the following groups: alkyl, alkoxy, aryl, hydroxyl, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy, and oxy-C2-C3-alkylene; all of these may optionally be further substituted, for example, as defined above; or the aryl group may be 1- or 2-naphthyl; or 1- or 2-phenanthrene. Alkylenedioxy is a divalent substituent attached to two adjacent carbon atoms of a phenyl group, such as methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms of a phenyl group, such as oxoethylene or oxopropylene. An example of oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0108] The aryl group may include substituted aryl groups. Substituted aryl groups include, for example, but not limited to, naphthyl or phenyl, optionally mono- or di-substituted with: alkoxy, phenyl, halogen, alkyl or trifluoromethyl, hydroxyl, C1-C... 12 Alkyl, C2-C 12 Olefins, C2-C 12 Alkynes, C3-C 12 cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy group, C2-C 18 (Miscellaneous) arylamino, carboxylates / esters, carboxylic acids, C2-C 12 Alkyl carboxylic acids, C2-C 12Alkyl carboxylates / esters, C2-C 12 Alkyl carboxylic esters, C1-C 12 Alkoxy groups, water-solubilizing groups (WSG), functional groups, sulfonic acids, sulfonates / esters, C1-C 12 Alkyl sulfonates / esters. In some cases, the substituted aryl group, such as naphthyl or phenyl, may be monosubstituted or disubstituted with a functional group, a WSG (optionally containing a functional group, an alkoxy group, a halogen, or a trifluoromethyl group). The WSG may be a branched WSG that optionally contains a functional group, such as a WSG containing PEG and a functional group.

[0109] As used herein, the term "aryloxy group" refers, either on its own or as part of another substituent, to an O-aryl group, where the aryl group is as defined above. An aryloxy group may be unsubstituted or substituted with one or two suitable substituents. The term "phenoxy group" refers to an aryloxy group in which the aryl moiety is a benzene ring. As used herein, the term "(hetero)aryloxy group" means -O-heteroaryl, where the heteroaryl group is as defined below. The term "(hetero)aryloxy group" is used to indicate that the moiety is an aryloxy group or a (hetero)aryloxy group.

[0110] As used herein, the term "aralkyl" refers to an alkyl group in which the hydrogen or carbon bonds of the alkyl group are replaced by bonds of an aryl group as defined herein. Representative aralkyl groups include benzyl and phenethyl, as well as fused (cycloalkylaryl)alkyl groups, such as 4-ethyl-indenyl. Arylene is an alkenyl group in which the hydrogen or carbon bonds of the alkyl group are replaced by bonds of an aryl group as defined herein.

[0111] As used herein, the term "amine," whether used alone or as part of another substituent, refers to an alkyl group as defined herein having one or more amino groups. The amino group can be a primary, secondary, or tertiary amino group. Alkylamines may optionally be further substituted, for example, by a hydroxyl group. Amines that can be used in this disclosure include, but are not limited to, ethylamine, propylamine, isopropylamine, ethylenediamine, and ethanolamine. The amino group may connect the alkylamine to the remaining portion of the compound at the ω-position of the alkyl group, or connect at least two carbon atoms of the alkyl group together. Those skilled in the art will understand that other alkylamines can be used in this disclosure.

[0112] As used herein, the term "amino" refers to substituents in the following forms: -NH2, -NHR, -NR2, -NR3. + (where each R is chosen independently), and the protonated form of each, except -NR3. +Furthermore, it cannot be protonated. Therefore, any compound substituted with an amino group can be considered an amine. In this context, "amino" can refer to a primary, secondary, tertiary, or quaternary amino group. An "alkylamino" group can include monoalkylamino, dialkylamino, or trialkylamino groups.

[0113] The term "amide" refers to a functional group having a carbonyl group attached to an amine group, having the general formula RC(=O)NR'R", where R, R', and R" represent organic groups or hydrogen atoms. The term "amide group" refers to a substituent containing an amide group.

[0114] As used herein, the term "carbamate" refers, either on its own or as part of another substituent, to a functional group having the structure -NR"CO2R', wherein R' and R" are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl. Some examples of carbamates include t-Boc, Fmoc, benzyloxy-carbonyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-sulfonyl)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, Tbfmoc, Climoc, Bimoc, DBD-Tmoc, Bsmoc, Troc, Teoc, 2-phenylethyl carbamate, Adpoc, 2-chloroethyl carbamate, 1,1-dimethyl-2-haloethyl carbamate, DB-t-BOC, TCBOC, Bpoc, t-Bumeoc, Pyoc, Bnpeoc, V-(2-neopentylamino)-1,1-dimethylethyl carbamate, and NpSSPeoc.

[0115] The term “carboxylic acid” as used in this article, either on its own or as part of another substituent, refers to the structure R-COOH, where R is a carbon-containing atomic group.

[0116] The term "carboxylate / ester" as used herein, either on its own or as part of another substituent, refers to the conjugate base of a carboxylic acid, which is typically derived from the formula RCOO. - For example, the term "magnesium carboxylate" refers to the magnesium salt of a carboxylic acid. The term "carboxylic acid ester" as used herein, either alone or as part of another substituent, refers to a compound derived from a carboxylic acid, which is generally represented by the formula RCOOR', where R' can be an alkyl, alkene, alkyne, haloalkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, (unsubstituted aryl)alkyl, and (unsubstituted aryl)oxy-alkyl or other carbon-containing atomic groups. R' may optionally contain a functional group.

[0117] As used herein, the term "cycloalkyl" itself, or as part of another substituent, refers to a saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic cyclic assembly comprising 3 to 12 ring atoms or the indicated number of atoms. Cycloalkyl groups may contain any number of carbons, such as C10. 3-6 C 4-6 C 5-6 C 3-8 C 4-8 C 5-8 C 6-8 C 3-9 C 3-10 C 3-11 , and C 3-12 Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2]bicyclooctane, decahydronaphthalene, and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative partially unsaturated cycloalkyl groups include, but are not limited to: cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene. When the cycloalkyl group is a saturated monocyclic C 3-8 When cycloalkyl is used, some exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. When the cycloalkyl group is a saturated monocyclic C 3-6 When cycloalkyl is used, some exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The cycloalkyl group can be substituted or unsubstituted. Unless otherwise stated, a "substituted cycloalkyl" group can be substituted with one or more groups selected from halogens, hydroxyl, amino, alkylamino, amide, acyl, nitro, cyano, and alkoxy. The cycloalkyl group can be KR... 13 H, halogens, OC 1-6 Alkyl, SC 1-6 Alkyl, O-aryl, S-aryl, NHC 1-6 Alkyl groups, Ph-NCS, Ph-CO2H, and Ph-(CH2) 1-4 CO2H substitution. The term "lower cycloalkyl" refers to cycloalkyl groups having three to seven carbons, including, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Monocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, norbornene, decahydronaphthalene, and adamantane. For example, C 3-8 Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornene. Polycyclic systems can be substituted or unsubstituted.

[0118] As used herein, the term "cycloalkylene" refers to a cycloalkyl group as defined above (i.e., a divalent cycloalkyl group) that is attached to at least two other groups. The two parts attached to the cycloalkylene group may be attached to the same atom or different atoms of the cycloalkylene group.

[0119] As used herein, the term "haloalkyl" itself, or as part of another substituent, refers to an alkyl group as defined above in which some or all of its hydrogen atoms are replaced by halogen atoms. Halogen (halogenated) preferably means chlorinated or fluorinated, but can also be brominated or iodinated. For example, haloalkyl groups include trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluorophenyl, etc. The term "perfluoro" defines a compound or group having at least two available hydrogen atoms replaced by fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0120] The term "halogen" as used in this article, either on its own or as part of another substituent, refers to fluorine, chlorine, bromine, and iodine.

[0121] As used herein, the term "heteroaryl" refers, either on its own or as part of another substituent, to a monocyclic or fused polycyclic aromatic ring, such as a bicyclic, tricyclic, tetracyclic, or pentacyclic aromatic ring assembly, comprising, for example, 5 to 22, 14 to 22, 17 to 22, 6 to 16, or 5 to 16 ring atoms, wherein 1 to 4 ring atoms may be heteroatoms, such as N, O, or S. Additional heteroatoms may also be useful, including but not limited to B, Al, Si, or P. Heteroaryl groups may be substituted or unsubstituted. Substituted heteroaryl groups may contain one or more KRs. 13 Halogen, OC 1-6 Alkyl, SC 1-6 Alkyl, O-aryl, S-aryl, NHC 1-6 Alkyl groups, Ph-NCS, Ph-CO2H, Ph-(CH2) 1-4 CO2H substituent.

[0122] As used herein, the terms “heteroalkyl” or “heteroalkoxy” refer, either on their own or as part of another substituent, to an alkyl or alkoxy group, preferably C1-C. 12 Alkyl or C1-C 12Alkoxy groups, wherein the carbon atom is substituted with a heteroatom (e.g., N, O, or S). For example, heteroalkyl or heteroalkoxy groups may include ethers, thioethers, and alkylamines. Additional heteroatoms may also be useful, including but not limited to B, Al, Si, or P. Heteroatoms may be oxidized to form, for example, but not limited to, -S(O)-, -S(O)2-, sulfinates / esters, and sulfonamide moieties. The heteroatom moiety of a heteroalkyl group may substitute the hydrogen atom of the alkyl group to form a hydroxyl, thio, or amino group. Alternatively, the heteroatom moiety may be a linking atom or inserted between two carbon atoms. For example, heteroaryl groups may include pyridinyl, indolyl, indazole, quinoxalinyl, quinolinyl, isoquinolinyl, benzothiophene, benzofuranyl, furanyl, pyrroleyl, thiazolyl, benzothiazolyl, etc. azole group, iso The group may contain azole, triazolyl, tetrazolyl, pyrazolyl, imidazole, thiophene, or any other substituted group, especially those mono- or di-substituted by, for example, alkyl, nitro, or halogen. Pyridyl may represent 2-, 3-, or 4-pyridyl, advantageously 2- or 3-pyridyl. Thiophene may represent 2- or 3-thienyl. Quinolinyl may preferably represent 2-, 3-, or 4-quinolinyl. Isoquinolinyl may preferably represent 1-, 3-, or 4-isoquinolinyl. Benzopyranyl and benzothiopyranyl may preferably represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl may preferably represent 2- or 4-thiazolyl, and most preferably 4-thiazolyl. Triazolyl may preferably represent 1-, 2-, or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl. The heteroaryl group may include aryloxy or arylamino groups. In some embodiments, the heteroaryl group is pyridyl, indolyl, quinolinyl, pyrroleyl, thiazolyl, isoaryl, etc. Azolyl, triazolyl, tetrazolyl, pyrazolyl, imidazole, thiophenyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothiaphenyl Azolium, indazole, or any substituted group, especially monosubstituted or disubstituted groups.

[0123] The term "heterocyclic alkyl" itself, or as part of another substituent, refers to a monocyclic or fused polycyclic compound, such as a bicyclic, tricyclic, tetracyclic, or pentacyclic aromatic ring assembly, comprising, for example, 5 to 22, 14 to 22, 17 to 22, 6 to 16, or 5 to 16 ring atoms, wherein 1 to 4 ring atoms may be heteroatoms, each independently selected from N, O, or S. Additional heteroatoms may also be useful, including but not limited to B, Al, Si, or P. Heterocyclic alkyl groups may be substituted or unsubstituted. Substituted heterocyclic alkyl groups may contain one or more KRs. 13 Halogen, OC 1-6 Alkyl, SC 1-6 Alkyl, O-aryl, S-aryl, NHC 1-6Alkyl groups, Ph-NCS, Ph-CO2H, Ph-(CH2) 1-4 CO2H substituent.

[0124] As used herein, the term "heteroalkylene" refers to a heteroalkylene group (i.e., a divalent heteroalkylene group) as defined above, which is attached to at least two other groups. The two parts attached to the heteroalkylene group may be attached to the same atom or different atoms of the heteroalkylene group.

[0125] As used herein, the term "(hetero)arylamino" refers, either on its own or as part of another substituent, to an amine group substituted with an aryl group (e.g., -NH-aryl). Arylamino can also be an aryl group substituted with an amine group (e.g., -aryl-NH2). Arylamino can be substituted or unsubstituted.

[0126] In some embodiments, the substituents of the aryl, heteroaryl, and heteroalkyl groups are different and selected from: -halogen, -OR′, -OC(O)R′, -C(O)R′, -NR′R″, -SR′, -R′, -CN, -NO2, -CO2R′, -CONR′R″, -C(O)R′, -OC(O)NR′R″, -NR″C(O)R′, -NR″C(O)2R′, -NR′-C(O)NR″R″′, -NH-C(NH2)=NH, -NR′C(NH2)=NH, -NH-C(NH2)=NR′, -S(O)R′, -S(O)2R′, -S(O)2NR′R″, -N3.-CH(Ph)2,

[0127] The number of perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl compounds ranges from zero to the total number of open valences on the aromatic ring system; and wherein R', R” and R”' are independently selected from hydrogen, (C1-C5)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0128] The two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CH2). q Substituents of -U- are used, where T and U are independently -NH-, -O-, -CH2-, or single bonds, and q is an integer from 0 to 2. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by the formula -A-(CH2). rThe substituents -B- are replaced, where A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR′- or single bonds, and r is an integer from 1 to 3. One of the single bonds in the newly formed ring may optionally be replaced by a double bond. Alternatively, the two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by the formula -(CH2). s -X'-(CH2) t - Substituent substitution, wherein s and t are independently integers from 0 to 3, and X' is -O-, -NR′-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR′-. The substituent R' in -NR′- and -S(O)2NR′- is selected from hydrogen or unsubstituted (C1-C6) alkyl groups.

[0129] As used herein, the term "oligoether" is understood to mean an oligomer containing a repeating structural unit with ether functionality. As used herein, "oligomer" is understood to mean a molecule containing one or more identifiable repeating structural units of the same or different formulas.

[0130] The terms “polyethylene oxide,” “PEO,” “polyethylene glycol,” and “PEG” are used interchangeably and refer to substances containing the formula -(CH2-CH2-O). n- or its derivatives, describing the polymeric portion of the chain. PEG can be linear or branched. In some embodiments, "n" is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 3 to 15 or 10 to 15. The PEG group used herein includes, but is not limited to, PEG, modified PEG, linked PEG, amide PEG, sulfonamide PEG, phosphoramide-PEG, alkylsulfonamide-PEG, and alkoxysulfonamide-PEG. It should be understood that the PEG polymeric portion can have any convenient length and can contain a variety of terminal groups and / or other substituent groups, including, but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amide terminal groups and / or substituent groups. PEG groups applicable to the subject compound include those described by S. Zalipsky in “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6(2), 150-165; by Zhu et al in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112(8), pp. 4687-4735; by J.M. Harris in “Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications”, Plenum Press, New York, NY (1992); and by J.M. Harris and S. Zalipsky in “Poly(ethylene glycol) Chemistry and Biological Applications”, ACS (1997). In some cases, the PEG and modified PEG moiety can be those taught in, for example, the following international patent applications:

[0131] WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951, WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964 U.S. Patent Nos. 4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990, 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966,

[0132] which are each incorporated herein by reference.

[0133] As used herein, the terms “sulfonate / ester functional group” or “sulfonate / ester” refer, either on their own or as part of another substituent, to both the free sulfonate anion (-S(=O)₂O⁻) and its salt. Therefore, the term sulfonate / ester encompasses sulfonates such as sodium sulfonate, lithium sulfonate, potassium sulfonate, and ammonium sulfonate.

[0134] As used herein, the term "sulfonamide" itself, or as part of another substituent, refers to a group of the formula -SO2NR2, wherein each R can independently be, for example, a water-soluble moiety, hydrogen, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, aryl, or functional group, and may contain a carboxylic acid group. R can be a water-soluble polymer, including but not limited to polymers comprising six or more monomer units, nonionic water-soluble polymers, such as PEG or modified PEG terminated with a carboxylic acid or carboxylic acid ester. "Sulfanamide" is linked to another molecule via a linker or bond. "Sulfanamide" can be, for example, sulfonamide-PEG, alkylsulfonamide, alkoxysulfonamide, alkylsulfonamide PEG, alkoxysulfonamide PEG, alkylsulfonamide PEG carboxylate / ester, and alkoxysulfonamide PEG carboxylate / ester.

[0135] As used herein, the term "sulfonamide group" refers, either on its own or as part of another substituent, to a group of the formula -SO2NR-, where R can be, for example, a water-soluble moiety, hydrogen, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, aryl, or functional group, and may contain a carboxylic acid group. R can be a water-soluble polymer, including but not limited to polymers containing six or more monomer units, nonionic water-soluble polymers, such as PEG or modified PEG terminated with a carboxylic acid or carboxylic acid ester. The "sulfonamide group" can be linked to another molecule via a linker or bond. The "sulfonamide group" can be, for example, sulfonamide-PEG, alkylsulfonamide, alkoxysulfonamide, alkylsulfonamide PEG, alkoxysulfonamide PEG, alkylsulfonamide PEG carboxylate / ester, and alkoxysulfonamide PEG carboxylate / ester.

[0136] As used herein, the term "sulfinamide" itself, or as part of another substituent, refers to a group of the formula -SONR2, wherein each R can independently be, for example, a water-soluble moiety, hydrogen, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, aryl, or functional group, and may contain a carboxylic acid group. R can be a water-soluble polymer, including but not limited to polymers comprising six or more monomer units, nonionic water-soluble polymers, such as PEG or modified PEG terminated with a carboxylic acid or carboxylic acid ester. "Sulfanamide" can be linked to another molecule via a linker or bond. "Sulfanamide" can be, for example, sulfinamide-PEG, alkylsulfinamide, alkoxysulfinamide, alkylsulfinamide PEG, alkoxysulfinamide PEG, alkylsulfinamide PEG carboxylate / ester, and alkoxysulfinamide PEG carboxylate / ester.

[0137] As used herein, the terms “hydrazine” and “acylhydrazine”, either on their own or as part of another substituent, refer to compounds containing a single-bonded nitrogen, one of which is a primary amine functional group. For example, the term “hydrazine” refers to a moiety having a -NHNH2 structure.

[0138] As used herein, the term "thiol" itself, or as part of another substituent, refers to a compound containing a functional group consisting of a sulfur-hydrogen bond. The general chemical structure of the thiol functional group is R-SH, where R represents an alkyl, alkene, aryl, or other carbon-containing atomic group.

[0139] The term "silyl" as used in this article, either on its own or as part of another substituent, refers to Si(R) z )3, where each R z It is independently an alkyl, aryl, or other carbon-containing atomic group.

[0140] The term "diazonium salt" as used in this article, either on its own or as part of another substituent, refers to a salt with the structure R-N2. + X - The group of an organic compound, wherein R can be any organic group (e.g., alkyl or aryl), and X is an inorganic or organic anion (e.g., halogen).

[0141] The term “trifluoromethanesulfonate” as used in this article, either on its own or as part of another substituent, refers to trifluoromethanesulfonate, which is a group having the formula CF3SO3.

[0142] The term "boronic acid" as used herein, either on its own or as part of another substituent, refers to the structure -B(OH)2. Those skilled in the art will recognize that boric acid can exist as a boronic ester. Boronic acid means esters that include such esters. The terms "boronic ester" or "boronate ester" as used herein refer to esters containing -B(OH)2. 1 (Z) 2 ) of the compounds, wherein Z 1 and Z 2 Together, they form a portion in which, in each case, the atom bonded to boron is an oxygen atom. In some embodiments, the borate ester portion is a 5-membered ring. In other embodiments, the borate ester portion is a 6-membered ring. In still other embodiments, the borate ester portion is a mixture of 5-membered and 6-membered rings.

[0143] The term "maleimide" as used in this article, either on its own or as part of another substituent, refers to the structure R can be, for example, a water-soluble moiety, hydrogen, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, aryl, halogen, or other group, and may include a carboxylic acid group. R can be a water-soluble polymer, including but not limited to Y polymers containing six or more monomer units, nonionic water-soluble polymers, PEG, and modified PEGs terminated with carboxylic acid or carboxylic acid esters.

[0144] The term “hydrazone” as used in this article, either on its own or as part of another substituent, refers to the structure R can be, for example, a water-soluble moiety, hydrogen, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, aryl, or other group, and may contain a carboxylic acid group. R can be a water-soluble polymer, including but not limited to polymers containing six or more monomer units, nonionic water-soluble polymers, PEG, and modified PEG terminated with carboxylic acid or carboxylic acid esters.

[0145] The term “azide” as used in this article, either on its own or as part of another substituent, refers to the structure -N3.

[0146] The term “N-hydroxysuccinimide group” as used in this article, either on its own or as part of another substituent, refers to the structure R can be, for example, a water-soluble moiety, hydrogen, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, aryl, or other group, and may contain a carboxylic acid group. R can be a water-soluble polymer, including but not limited to polymers containing six or more monomer units, nonionic water-soluble polymers, PEG, and modified PEG terminated with carboxylic acid or carboxylic acid esters.

[0147] The term "phosphoramide" as used in this article, either on its own or as part of another substituent, refers to the structure R can be, for example, a water-soluble moiety, hydrogen, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, aryl, or other group, and may contain a carboxylic acid group. R can be a water-soluble polymer, including but not limited to polymers containing six or more monomer units, nonionic water-soluble polymers, such as PEG or modified PEG terminated with carboxylic acid or carboxylic acid esters. "Phosphoramide" can be linked to another molecule via a linker or bond. "Phosphoramide" can be, for example, phosphoramide-PEG, alkylphosphoramide, alkoxyphosphoramide, alkylphosphoramide PEG, alkoxyphosphoramide PEG, alkylphosphoramide PEG carboxylate / ester, alkoxyphosphoramide PEG carboxylate / ester.

[0148] The term "phosphonamide ester" as used in this article, either on its own or as part of another substituent, refers to the structure R can be, for example, a water-soluble moiety, hydrogen, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, aryl, or other group, and may include a carboxylic acid group. R can be a water-soluble polymer, including but not limited to polymers containing six or more monomer units, nonionic water-soluble polymers, such as PEG or modified PEG terminated with carboxylic acid or carboxylic acid ester. "Fosmine" can be linked to another molecule via a linker or bond. "Fosmine" can be, for example, phosmine-PEG, alkylphosmine, alkoxyphosmine, alkylphosmine PEG, alkoxyphosmine PEG, alkylphosmine PEG carboxylate / ester, alkoxyphosmine PEG carboxylate / ester.

[0149] The term "phosphatidylamide" as used in this article, either on its own or as part of another substituent, refers to the structure R can be, for example, a water-soluble moiety, hydrogen, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, aryl, or other group, and may contain a carboxylic acid group. R can be a water-soluble polymer, including but not limited to polymers containing six or more monomer units, and nonionic water-soluble polymers, such as PEG, modified PEG terminated with carboxylic acid or carboxylic ester. "Syringamide" can be linked to another molecule via a linker or bond. "Syringamide" can be, for example, phosringamide-PEG, alkylphosringamide, alkoxyphosringamide, alkylphosringamide PEG, alkoxyphosringamide PEG, alkylphosringamide PEG carboxylate /

[0150] Ester, alkoxyphosphine PEG carboxylate / ester.

[0151] As used in this article, the term "fluorescence" refers to the emission of light (typically) of a different wavelength when a compound is irradiated with light of a wavelength it absorbs. Fluorescence is the light emitted by a substance that has absorbed light or other electromagnetic radiation. In most cases, the emitted light has a longer wavelength than the absorbed light.

[0152] The terms “maximum absorbance” or “Absλmax” or “maxλabs” refer to the wavelength at which the maximum absorbance is measured by UVVis spectroscopy.

[0153] The term "excitation wavelength" or "λex" refers to the wavelength at which a compound can be excited to induce fluorescence emission, and it does not necessarily have to be at Absλmax.

[0154] The term "chromophore" refers to a molecular entity or part thereof consisting of atoms or groups of atoms, in which the electronic transitions responsible for a given spectral band are approximately located. In some cases, the "chromophore" itself can be fluorescent. The terms "fluorescent chromophore" and "fluorescent dye" are used interchangeably herein and refer to compounds having a structure capable of collecting light with a specific wavelength of maximum absorption and converting it into emitted light with a longer wavelength of maximum emission. Chromophores may have covalently bondable reactive groups (e.g., carboxylate / ester moieties, amino moieties, haloalkyl moieties, etc.). Examples of suitable chromophores include, but are not limited to, those in U.S. Patent Nos. 7,687,282; 7,671,214; 7,446,202; 6,972,326; 6,716,979; 6,579,718; 6,562,632; 6,399,392; 6,316,267; 6,162,931; 6,130,101; and 6,005,113. The patents described in ; 6,004,536; 5,863,753; 5,846,737; 5,798,276; 5,723,218; 5,696,157; ​​5,658,751; 5,656,449; 5,582,977; 5,576,424; 5,573,909; and 5,187,288, are incorporated herein by reference in their entirety.

[0155] The term "part" refers to a group that is part of a molecule, which may be a functional group, or part of a molecule having multiple groups that share common structural and / or functional aspects. Some examples of groups or parts include, but are not limited to, linker portions, functional groups, water-soluble portions, and PEG portions according to this disclosure.

[0156] The terms "linkage," "connection," or "linkage" refer to a connecting portion of two groups having a main chain of 100 atoms or fewer in length. A linker or linker can be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, such as a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or more carbon atoms, wherein the linker can be linear, branched, cyclic, or a single atom. In some embodiments, the linker is a branched linker, which refers to a connecting portion connecting three or more groups. In some cases, one, two, three, four, or five or more carbon atoms in the linker main chain may optionally be replaced by sulfur, nitrogen, or oxygen heteroatoms. In some embodiments, the linker backbone includes a linking functional group, such as ether, thioether, amino, amide, carbonyl, acyl, sulfonamide, sulfinamide, disulfonamide, disulfinamide, sulfonolactam, amide, secondary amine, phosphonamide, hypophosphamide, phosphonamide ester, selenamide, selenite, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. In some embodiments, the linker backbone includes a linking functional group, such as amino, amide, carbonyl, sulfonamide, sulfinamide, disulfonamide, disulfinamide, sulfonolactam, amide, secondary amine, phosphonamide, hypophosphamide, phosphonamide ester, selenamide, or selenite. The bonds between backbone atoms can be saturated or unsaturated, and in some cases, the linker backbone contains no more than one, two, or three unsaturated bonds. The linker may contain one or more substituent groups, such as alkyl, aryl, or alkenyl groups. The linker may include, but is not limited to, polyethylene glycol, ether, thioether, tertiary amine, and alkyl groups, which may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone may contain cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, wherein two or more atoms of the cyclic group, such as two, three, or four atoms, are included in the backbone.

[0157] The phrase "conjugated water-soluble fluorescent compound" refers to a water-soluble fluorescent compound having a binding pair with which it is conjugated.

[0158] In chemical structure, Indicates a single bond or a double bond.

[0159] In chemical structure, The aryl group represents, for example, substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl, polycyclic aryl, monocyclic heteroaryl, or polycyclic heteroaryl.

[0160] In chemical structure, The substituted cycloalkenyl or polycyclic alkenyl moiety may be partially unsaturated. The optionally substituted cycloalkenyl group may be an optionally substituted cyclohexyl-1-en-1-yl group. The optionally substituted cycloalkenyl group may be substituted with a halogen (e.g., chloro or phenylthio). The optionally substituted polycyclic alkenyl moiety may be an optionally substituted naphthyl moiety. The optionally substituted cycloalkenyl or polycyclic alkenyl moiety may be partially unsaturated.

[0161] The phrase "binding partner" refers to any molecule or molecular complex capable of specifically binding to a target analyte. Binding partners in this disclosure include, for example, proteins (e.g., antibodies or antibody fragments), small organic molecules, carbohydrates (e.g., polysaccharides), oligonucleotides, polynucleotides, lipids, affinity ligands, aptamers, etc. In some embodiments, the binding partner is an antibody or a fragment thereof. In the context of this disclosure, specific binding refers to a binding reaction that identifies the presence of a target analyte in the presence of a heterogeneous population. Thus, under certain assay conditions, a particular binding partner preferentially binds to a specific protein or a specific isoform of a protein, rather than binding in significant amounts to other proteins or other isoforms present in the sample.

[0162] In some cases, antibodies include intravenous immunoglobulin (IVIG) and / or antibodies derived from (e.g., enriched from, purified from, or affinity purified from) IVIG. IVIG is a blood product containing IgG (immunoglobulin G) conjugated from plasma (e.g., in some cases, free of any other proteins) of many (e.g., sometimes between 1,000 and 60,000) normal and healthy blood donors. IVIG is commercially available. For example, aspects of IVIG are described in U.S. Patent Application Publications Nos. 2010 / 0150942; 2004 / 0101909; 2013 / 0177574; 2013 / 0108619; and 2013 / 0011388.

[0163] In some cases, the antibody is a monoclonal antibody that defines a specific subclass (e.g., IgG1, IgG2, IgG3, or IgG4). If a combination of antibodies is used, the antibodies may be from the same subclass or from different subclasses. For example, the antibody may be an IgG1 antibody. In some embodiments, the monoclonal antibody is humanized.

[0164] The phrase "water-soluble fluorescent complex" refers to the water-soluble fluorescent compound of this disclosure conjugated with a binding partner.

[0165] The phrase “protected group” (also known as “protecting group” or “protected”) refers to the reversible formation of derivatives from existing functional groups in a molecule by linking them together to reduce reactivity, such that the protected functional group does not react under the synthetic conditions subjected to the molecule. Some examples of amine protecting groups include, but are not limited to, benzyloxycarbonyl; 9-fluorenylmethoxycarbonyl (Fmoc); tert-butoxycarbonyl (Boc); allyloxycarbonyl (Alloc); p-toluenesulfonyl (Tos); 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc); 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf); mesitylene-2-sulfonyl (Mts); 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr); acetamyl; phthalimido, etc. These and other protecting groups of amines, carboxylic acids, alcohols and other functional groups can be added to and removed from the polymers of this disclosure using known techniques, such as those described by Green and Wuts (Protective Groups in Organic Synthesis, 4th Edition, 2007, Wiley-Interscience, New York).

[0166] The term "sample" refers to a material or mixture of materials containing one or more analytes for a specific purpose, in some cases in liquid form. In some embodiments, when used in its broadest sense, the term refers to any plant, animal, or bacterial material containing cells or producing cellular metabolites, such as tissues or fluids isolated from an individual (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or from components of in vitro cell cultures, as well as samples from the environment. The term "sample" can also refer to a "biological sample." As used herein, the term "biological sample" refers to a whole organism or a subgroup of its tissues, cells, or components (e.g., bodily fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic sac blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to a homogenate, lysate, or extract prepared from a whole organism or a subgroup or fraction thereof of its tissues, cells, or components, including but not limited to plasma, serum, cerebrospinal fluid, lymph, secretions from the skin, respiratory tract, intestines, and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. In some embodiments, the sample has been taken from an animal or plant. Biological samples may include cells. The term "cell," used in its conventional sense, refers to the basic structural unit of a living organism (both eukaryotic and prokaryotic) that has at least a nucleus and a cell membrane. In some embodiments, cells include prokaryotic cells, such as prokaryotic cells from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples from animals, plants, or fungi.

[0167] The term "substrate" refers to a solid material having various configurations. Substrates can be, for example, sheets, beads, or other structures such as porous plates, polymers, particles, semiconductor surfaces, nanotubes, fibrous webs, hydrogels, porous matrices, needles, microarray surfaces, chromatographic supports, etc. In some cases, the substrate is selected from particles, flat solid substrates, fibrous webs, hydrogels, porous matrices, needles, microarray surfaces, and chromatographic supports.

[0168] As used herein, the term "water" refers to any aqueous solution that is primarily water and compatible with physiological conditions. In some cases, an aqueous solution contains more than 50% water, such as more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% water. The term "water" includes, for example, biological buffers and other aqueous solutions that may contain additives such as salts, detergents, stabilizers, and other water-soluble components (e.g., sugars, proteins, amino acids, and nucleotides). In some cases, "water" can be an aqueous solution containing up to 10% miscible organic solvent (e.g., up to 10% DMSO in water). The term "water" does not include pure solvents or combinations of solvents other than water, such as pure alcohols, such as pure methanol or ethanol; pure ethers, such as pure diethyl ether or tetrahydrofuran; or any other pure solvent that is miscible or immiscible with water.

[0169] As used herein, the term "water-soluble moiety" or "water-soluble group" (WSG or W), either on its own or as part of another group, refers to any hydrophilic group that is readily solubilized in an aqueous environment (e.g., under physiological conditions) and is capable of enhancing the water solubility of the molecule to which it is attached. Dyes described herein may contain any convenient WSG to provide enhanced water solubility. The water-soluble moiety enhances the solubility of the compound in a predominantly aqueous solution compared to a control compound lacking a water-soluble moiety. The water-soluble moiety can be any convenient hydrophilic moiety that is readily solubilized in an aqueous environment. In some cases, the water-soluble moiety may be able to impart solubility in water (e.g., an aqueous buffer) of >1 mg / mL, >2 mg / mL, >3 mg / mL, >4 mg / mL, >5 mg / mL, >6 mg / mL, >7 mg / mL, >8 mg / mL, >9 mg / mL, or >10 mg / mL. In some cases, the water-soluble fraction may be able to impart solubility in water of >10 mg / mL, >20 mg / mL, >30 mg / mL, >40 mg / mL, >50 mg / mL, >60 mg / mL, >70 mg / mL, >80 mg / mL, >90 mg / mL, or >100 mg / mL.

[0170] The increase in water solubility of a molecule can vary depending on the attached moiety. In some cases, the increase in water solubility (compared to the solubility of a molecule without the attached moiety) is 2 to 5 times, 10 to 25 times, 50 to 100 times, or 100 to 100 times. In some cases, the water-soluble moiety is charged, such as a positively or negatively charged hydrophilic moiety. In some cases, the water-soluble moiety is a neutral hydrophilic moiety. In some cases, the water-soluble moiety is branched (e.g., as described herein). In some cases, the water-soluble moiety is linear. Water-soluble moieties include, but are not limited to, those taught in U.S. Patent Publication No. 2022 / 0348770, which is incorporated herein by reference in its entirety.

[0171] At ambient room temperature, a "water-soluble compound" may exhibit solubility in water (e.g., aqueous buffer solutions) of >1 mg / mL, >2 mg / mL, >3 mg / mL, >4 mg / mL, >5 mg / mL, >6 mg / mL, >7 mg / mL, >8 mg / mL, >9 mg / mL, or >10 mg / mL. In some cases, at ambient room temperature, a water-soluble compound may exhibit solubility in water of >10 mg / mL, >20 mg / mL, >30 mg / mL, >40 mg / mL, >50 mg / mL, >60 mg / mL, >70 mg / mL, >80 mg / mL, >90 mg / mL, and / or >100 mg / mL.

[0172] The dyes described herein may contain any convenient WSG to provide improved water solubility. WSG may be, but is not limited to, carboxylates / esters, carboxylic acids, phosphonates / esters, phosphates / esters, sulfonates / esters, sulfates / esters, sulfinates / esters, sulfonates, sulfonates, esters, polyethylene glycol (PEG) and modified PEG, linear PEG groups, branched PEG groups, hydroxyl groups, amines, amino acids, ammonium, guanidines. Salt, pyridine Polyamines and sulfonates, polyols, straight-chain or cyclic sugars, primary amines, secondary amines, tertiary or quaternary amines and polyamines, phosphonates / ester groups, hypophosphonates / ester groups, ascorbate / ester groups, ethylene glycols including polyethers, zwitterionic derivatives, peptide sequences, nucleotides (DNA and RNA), peptide-like substances, carbohydrates, Azoline, polyol, dendrite, dendritic polyglycerol, cellulose, chitosan, -COOM', -SO3M', -PO3M', -NR 3 + Y', (CH2CH2O) pR and mixtures thereof, wherein Y' can be any halogen, sulfate / ester, sulfonate / ester, or oxyanion, p can be 1 to 500, each R can be independently H or alkyl (e.g., methyl), and M' can be a cationic counterion (e.g., Na). + K + (etc.) or hydrogen, -(CH2CH2O) yy CH2CH2XR yy -(CH2CH2O) yy CH2CH2X-, -X(CH2CH2O) yy CH2CH2-, ethylene glycol and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S and NR. ZZ And R ZZ and R YY Independently selected from H and C1-3 alkyl groups, and combinations thereof or derivatives thereof. In some cases, WSG includes, but is not limited to, PEG, modified PEG, peptide sequences, peptide-like substances, carbohydrates, Azoline, polyol, dendrite, dendritic polyglycerol, cellulose, chitosan or derivatives thereof. WSG can be unsubstituted or substituted.

[0173] In some cases, WSG can be a hydrophilic polymer. For example, hydrophilic polymers that can be used for WSG include, but are not limited to, polymers based on polyepoxides containing the formula -(CH2-CH2-O). n -or-(O-CH2-CH2) n -The repeating ethylene oxide unit, such as PEG, polyamide epoxide, or derivatives thereof. Other examples of the target polymer include polyamides with a molecular weight greater than 1,000 Daltons, having the formula: or Wherein X and Y are divalent groups that may be the same or different, and may be branched or linear, and n is a discrete integer from 2 to 100 (e.g., 2 to 50), and wherein one or both of X and Y comprises a biocompatible, substantially non-antigenic, water-soluble repeating unit, which may be linear or branched. The number of such water-soluble repeating units can vary significantly, wherein the number of such units is 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6 to 100, for example 2 to 50 or 6 to 50. One example of an embodiment is one in which one or both of X and Y are selected from: -((CH2) n1 -(CH2-CH2-O) n2 -(CH2)- or -((CH2) n1 -(O-CH2-CH2) n2 -(CH2) n1-), where n1 is 1 to 6, 1 to 5, 1 to 4, or 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, or 2 to 5. In some cases, the water-soluble polymer is a group of 1 to 50 monomer units, such as 1 to 40, 1 to 30, 1 to 20, 2 to 24, 2 to 20, 2 to 10, or 2 to 6 monomer units. Another example of an embodiment is one in which X is -(CH2-CH2)-, and Y is -(CH2-(CH2-CH2-O)3-CH2-CH2-CH2)- or -(CH2-CH2-CH2-(O-CH2-CH2)3-CH2)-. In some cases, as is known in the art, any of the formulas described herein may be replaced by a water-soluble portion of a dendritic structure.

[0174] In some cases, hydrophilic polymers can be, for example, PEG, peptide sequences, peptide-like substances, carbohydrates, Azoline, polyol, dendrite, dendritic polyglycerol, cellulose, chitosan or its derivatives.

[0175] In some cases, WSG is (CH2). x (OCH2CH2) y OCH3, where each x is an independent integer from 0 to 20, and each y is an independent integer from 0 to 50. In some cases, the water-soluble polymer is a PEG group or modified PEG polymer with 6 to 24 monomer units, such as 10 to 30, 10 to 24, 10 to 20, 12 to 24, 12 to 20, 12 to 16, or 16 to 20 monomer units.

[0176] In some cases, WSG comprises a nonionic polymer (e.g., a PEG polymer) whose ends are replaced by ionic groups (e.g., sulfonate groups). In some embodiments of the formula, WSG comprises a group selected from (CH2). x (OCH2CH2) y The substituents of OCH3, wherein each x is an integer from 0 to 20 independently, and each y is an integer from 0 to 50 independently; and the benzyl group is optionally replaced by one or more halogens, hydroxyl groups, C1-C... 12 Alkyl group or (OCH2CH2) z OCH3 substitution, where each z is an independent integer from 0 to 50. In some cases, WSG is (CH2)3(OCH2CH2). 11 OCH3. In some embodiments, one or more of the substituents are selected from (CH2). x (OCH2CH2) yThe benzyl group substituted with a WSG group (e.g., one or two WSG groups) in OCH3, wherein each x is an integer independently from 0 to 20, and each y is an integer independently from 0 to 50. It should be understood that hydroxyl-terminated polymer chains (e.g., PEG chains) rather than methoxy-terminated polymer chains (e.g., PEG chains) can be used in any water-soluble moiety.

[0177] The term modified polymer, such as modified PEG, refers to a water-soluble polymer that has been modified or derivatized at one or both ends, for example, to include terminal substituents (e.g., terminal alkyl, substituted alkyl, alkoxy, or substituted alkoxy groups) and / or terminal linking functional groups (e.g., amino or carboxylic acid groups suitable for linking the polymer to a target molecule (e.g., via branched groups to light-harvesting chromophores) suitable for linking the polymer to the target molecule. The subject water-soluble polymer may include any convenient linking group. It should be understood that in some cases, depending on the method of preparation and / or purification of the polymer starting material, the water-soluble polymer may exhibit some dispersibility relative to the polymer length. In some cases, the water-soluble polymer is monodisperse.

[0178] Water-soluble polymers may contain one or more spacers or connectors. Some examples of spacers or connectors include straight-chain or branched portions containing one or more repeating units used in the water-soluble polymer, diamino and / or diacid units, natural or non-natural amino acids or derivatives thereof, and aliphatic portions including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., which may contain, for example, up to 18 carbon atoms or even other polymer chains.

[0179] The water-soluble polymer moiety, or one or more spacers or connectors of the polymer moiety (when present), may comprise biostable or biodegradable polymer chains or units. For example, polymers with repeating linkages exhibit varying degrees of stability under physiological conditions, depending on bond instability. Based on the known hydrolysis rates of low molecular weight analogs, polymers with such bonds can be classified according to their relative hydrolysis rates under physiological conditions, for example, from less unstable to more stable: e.g., polyurethane (-NH-C(O)-O-) > polyorthoester (-OC((OR)(R'))-O-) > polyamide (-C(O)-NH-). Similarly, the linkage system connecting the water-soluble polymer to the target molecule can be biostable or biodegradable, for example, from less unstable to more stable: carbonate (-OC(O)-O-) > ester (-C(O)-O-) > urethane (-NH-C(O)-O-) > orthoester (-OC((OR)(R'))-O-) > amide (-C(O)-NH-). Generally, given the instability of sulfate groups, the use of sulfated polysaccharides can be avoided. Additionally, the use of polycarbonates and polyesters may be less desirable. These bonds are provided by way of example and are not intended to limit the linking systems of the water-soluble polymers available in the WSGs disclosed herein or the types of bonds employed in the polymer chains.

[0180] In some cases, the water-soluble moiety includes, but is not limited to, hydroxyl, alkoxy, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylcarboxylate / ester, alkylsulfonate, alkoxysulfonate, oligoether sulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylcarboxylate / ester, alkylamide, alkoxysulfonate / ester, alkylsulfonate / ester, alkylsulfonate,

[0181]

[0182]

[0183] In some cases, the subject compound may comprise multiple water-soluble moieties linked to a single position in the subject compound, for example, via a branching linker (e.g., an aralkyl substituent further disubstituted with a water-soluble group). Thus, in some cases, the branching linker group is a substituent of the dye, which links the dye to two or more water-soluble groups. In some cases, multiple water-soluble moieties may be linked to the subject compound via groups having, for example, the following formula:

[0184]

[0185] Where X 1 X 2 It is a branch point, L 1 L 2 L 3 It is a connector, where m' is an integer of 1, 2, or 3; W 1 It is the water-soluble part.

[0186] In some cases, one or more water-soluble moieties may be linked to the subject compound via groups comprising a linker according to this disclosure, for example, as taught in U.S. Publication No. 2020 / 0190253A1, which is incorporated herein by reference in its entirety. The linker moieties may be linked to a cyanine bridge or a heterocyclic aryl group of a fluorescent compound of this disclosure. The linker may be cleavable or non-cleavable.

[0187] One or more water-soluble portions may also be linked to the subject compound via groups comprising a linker, such as, but not limited to, the following linker formula (VIe):

[0188] -(L 3 ) m -(X 1 ) m’ -((L 1 ) m” -(W 1 ) s ) t -R 3 (Vle)

[0189] in:

[0190] Each optional L 1 and L 3 It is an independently selectable connector part;

[0191] Any X that exists 1 It is a branch point;

[0192] W 1 It is the water-soluble portion, including but not limited to water-soluble polymers containing 2 to 50, 4 to 30, or 6 to 24 monomer units;

[0193] Each m is independently 0 or 1; each m' is independently 0 or 1; each m” is independently 0 or 1;

[0194] Each s is independently 1 or 2;

[0195] Each t is independently 0, 1, 2, or 3; and

[0196] R 3 As defined in this article.

[0197] In some cases, L 1 L 3 And X does not exist, and W 1 It is a water-soluble moiety, for example, a water-soluble polymer containing 2 to 50, 4 to 30, or 6 to 24 monomer units, such as 10 to 30, 10 to 24, 10 to 20, 12 to 24, 12 to 20, 12 to 16, or 16 to 20 monomer units. In some cases, the water-soluble moiety can be a linear water-soluble moiety. For example, L 1 And X can be non-existent, L 3 It is a connector (e.g., as disclosed herein), and W 1 It is the water-soluble part.

[0198] In some cases, L 1 L 2 and / or L 3 At least one, at least two, or all three of the connectors may be selected from alkyl or substituted alkyl connectors, alkenyl or substituted alkenyl connectors, alkoxy or substituted alkoxy connectors, PEG connectors, sulfonamide-alkyl or substituted sulfonamide-alkyl connectors, amamide-alkyl or substituted amamide-alkyl connectors, and alkyl-amido-alkyl or substituted alkyl-amido-alkyl connectors. In some cases, the connector contains a carbonyl group. The connector moiety may be covalent, alkoxy, sulfonamide, disulfonamide, selenamide, sulfinamide, sulfonolactam, disulfinamide, amide, carbonyl, selenamide, phosphonamide, hypophosphamide, phosphonamide ester, or secondary amine.

[0199] In some cases, L 2 and L 3 Each can be independently selected from the following junction portions: covalent bond, C 1-8 Alkylenes, 2- to 8-membered heteroalkylenes, and chains with a length of 2 to 200 main chain atoms, wherein the chains comprise straight chains, branched chains, and / or cyclic portions.

[0200] In some cases, L 1 It can be sulfonamide, sulfinamide, disulfonamide, disulfinamide, sulfolacamide, amide, secondary amine, phosphonamide, hypophosphonamide, phosphonamide ester, selenamide, or selenamide.

[0201] In some cases, L 3 It can be a connector with a main chain of 20 atoms or less in length, and W 1 It is the water-soluble portion (e.g., as described herein). In some cases, L 3The connector can be selected from alkyl or substituted alkyl links, alkenyl or substituted alkenyl links, alkynyl or substituted alkynyl links, acyl or substituted acyl links, alkoxy or substituted alkoxy links, PEG links, sulfonamide-alkyl or substituted sulfonamide-alkyl links, amamide-alkyl or substituted amamide-alkyl links, and alkyl-amido-alkyl or substituted alkyl-amido-alkyl links. In some cases, L 3 It can be a key. In some cases, L 3 It can be an alkyl or substituted alkyl linker, an alkenyl or substituted alkenyl linker, an alkoxy or substituted alkoxy linker, and X can be an aryl linker.

[0202] In some cases, L 1 and L 3 Each is independently selected from C1-C 12 Alkyl or substituted alkyl linker, C1-C 12 Alkenyl or substituted alkenyl linkers, C1-C 12 Alkyne or substituted alkyne linkers, C1-C 12 Acyl or substituted acyl linker, C1-C 12 Alkoxy or substituted alkoxy linkers, C1-C 12 Amide-alkyl or substituted amide-alkyl linker, C1-C 12 Alkyl-amido-alkyl or substituted alkyl-amido-alkyl joints, sulfonamides, sulfinamides, disulfonamides, disulfinamides, sulfonolamides, amides, secondary amines, phosphonamides, hypophosphamides, phosphonamide esters, selenamides, and selenides. In some cases, L 3 Contains carbonyl or alkoxy groups, and L 1 It is C1-C 12 Alkyl or substituted alkyl groups, sulfonamides, sulfinamides, disulfonamides, disulfinamides, sulfonolamides, amides, secondary amines, phosphonamides, phosphinoamides, phosphonamide esters, selenamides, and selenides. In some cases, L... 3 It can be an alkoxy or a substituted alkoxy linker, X may be absent, and L 1 It can be sulfonamide, sulfinamide, disulfonamide, disulfinamide, sulfolacamide, amide, secondary amine, phosphonamide, hypophosphonamide, phosphonamide ester, selenamide, or selenamide.

[0203] In some cases, the branch point X 1 Selected from N, CR', C(=O)N, SO2N, trisubstituted aryl moieties (e.g., 1,3,5-phenyl), tetrasubstituted aryl moieties (e.g., 1,3,4,5-phenyl), and trisubstituted heteroaryl moieties. In some cases, the branch point X... 1It is a nitrogen atom. In other cases, the branch point X 1 It is CR', where R' is selected from hydrogen, alkyl, substituted alkyl, or -L. 3 -W 1 (For example, as described in this article).

[0204] The term "sulfonamide" refers to a portion of -S(O)2NR-; the term "disulfonamide" refers to a portion of -S(O)2NRS(O)2-; the term "selenamide" refers to a portion of -Se(O)2NR-; the term "sulfinamide" refers to a portion of -S(O)NR2; the term "disulfinamide" refers to a portion of -S(O)NRS(O)-; the term "selenamide" refers to a portion of -Se(O)NR-; the term "phosphonamide" refers to a portion of -NR-PR(O)NR-; the term "phosphonamide ester" refers to a portion of -O-PR(O)NR-; and the term "sulfonamide" refers to a cyclic sulfonamide (e.g., where the R group is bonded to a sulfur atom via an alkylene moiety); wherein for each term, the R group is independently H, alkyl, haloalkyl, or aryl.

[0205] As used herein, the term "cyanine" refers to a substituted or unsubstituted bridging unit that allows delocalization across the BtCy molecule of the present invention. In some embodiments, "cyanine" is a substituted or unsubstituted methine or polymethyl unit, such as a trimethyl, pentamethyl, or heptamethyl unit. For example, in some embodiments, "cyanine" refers to substituted and unsubstituted groups, such as the following:

[0206] In some implementation schemes, cyanide is In other implementations, cyanide is In some implementation schemes, cyanide is In some other implementation schemes, cyanide is... In some implementation schemes, cyanide is In some embodiments, "cyanine" is a substituted or unsubstituted "cyclic group", such as the cycloalkenyl or polycyclic alkenyl moiety of "squaricine", which may be substituted or unsubstituted.

[0207] As used herein, the term "cucurbitacin" refers to a substituted or unsubstituted cyclic group, such as a 4-, 5-, or 6-membered ring. Additionally, the molecules of this invention include other cyanine cyclic groups and heterocyclic groups, including but not limited to substituted or unsubstituted five- or six-membered cyclic groups and heterocyclic groups. For example, cyanine cyclic groups include, but are not limited to, substituted or unsubstituted cyanine cyclic groups and heterocyclic groups, such as the following:

[0208]

[0209] Wherein C is a four-, five-, or six-membered cyclic group, a heterocyclic group, or a fused polycyclic group. In some embodiments, the anthocyanin cyclic group is a squaric acid cyanine group, for example... In some implementations, the cyanine cyclic group is In some implementations, the cyanine cyclic group is In other embodiments, the cyanine cyclic group is In some other embodiments, the cyanine cyclic group is In some implementations, the cyanine cyclic group is In some implementations, the cyanine cyclic group is In some implementations, the cyanine cyclic group is In some implementations, the cyanine cyclic group is In some implementations, the cyanine cyclic group is

[0210] III. Benzothiophene-pyrrole-cyanine compounds

[0211] This disclosure provides benzothiophene-pyrrole-cyanine (BtCy) compounds and methods for preparing BtCy compounds. The BtCy compounds can be fluorescent water-soluble dyes. This disclosure provides BtCy tandem dyes comprising a BtCy compound and a donor or acceptor dye. This disclosure also provides labeled specific binding pairs comprising a BtCy compound or a BtCy tandem dye and a specific binding pair covalently linked to the BtCy compound or BtCy tandem dye. The BtCy tandem dye comprises a BtCy compound or a labeled specific binding pair having an acceptor or donor chromophore linked thereto. This disclosure also provides methods for detecting target analytes in samples using the labeled specific binding pairs or tandem dye-labeled specific binding pairs of the present invention. This disclosure also provides kits containing BtCy compounds according to the present invention, labeled specific binding pairs, and / or tandem dyes. In addition to the specific structures disclosed herein, structural isomers of the disclosed structures are also included.

[0212] Known cyanine compounds contain a cyanine bridge and two indole moieties. The structures of known Cy3, Cy5, and Cy7 compounds are... Figure 1As shown in the figure, the prior art Cy3 exhibits typical excitation wavelengths (λex) and emission wavelengths (λem) of approximately 555 nm and 569 nm, respectively (λex / λem, PBS). The prior art Cy5 exhibits λex and λem of approximately 651 nm and 670 nm, respectively (λex / λem, PBS). The prior art Cy7 exhibits λex and λem of approximately 750 nm and 780 nm, respectively (λex / λem, PBS).

[0213] The BtCy compounds disclosed herein each comprise at least one benzothiophene-pyrrole moiety or a derivative thereof and exhibit λex of about 500 nm to about 1100 nm and λem of about 550 nm to about 1200 nm.

[0214] In some cases, the BtCy compounds of this disclosure each comprise at least one benzothiophene-pyrrole moiety or a derivative thereof and exhibit λex of about 600 nm to about 1100 nm and λem of about 650 nm to about 1300 nm.

[0215] In some cases, the BtCy compounds of this disclosure exhibit λex at about 625 nm to about 1000 nm and λem at about 660 nm to about 1050 nm.

[0216] In some cases, the BtCy3 compounds of this disclosure exhibit λex at about 600 nm to about 775 nm and λem at about 650 nm to about 800 nm.

[0217] In some cases, the BtCy5 compounds of this disclosure exhibit λex at about 725 nm to about 875 nm and λem at about 750 nm to about 900 nm.

[0218] In some cases, the BtCy7 compounds of this disclosure exhibit λex at about 825 nm to about 975 nm and λem at about 850 nm to about 1000 nm.

[0219] The BtCy compounds according to this disclosure may comprise a structure according to formula (I):

[0220] in

[0221] Selected from substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl, polycyclic aryl, monocyclic heteroaryl and polycyclic heteroaryl;

[0222] Selected from substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl and polycyclic aryl;

[0223] Each T is independently for NR4’ CR 1 CR 1 R 2 , O, S, Se or Te;

[0224] V is NR 11 CR 8 CR 8 R 9 O, S, Se or Te, or each TV together can represent SO2, -CR 1 -O-,-O-CR 1 -,-CO-O-,-O-CO-,-CO-NR 11 -, or -NR 4’ -CO- structural elements;

[0225] G is either C or N;

[0226] Each R 1 R 2 R 8 and R 9 Independently selected from water-soluble moieties, linked water-soluble moieties, linker moieties, E, linked E, reactive groups, linked reactive groups, conjugated tags, linked conjugated tags, conjugated mating bodies, linked conjugated mating bodies, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonic acid, sulfonate / ester, alkylsulfonate / ester, alkylsulfonate, alkoxysulfonate / ester, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, alkoxycarboxylate / ester, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylsulfonamide PEG, alkylamide.

[0227]

[0228]

[0229] Each R 4 R 4’ R 10 and R 11Independently selected from the following: linker portion, chromophore, linked chromophore, reactive group, linked reactive group, conjugated tag, linked conjugated tag, water-soluble portion, linked water-soluble portion, conjugated mating body, linked conjugated mating body, E, linked E, H, halogenated alkyl, alkenyl, alkynyl, PEG group, linked PEG group, cycloalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, sulfonic acid, sulfonate / ester, alkyl sulfonate / ester, alkyl sulfonate, carboxylic acid, carboxylate / ester, alkyl carboxylate / ester, alkyl sulfonamide, alkyl sulfonamide PEG, alkylamide, alkylamide-PEG.

[0230]

[0231] Or its protected group; or R 4 and R 12 Together, R 14 and R 10 Together, R 4 and R 13 Together, R 13 and R 10 Together, R 13 and R 11 Together, R 12 and R 14 Together, R 4’ and R 13 Together, R 4’ and R 12 Together, R 13 and R 10 Together, R 4 R 12 R 14 and R 10 Together, R 4 R 13 and R 10 Together, R 4 R 13 and R 11 Together, R 4 R 12 and R 14 Together, R 4’ R 13 and R 11 Together, and R 12 R 14 and R 10 One, two, three, or four together form unsubstituted or substituted unsaturated or partially unsaturated C3-C atoms. 10 cycloalkyl; unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 10 Heterocyclic alkyl groups; unsubstituted or substituted unsaturated or partially unsaturated C3-C14 C3-C 10 Or C3-C8 polycyclic alkyl; or unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polyhedraloyl groups;

[0232] Each R 3 Independently selected from H, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, alkyl sulfonate / ester, alkyl carboxylate / ester, water-soluble moiety, linked water-soluble moiety, chromophore, linked chromophore, functional moiety, linked functional moiety, conjugated tag, linked conjugated tag, conjugated partner, linked conjugated partner, PEG group and linked PEG group;

[0233] Each Q is independently a bond, O, NH, NR 4 C1-C 12 Alkylene, CHR 4 Or CH2;

[0234] Each Z is independently CH2, CHR 4 O, NR 4 Or NH;

[0235] Each W 1 It is a water-soluble component on its own.

[0236] L 1 L 2 and L 3 Each connector is selected independently.

[0237] Each E is independently selected from chromophores, functional moieties, substrates, reactive groups, conjugation tags, linked conjugation tags, and binding partners;

[0238] Each R 7 Independently selected from H, hydroxyl, C1-C 12 Alkyl, C1-C 12 Heteroalkyl, C2-C 12 Olefins, C2-C 12 Alkynes, C3-C 12 cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy group, C2-C 18 (Miscellaneous) arylamino, carboxylates / esters, carboxylic acids, C2-C 12 Alkyl carboxylic acids, C2-C 12 Alkyl carboxylates / esters, C2-C12 Alkyl carboxylic acid esters, aryl carboxylic acids, aryl carboxylic acid esters, C1-C 12 Alkyl groups, water-soluble moiety, PEG moiety, protected or unprotected functional groups, conjugated labels, linked conjugated labels, chemoselective functional groups, linkers, sulfonic acids, sulfonates / esters, C1-C 12 Alkyl sulfonates / esters, sulfonamides;

[0239] Each R 12 R 13 and R 14 Independently selected from hydrogen, halogen, one or more heteroatoms, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C1-C6 olefin, substituted or unsubstituted C3-C 10 Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C3-C 10 Heterocyclic alkyl groups, CO2R 1 CONR 1 R 2 O-aryl, S-aryl, N-aryl, -O-alkyl, S-alkyl, N-alkyl, wherein each alkyl or aryl group may optionally be represented by one or more R groups. 7 PEG or PEG-R 7 Replace, optionally, each of R 12 R 13 and R 14 Independently replaced by one or more R 7 Group; or R 1 R 2 R 4 R 4’ R 8 R 9 R 10 R 11 R 12 R 13 , and R 14 At least two of them together, including but not limited to R 1 R 2 R 4 R 4' R 8 R 9 R 10 R 11 R 12 R 13 and R 14 Two, three, or four of them together, optionally including but not limited to R 1 R 2 R4 R 4' R 8 R 9 R 10 R 11 R 12 R 13 , and R 14 One, two, three or four together, including but not limited to R 4 and R 12 Together, R 14 and R 10 Together, R 4 and R 13 Together, R 13 and R 10 Together, R 13 and R 11 Together, R 12 and R 14 Together, R 4' and R 13 Together, R 4’ and R 12 Together, R 13 and R 10 Together, R 4 R 12 R 14 and R 10 Together, R 4 R 13 and R 10 Together, R 4 R 13 and R 11 Together, R 4 R 12 and R 14 Together, R 4’ R 13 and R 11 Together, and R 12 R 14 and R 10 Formation of unsaturated or partially unsaturated C3-C, whether substituted or substituted. 10 Cycloalkyl, unsubstituted or optionally O- or N-substituted, substituted unsaturated or partially unsaturated C3-C 10 Heterocyclic alkyl groups, unsubstituted or substituted, unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polycyclic alkyl groups, or unsubstituted or optionally O- or N-substituted substituted unsaturated or partially unsaturated C3-C groups. 14 C3-C 10 Or C3-C8 polyhedraloyl groups;

[0240] Each K is independently a covalent bond, O, S, Se, P, NR 1 or CR 1 R 2 ;

[0241] Each f is an independent integer from 0 to 50, 1 to 30, or 2 to 20;

[0242] Each m and m' is independently 0, 1, 2, or 3;

[0243] Each n is an independent integer from 1 to 20; from 1 to 10; or 0, 1, 2, or 3.

[0244] Each p is independently 1, 2, 3 or 4;

[0245] Each s is independently 1 or 2;

[0246] Each t is independently 0, 1, 2, 3, or 4; and

[0247] X is a counter ion.

[0248] In some cases, p is 1, 2, or 3. In some cases, p is 1. In some cases, p is 2. In some cases, p is 3. In some cases, p is 4.

[0249] In some cases, m is 0. In some cases, m is 1. In some cases, when m is 0, each T is independently CR. 1 or CR 1 R 2 In some cases, when m is 0, T is not NR. 4’ S or O. In some cases, when m is 1, each T is independently NR. 4’ O, S, Se, or Te. In some cases, when m is 1, T is not CR. 1 or CR 1 R 2 .

[0250] The BtCy compounds according to this disclosure may comprise structures according to any one of formulas (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), (IIg), (IIh), (IIi), (IIj), (IIk), (IIl), (IIm), (IIIn), and (IIo):

[0251]

[0252]

[0253]

[0254]

[0255] in

[0256] T is NR 4’ CR 1 CR 1 R 2 , O or S;

[0257] V is NR 11 CR 8 CR 8 R 9 , O or S;

[0258] Each R 5 and R 6 Independently selected from water-soluble portion, linked water-soluble portion, linker portion, E, linked E, reactive group, linked reactive group, conjugation tag, linked conjugation tag, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 Independently selected from hydrogen, sulfonate / ester, alkyl sulfonate / ester, alkyl sulfonamide, alkyl sulfonamide-PEG, phosphate group, carboxylic acid, carboxylate / ester, amide, alkylamide, amide-PEG and water-soluble group;

[0259] R 16 It's KR 13 Optional H, halogen, OC 1-6 Alkyl, SC 1-6 Alkyl, O-aryl, S-aryl, NHC 1-6 Alkyl groups, Ph-NCS, Ph-CO2H, Ph-(CH2) 1-4 CO2H; and

[0260] It is an optional substituted cycloalkenyl or polycycloalkenyl moiety.

[0261] The BtCy compounds according to this disclosure may comprise structures according to any one of formulas (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), (IIIj), (IIIk), (IIIl), (IIIm), and (IIIn):

[0262]

[0263]

[0264]

[0265]

[0266]

[0267] in

[0268] Each R 5 and R 6 Independently selected from water-soluble portion, linked water-soluble portion, linker portion, E, linked E, reactive group, linked reactive group, conjugation tag, linked conjugation tag, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 Independently selected from hydrogen, sulfonates / esters, alkyl sulfonates / esters, alkyl sulfonamides, alkyl sulfonamide-PEG, phosphate groups, carboxylic acids, carboxylates / esters, amides, alkylamides, amide-PEG, and water-soluble groups; and

[0269] It is an optional substituted cycloalkenyl or polycycloalkenyl moiety.

[0270] BtCy compounds according to this disclosure may comprise structures according to any one of the formulas (IVa), (IVb), (IVc), (IVd), (IVe), (IVf), (IVg), (IVh), (IVVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVu), (IVv), (IVw), and (IVx):

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277] in

[0278] Each R 5 and R 6 Independently selected from water-soluble portion, linked water-soluble portion, linker portion, E, linked E, reactive group, linked reactive group, conjugation tag, linked conjugation tag, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 Independently selected from hydrogen, sulfonates / esters, alkyl sulfonates / esters, alkyl sulfonamides, alkyl sulfonamide-PEG, phosphate groups, carboxylic acids, carboxylates / esters, amides, alkylamides, amide-PEG, and water-soluble groups; and

[0279] It is an optional substituted cycloalkenyl or polycycloalkenyl moiety.

[0280] The BtCy compounds according to this disclosure may comprise structures according to any one of the formulas (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi), (Vj), (Vk), and (Vl):

[0281]

[0282]

[0283]

[0284] Among them, Ar 1 Ar 2 R 1 R 2 R 3 R 4 R 5 5, R 6 R 8 R 9 R 10 R 11 R 12 R 13 R 14 K, T, V, X, and m are defined as in this paper, and Y is selected from NR. 4 CR 1 CR 1 R 2 O and S. In some cases, polycyclic cyanine bridges can be substituted or unsubstituted. In some cases, polycyclic cyanine bridges can be via one or more R... 16 Group substitution. Polycyclic alkenyl anthocyanin bridges can be prepared according to the methods described in Michie et al. 2017 JACS, 139, 12406-12409 or Bandi et al. 2022, Nature Methods, 19, 353-358, each of which is incorporated herein by reference in its entirety.

[0285] The BtCy compounds of this disclosure may comprise structures according to formula (I), such as those of formulas (IIa) to (IIo), (IIIa) to (IIIn), (Iva) to (IVx), (Va) to (Vk) and (Vl), or modified structures thereof, wherein Ar 1 and Ar 2 One or both of the aryl rings can be fused at any two adjacent carbons in the terminal heteroaryl ring system.

[0286] In some instances, R 1 R 2 R 3 R4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains E or is connected to E.

[0287] In some instances, R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains a combined spouse or a connected combined spouse.

[0288] In some instances, R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one, at least two, at least three, or at least four of the components contain water-soluble portions or linked water-soluble portions. The water-soluble portions or linked water-soluble portions may be selected from, for example, carboxylates / esters, carboxylic acids, phosphonates / esters, phosphates / esters, sulfonates / esters, sulfonamides, sulfates / esters, sulfinates / esters, sulfonium, esters, polyoxyethylene, polyethylene oxide containing repeating ethylene oxide units of the formula -(CH2-CH2-O)n-, polyamide-ethylene oxide containing repeating ethylene oxide units of the formula -(CH2-CH2-O)n-, polyethylene glycol (PEG), modified PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, hydroxyl groups, amines, amino acids, ammonium, guanidine salts, pyridine, polyamines and sulfonium, polyols, linear or cyclic sugars, primary amines, secondary amines, tertiary amines or quaternary amines and polyamines, ethylene glycol, polyethers, -COOX, -SO3X, -PO3X, -NR 3+ X, (CH2CH2O) f R 15 and its mixtures, wherein R 15 X is hydrogen, a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted C1-C6 alkoxy group, and X is a counterion.

[0289] In some instances, R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains a reactive group, a linked reactive group, a conjugate tag, or a linked conjugate tag.

[0290] In some instances, R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains a chromophore or a connected chromophore.

[0291] In some instances, p is 1, 2, or 3. In some cases, p is 1. In some cases, p is 2. In some cases, p is 3. In some cases, p is 4.

[0292] In some instances, m is 0. In some instances, m is 1. In some instances, when m is 0, each T is independently CR. 1 or CR 1 R 2 In some instances, when m is 0, T is not NR. 4’ S or O. In some instances, when m is 1, each T is independently NR. 4’ O, S, Se, or Te. In some instances, when m is 1, T is not CR. 1 or CR 1 R 2 .

[0293] In some instances, the fluorescent compounds according to this disclosure are symmetrical.

[0294] In some instances, the fluorescent compounds according to this disclosure are asymmetric.

[0295] In some instances, X is selected from the following counterions:

[0296] F - Cl - , Br - I -ClO4 - CF3CO2 - CH3CO2 - PO4 3- SO4 2- BF4 - Na + K + Mg ++ , and Ca ++ .

[0297] In some instances, the reactive group or conjugation tag is selected from thiols, maleimides, halogenated maleimides, iodoacetamides, amines, alkyl carboxylates / esters, alkyl sulfonates / esters, carboxylic amines, carbamates, carboxylic esters, N-hydroxysuccinimides, halogens, borate esters, boric acids, hydrazones, carboxylic acids or their active esters, azides, alkynes, cyclooctyne, cyclooctene, tetrazines, cyclooctene, dienes, dienophiles, sulfo(VI) fluorine (SuFEX), sulfonyl fluoride, hydroxyl groups, hydrazines, hydrazines, aldehydes, ketones, azides, alkynes, phosphine, epoxides, and their protected groups.

[0298] In some cases, when m is 0, each T is independently CR. 1 or CR 1 R 2 In some cases, when m is 0, T is not NR. 4’ S or O. In some cases, when m is 1, each T is independently NR. 4’ O, S, Se, or Te. In some cases, when m is 1, T is not CR. 1 or CR 1 R 2 .

[0299] Fluorescent compounds according to this disclosure may comprise structures selected from the following:

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306] In this disclosure, benzothiophene-pyrrole intermediate compounds can be prepared via a variety of synthetic routes, for example, such as Figure 2 Schemes 1 and 2 Figure 3 Schemes 3A, 3B and 4 Figure 9 Option 14 Figure 16 Option 18 Figure 17 Option 20 Figure 19 As shown in Scheme 23, and as detailed in Embodiments 1A, 1B, 1C, 1D, 3F, 3I, 3J and 3L.

[0307] As shown in Scheme 1, tert-butyl hydrazine carboxylate and 2-iodobenzo[b]thiophene in DMF can be treated with Cs₂CO₃ and 1,10-phenanthroline and CuI to provide benzothiophene hydrazine compounds. 1 1-(benzo[b]thiophene-2-yl)hydrazine-1-carboxylic acid tert-butyl ester. (Compound) 1 The compound can be obtained by treatment with 3-methylbut-2-one and para-tolunensulfonic acid (pTSA). 2 2,3,3-Trimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole. (Compound) 2 Treatment with 1,3-propanesulfonic acid lactone can yield benzothiophene-pyrrole intermediates. 3 3-(2,3,3-trimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- -1-yl)propane-1-sulfonate / ester.

[0308] As shown in Scheme 2, ethyl 2-methyl-3-oxobutyrate in tert-butanol can be treated with potassium tert-butoxide and 1,3-propanesulfonic acid lactone to provide the compound. 4 4-Methyl-5-oxohexane-1-sulfonic acid. (Compound) 4 Benzothiophene hydrazine compounds can be used 1 Processing to obtain compounds 5 3-(2,3-dimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrolo-3-yl)propane-1-sulfonic acid. (Compound) 5 Treatment with 1,3-propanesulfonic acid lactone can yield benzothiophene-pyrrole intermediates. 6 3-(2,3-dimethyl-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- -1-yl)propane-1-sulfonate / ester.

[0309] Benzothiophene-pyrrole intermediates can be further derivatized, for example, as... Figure 3 As shown in Schemes 3A and 3B, and detailed in Examples 1C and 1D. Scheme 3A illustrates the compound being processed by treatment with 3-iodopropionic acid. 2Derivatization to provide benzothiophene-pyrrole (Bt) intermediate compounds 7 1-(2-Carboxyethyl)-2,3,3-trimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Iodides. Scheme 3B illustrates the derivatization of compound 5 by treatment with 3-iodopropionic acid to provide the Bt intermediate compound 8: 1-(2-carboxyethyl)-2,3-dimethyl-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Iodides.

[0310] Various indole intermediates can also be prepared for the synthesis of asymmetric BtCy dyes, for example, Figure 3 As shown in Scheme 4 and detailed in Example 1E, an indole intermediate compound is illustrated. 10 5-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)oxy)-1-(2-carboxyethyl)-2,3,3-trimethyl-3H-indole-1- An exemplary synthetic route for the iodide is carried out in two steps, which involves treating 2,3,3-trimethyl-3H-indole-5-ol with PEG-toluenesulfonyl to provide a PEGylated intermediate compound 9. Compound 9 can be treated with 3-iodopropionic acid to provide an indole intermediate compound 10.

[0311] Symmetrical BtCy compounds can be prepared, for example, such as Figure 4 and 5 As shown in schemes 5, 6, 7 and 8, and as detailed in embodiments 2A to 2D.

[0312] like Figure 4 As shown in Scheme 5, the benzothiophene-pyrrole intermediate compound can be treated with N,N'-diphenylformamidinium, acetic anhydride, and NaOAc to obtain a symmetrical BtCy3 fluorescent dye, such as compound 11 .

[0313] like Figure 4 and 5 As shown in schemes 6 and 8, the benzothiophene-pyrrole (Bt) intermediate compound can be treated with N-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline, acetic acid, and NaOAc to obtain symmetrical BtCy5 fluorescent dyes, such as compound 12 and 14 .

[0314] like Figure 4As shown in Scheme 7, the benzothiophene-pyrrole (Bt) intermediate compound can be treated with N-((1E,3E,5Z)-5-(phenylimino)pent-1,3-dien-1-yl)aniline, acetic acid, and NaOAc to obtain symmetrical ByCy3, BtCy5, and BtCy7 fluorescent dye compounds, respectively. 13 to 15 .

[0315] Asymmetric BtCy compounds can be prepared, for example, such as Figure 6 , 7 Schemes 9 to 15 and 17 to 22 of 8 and 9 are shown and detailed in Examples 3A to 3K.

[0316] like Figure 6 As shown in Scheme 9, the indole intermediate 1,2,3,3-tetramethyl-3H-indole-1- Iodides can be produced using acetic anhydride, followed by N-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline, and then by benzothiophene-pyrrole intermediates. 3 Treatment to provide asymmetric benzothiophene-pyrrole-Cy5 compounds 15 Indole and benzothiophene pyrrole monomers in a molar ratio of approximately 1:1 can be used.

[0317] like Figure 6 As shown in Scheme 10, indole intermediate 16 Acetic anhydride can be used, followed by N-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline, and then by benzothiophene-pyrrole intermediate compounds. 3 Treatment to provide asymmetric benzothiophene-pyrrole-Cy5 compounds 17 .

[0318] like Figure 7 As shown in Scheme 11, indole intermediate 18 Acetic anhydride can be used, followed by N-((1E,3E,5Z)-5-(phenylimino)pent-1,3-dien-1-yl)aniline, and then by benzothiophene-pyrrole intermediate compounds. 6 Treatment to provide asymmetric benzothiophene-pyrrole-Cy5 compounds 19 .

[0319] like Figure 7 As shown in Scheme 12, indole intermediate 10 Acetic anhydride can be used, followed by N-((1E,3E,5Z)-5-(phenylimino)pent-1,3-dien-1-yl)aniline, and then by benzothiophene-pyrrole intermediate compounds. 6 Treatment to provide asymmetric benzothiophene-pyrrole-Cy5 compounds 20 .

[0320] like Figure 8 As shown in Scheme 13, the compound 21 1-(2-Carboxyethyl)-3,3-dimethyl-2-((1E,3Z)-3-(3-methyl-1,3-bis(3-sulfopropyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-ylidene)prop-1-en-1-yl)-3H-indole-1- Iodides can be derived from intermediate compounds 18 It is prepared by adding acetic anhydride and N,N'-diphenylmethanemidine, followed by the addition of a compound. 6 To provide compound 21.

[0321] like Figure 9 As shown in Scheme 15, the asymmetric BtCy compound 24 Can be derived from intermediate compounds 23 It is prepared by adding acetic anhydride, N-((1E,3E,5Z)-5-(phenylimino)pent-1,3-dien-1-yl)aniline and compound 6 to provide compound 24.

[0322] like Figure 15 As shown in Scheme 17, the asymmetric BtCy compound 25 It can be composed of 1,2,3,3-tetramethyl-3H-indole-1- It is prepared by adding acetic anhydride, N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohexyl-2-en-1-yl)methyl)aniline and intermediate compounds. 3 To provide asymmetric BtCy compounds 25 .

[0323] like Figure 16 As shown in Scheme 18, the intermediate compound 26 By using compounds 3 It is made by exposure to fuming sulfuric acid.

[0324] like Figure 16 As shown in Scheme 19, the asymmetric BtCy compound 27 It can be composed of 1,2,3,3-tetramethyl-3H-indole-1- It is prepared by adding acetic anhydride, N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohexyl-2-en-1-yl)methyl)aniline and intermediate compounds. 26 To provide asymmetric BtCy compounds 27 .

[0325] like Figure 17 As shown in scheme 20, the intermediate compound28 It can be prepared by treating 2,3,3-trimethylindole with 6-bromohexanoic acid.

[0326] like Figure 17 As shown in Scheme 21, the asymmetric BtCy compound 29 It can be produced by using acetic anhydride, N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohex-2-en-1-ylidene)methyl)aniline and intermediate compounds 26 Treatment of intermediate compounds 28 To prepare to provide asymmetric BtCy compounds 29 .

[0327] like Figure 18 As shown in scheme 22, asymmetric BtCy compound 30 can be prepared by treating BtCy compound 29 with thiophenol and Cs2CO3.

[0328] like Figure 19 As shown in scheme 23, intermediate compound 32 can be prepared by treating naphth-2-ylhydrazine HCl with 7-oxooctanoic acid in acetic acid in a sealed flask to provide intermediate compound 31, and then treating compound 31 with 1,3-propanesulfonic acid lactone to provide intermediate compound 32.

[0329] like Figure 19 As shown in scheme 24, the asymmetric BtCy compound 33 It can be produced by using acetic anhydride, N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohex-2-en-1-ylidene)methyl)aniline and intermediate compounds 26 Treatment of intermediate compounds 32 To prepare to provide asymmetric BtCy compounds 33 .

[0330] This disclosure provides labeled specific binding partners comprising a BtCy compound or a BtCy tandem dye according to this disclosure; and a specific binding partner covalently linked to a BtCy compound.

[0331] In some instances, specific binding couplers are selected from proteins, peptides, affinity ligands, antibodies, antibody fragments, carbohydrates, lipids, nucleic acids, and aptamers. In some instances, specific binding couplers are antibodies. Specific binding couplers can specifically bind to target analytes. Specific binding couplers can specifically bind to target antigens.

[0332] This disclosure provides BtCy tandem dyes comprising the BtCy compound of this disclosure or a labeled specific binding partner; and a chromophore, fluorophore, or acceptor chromophore covalently linked to the BtCy compound or the labeled specific binding partner. The BtCy compound may also be an acceptor dye linked to a fluorescent compound. In this case, the BtCy tandem dye may also comprise a fluorescent dye and the BtCy compound of this disclosure covalently linked to the fluorescent dye.

[0333] The BtCy compounds, labeled specific binding partners, or tandem dyes according to this disclosure can be water-soluble fluorescent dyes. The fluorescent compounds, labeled specific binding partners, or tandem dyes according to this disclosure can exhibit solubility in water of >1 mg / mL, >2 mg / mL, >3 mg / mL, >4 mg / mL, >5 mg / mL, >6 mg / mL, >7 mg / mL, >8 mg / mL, >9 mg / mL, >10 mg / mL, >20 mg / mL, >30 mg / mL, >40 mg / mL, >50 mg / mL, >80 mg / mL, or >100 mg / mL at ambient room temperature.

[0334] BtCy compounds, labeled specific binding partners, or tandem dyes according to this disclosure may exhibit excitation maxima (λex) in the wavelength range of >500 nm, >600 nm, >700 nm, >800 nm, >850 nm, >900 nm, >950 nm, >1,000 nm, or >1,050 nm, >1,100 nm, or in the wavelength range of about 500 nm to 1200 nm, about 500 nm to about 1,100 nm, about 550 nm to about 1150 nm, about 550 nm to about 1,075 nm, about 550 nm to about 1,050 nm, about 575 nm to about 1,000 nm, or about 600 nm to about 950 nm.

[0335] BtCy compounds, BtCy-labeled specific binding partners containing BtCy compounds or BtCy tandem dyes according to this disclosure, or BtCy tandem dyes containing BtCy compounds may exhibit emission maximum values ​​(λem) in the ranges of >550 nm, >650 nm, >750 nm, >850 nm, >900 nm, >1000 nm, >1050 nm, >1100 nm, >1150 nm, >1200 nm, or >1250 nm, or in the ranges of about 550 nm to about 1300 nm, about 600 nm to about 1300 nm, about 600 nm to about 1200 nm, about 650 nm to about 1150 nm, or about 550 nm to about 1050 nm, or about 650 nm to about 1050 nm.

[0336] The BtCy dyes, tandem dyes, labeled specific binding mates, compositions, methods, and systems described herein can be used in a variety of applications, including diagnostic and research applications, where labeling, detection, and / or analysis of a target analyte is desired. Such applications include, for example, methods such as cytology, microscopy, immunoassays (e.g., competitive or non-competitive), fluorescence in situ hybridization (FISH), cell tracing, acceptor labeling, fluorescence spectroscopy, evaluation of free analytes, evaluation of acceptor-binding ligands, etc. The compositions, systems, and methods described herein can be useful in any aspect of analyzing a wide variety of samples, including but not limited to biological fluids, cell culture samples, and tissue samples. In some aspects, the compositions, systems, and methods described herein can be used in methods in which fluorescent labeling is used to detect an analyte in a sample (if present), such as for fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, etc. In some cases, the compositions and methods can be used in applications in which the presence of a target analyte in a sample is evaluated. In some cases, the methods and compositions can be used in any assay in which a target is detected and / or analyzed from a target sample, including but not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography. In some cases, the methods and compositions can be used in any application in which the target molecule is fluorescently labeled. The subject composition can be adapted to any convenient application in which paired specific binding members, such as biotin-streptavidin and hapten-anti-hapten antibody, can be used.

[0337] This disclosure provides a method for detecting a target analyte in a sample, comprising: providing a sample suspected of containing the analyte; and contacting the sample with a specific binding coupler conjugated to a BtCy compound or tandem dye of this disclosure, wherein the specific binding coupler is capable of interacting with the target analyte. The specific binding coupler may be a target analyte-specific antibody or an antigen-binding fragment thereof.

[0338] In some instances, the method is configured for use in flow cytometry.

[0339] In some instances, water-soluble fluorescent compounds bind to the substrate.

[0340] In some instances, the analyte is a protein expressed on the cell surface.

[0341] In some instances, the method is configured as an immunoassay. In some instances, the method further includes providing an additional binding coupler for the simultaneous detection of another analyte.

[0342] This disclosure provides kits comprising at least one of a BtCy compound according to this disclosure, a labeled specific binding partner, or a tandem dye. Some aspects of the invention also include kits for carrying out the subject methods and compositions. The compositions of the invention may be included as reagents in the kits, as starting materials, or provided for use, for example, in the methods described above.

[0343] The kit may contain a BtCy dye, a BtCy tandem dye, or a BtCy-labeled specific binding member as described herein, and a container. Any convenient container may be used, such as tubes, bottles, wells in multi-well strips or plates, boxes, bags, insulated containers, etc. In some cases, the kit may contain one or more components selected from BtCy dyes, BtCy tandem dyes, fluorophores, chromophores, specific binding members, specific binding member conjugates, support-bound specific binding members, cells, supports, biocompatible aqueous elution buffers, and instructions for use. In some embodiments of the kit, the BtCy dye or BtCy tandem dye is covalently linked to the specific binding member.

[0344] In some cases, the subject kit may contain a “labeling kit” of BtCy dyes or BtCy tandem dyes containing side-chain chemiselective functional groups, such as BtCy-NHS esters (also known as “conjugated tags”), to which any convenient target motif (e.g., donor or acceptor dye, fluorophore, chromophore, specific binding partner, support) may be conjugated. The chemiselective functional group may include a reactive group (e.g., biotin) that targets a specific functional group on a biomolecule (e.g., a protein or antibody), such as a primary amine, thiol, carboxyl, or carbohydrate. The chemiselective functional group may be a functional group used for “click chemistry” reactions.

[0345] In some cases, the conjugation tag contains a maleimide functional group, and the target portion contains a thiol functional group, or vice versa. In some cases, the conjugation tag contains an alkyne (e.g., a cyclooctyne group) functional group, and the target portion contains an azide functional group, or vice versa, which can be conjugated via click chemistry. In some cases, the conjugation tag contains an alkene (e.g., a cyclooctene group) functional group, and the target portion contains a tetrazine functional group, or vice versa, which can be conjugated via a reverse-demand Diels-Alder cycloaddition reaction. In some cases, the conjugation tag contains an amine-reactive chemical group, such as an NHS ester (N-hydroxysuccinimide ester) or an imine ester functional group, and the target portion contains an NH2 functional group, or vice versa. In some cases, the conjugation tag contains a biotin-binding protein (e.g., avidin, streptavidin, or neutral avidin), and the target portion contains a biotin molecule, or vice versa, which can interact non-covalently.

[0346] In some cases, the target moiety is a specific binding partner. In some cases, the specific binding partner is an antibody. In some cases, the specific binding partner is an antibody fragment or its binding derivative. In some cases, the antibody fragment or its binding derivative is selected from Fab fragments, F(ab')2 fragments, scFv, diabody, and triabody.

[0347] In some cases, the target component is a fluorophore or chromophore. In others, the fluorophore or chromophore is an acceptor dye. In still others, the fluorophore or chromophore is a donor dye.

[0348] Table 1 illustrates the symmetric and asymmetric BtCy compounds of the exemplary invention of this disclosure and their fluorescence properties, including the maximum excitation wavelength (λex) and emission wavelength (λem). Compared to the common indole-cyanine compounds Cy3, Cy5, and Cy7, the BtCy3, BtCy5, and BtCy7 compounds exhibit a redshift of approximately 200 nm.

[0349] Table 1. BtCy compounds and excitation and emission data

[0350]

[0351]

[0352]

[0353]

[0354] n / a = Not yet available

[0355] Unless otherwise stated, the absorption / emission spectra shown in Table 1 were obtained in methanol. *Measured in DMF.

[0356] As shown in Table 1, the invented benzothiophene-pyrrole anthocyanin dye BtCy3 compound 11 BtCy5 compounds 12 and BtCy7 compounds 13 The benzothiophene-pyrrole cyanine dyes in Table 1 exhibit a redshift of approximately 200 nm Abs / Em compared to existing indole cyanine dyes Cy3, Cy5, and Cy7. The benzothiophene-pyrrole cyanine dyes in Table 1 show maximum absorbance values ​​from 642 to 972 nm. The benzothiophene-pyrrole cyanine dyes in Table 1 show maximum emission values ​​from 662 to 997 nm.

[0357] IV. Combination of the spouse body

[0358] The term "binding partner" or "specific binding partner" in this disclosure can refer to any molecule or molecular complex capable of specifically binding to a target analyte. Binding partners in this disclosure include, for example, proteins, small organic molecules, carbohydrates (including polysaccharides), oligonucleotides, polynucleotides, lipids, affinity ligands, antibodies, antibody fragments, aptamers, etc. In some embodiments, the binding partner is an antibody or a fragment thereof. In the context of this disclosure, specific binding refers to a binding reaction that identifies the presence of a target analyte in the presence of a heterogeneous population. Therefore, under specified assay conditions, a particular binding partner preferentially binds to a specific protein or a specific isoform of a protein and does not bind in significant amounts to other proteins or other isoforms present in the sample.

[0359] When the binding partner is an antibody, it can be a monoclonal or polyclonal antibody. As used herein, the term antibody refers to the immunoglobulin molecule and the immunoglobulin (Ig) molecule with immunoactivity. Such antibodies include, but are not limited to, polyclonal, monoclonal, monospecific polyclonal antibodies, antibody mimics, chimeric, single-chain, Fab, Fab' and F(ab')2 fragments, Fv and Fab expression libraries.

[0360] Generally, the water-soluble fluorescent compounds of this disclosure can be conjugated with binding couplers to form conjugated water-soluble fluorescent compound complexes using techniques known to those skilled in the art or by combining methods known in the art with those described herein.

[0361] A labeled specific binding coupler is provided, comprising: a fluorescent BtCy compound according to this disclosure; and a specific binding coupler covalently linked to the fluorescent compound. The specific binding coupler may be an antibody. The specific binding coupler may be specific to a target analyte.

[0362] In some embodiments, the fluorescent compounds of this disclosure can be conjugated to binding couplers using a direct modification method similar to that described in US2020 / 0190253 (which is incorporated herein by reference in its entirety). For example, BtCy dye-antibody complexes can be conjugated according to... Figure 14 The preparation is carried out according to the general scheme shown in Scheme 16.

[0363] For example, the preparation of BtCy-NHS ester can be performed as follows: Using a clean vial, dissolve 5 mg of BtCy in 1 mL of dry CH3CN. Add 2 mg of N,N,N',N'-tetramethyl-O-(N-succinimide)tetrafluoroborate (TSTU) and stir for more than 2 minutes. Add 20 μL of N,N-diisopropylethylamine (DIPEA) and continue stirring for about 1 hour, then seal the vial with a sealing film. Afterward, evaporate the organic solvent from the reaction mixture. The crude BtCy-NHS can be separated by extraction / purification and used immediately for antibody labeling.

[0364] The conjugation of BtCy NHS with anti-CD4 antibody can be performed as follows: Dissolve BtCy-NHS in approximately 10 μL of DMSO, add this to 0.6 mg of CD4, and mix with 100 μL of 0.5 M borate buffer (pH 9.0). Vortex rapidly for 30 seconds and allow to mix in a Coulter mixer for 3 to 4 hours.

[0365] The purification of BtCy-antibody conjugates can be performed as follows.

[0366] Method 1: Purification of BtCy-antibody conjugates via SEC column can be performed as follows. The crude conjugate, containing the free dye and the conjugate, is loaded onto a size exclusion column using 1×PBS. After examining the absorbance spectra, the tubes are combined and concentrated in an Amicon Ultra-15 centrifuge with a 30 kDa MWCO concentrator.

[0367] Method 2: The conjugation reaction mixture can be loaded into a pre-equilibrated (PBS 1×) size size size spin column with an appropriate cutoff value (10k, 20K or 40K Zeba) to obtain purified BtCy-antibody conjugates.

[0368] Method 3: Purification of BtCy-antibody conjugates via anti-mouse anti-H+L antibody-agarose beads can be performed as follows. At room temperature, mix the crude BtCy-antibody conjugate mixture with anti-mouse anti-H+L antibody-agarose beads in a biological buffer at a pH of approximately 6 to 8 for approximately 30 minutes. The anti-mouse anti-H+L antibody-agarose beads will bind to the BtCy-antibody conjugate. Remove unreacted BtCy by washing with the above biological buffer at 300 g for 3 minutes using a benchtop centrifuge. Repeat the washing process at least three times. To elute the BtCy-antibody conjugate, apply IgG elution buffer at a pH of approximately 2 to 4 to the washed antibody-agarose beads and incubate for approximately 10 to 15 minutes. Centrifuge to collect the runoff containing the antibody conjugate.

[0369] V. Tandem dyes

[0370] The compounds of this disclosure can be donor dyes or acceptor dyes. The compounds of this disclosure and labeled specific binding partners are capable of transferring energy to or receiving energy from a connected acceptor chromophore. When the compounds of this disclosure and labeled specific binding partners are donor dyes, the acceptor chromophore may be covalently linked to a DHP-bridged compound or labeled specific binding partner near the energy receiver, such that excitation of the donor DHP-bridged compound or labeled specific binding partner results in energy transfer to and emission from the covalently linked acceptor signaling chromophore. When the compounds of this disclosure and labeled specific binding partners are acceptor dyes, they may be covalently linked to a donor chromophore near the energy receiver, such that excitation of the donor results in energy transfer to and emission from the covalently linked acceptor signaling chromophore. The energy transfer mechanism between the compounds of this disclosure and labeled specific binding partners and the connected donor or acceptor chromophore includes, for example, resonant energy transfer (e.g., (or fluorescence) resonance energy transfer Chromophores can be involved in various processes, including fluorescence-resonant energy transfer (FRET) and quantum charge exchange (Dexter energy transfer). In some cases, a chromophore can be a fluorophore. In some cases, a chromophore can be an acceptor dye. In some cases, a chromophore can be a donor dye.

[0371] Therefore, in some embodiments, the fluorescent BtCy compound, water-soluble fluorescent BtCy compound, and labeled specific binding pair of this disclosure include an additional fluorophore, donor dye, acceptor dye, or chromophore linked to the fluorescent compound. In some cases, the BtCy compound is a donor dye. In some cases, the BtCy compound is an acceptor dye. When the donor compound is excited by a light source, the fluorophore, acceptor dye, or chromophore may absorb energy of an appropriate wavelength and emit or transfer energy.

[0372] The fluorophore (FP), chromophore, donor, or acceptor dyes associated with the fluorescent dyes of this invention may have absorption or emission spectra that overlap to some extent with the absorption or emission spectra of the BtCy compounds of this disclosure. The FP, chromophore, donor, or acceptor dyes associated with the fluorescent dyes of this invention may be fluorescent dyes with a maximum absorption length greater than 405 nm or 575 nm and a maximum emission length greater than 428 nm, 450 nm, or 600 nm, and optionally exhibit fluorescence quantum yields greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 10%. The fluorophore may be selected from coumarin, fluorescein, rhodamine, cyanine, bodipy, or other polycyclic aromatic hydrocarbons. Many fluorophores are commercially available and may be selected from, but are not limited to, any dye available from Beckman Coulter, Inc., including but not limited to SuperNova polymer dyes; any dye available from Becton Dickinson Biosciences, including but not limited to BD HorizonBrilliant. TM Polymer dyes; any dyes available from ThermoFisher Scientific, including but not limited to Super Bright polymer dyes and Alexa Fluor dyes, which include but are not limited to...

[0373] Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680; ATTO 390, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 532, ATTO 550, ATTO 565, ATTO590, ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740,

[0374] 5-Carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 5-carboxynapthofluorescein, 5-carboxytetramethylrhodamine (5-TAMRA), 5-FAM (5-carboxyfluorescein), 5-ROX, 6-TAMRA, 6-carboxyrhodamine 6G,

[0375] 6-CR6G、6-JOE、6-FAM、6-ROX、Bodipy 492 / 515、Bodipy 493 / 503、Bodipy 500 / 510、Bodipy 505 / 515、Bodipy 530 / 550、Bodipy 542 / 563、Bodipy 558 / 568、Bodipy 564 / 570、Bodipy 576 / 589、Bodipy 581 / 591、Bodipy 630 / 650-X、Bodipy 650 / 665-X、Bodipy665 / 676、Bodipy Fl、Bodipy R6G、Bodipy TMR、Bodipy TR、CF 488A、CF 555、CF 568,CF594ST,CF 633,CF 640R,CF 647、CF 660C、CF 680、CF680R、CF 750、CF 770、CF 790、CL-NERF、CMFDA、Cy2、Cy3、Cy3.5、Cy5、Cy5.5、Cy7、DDAO、DiA、DiD、DiI、DyLight 488、DyLight550、DyLight 594、DyLight 633、DyLight 650,DyLight 680,DyLight 755,DyLight 800, DiO, DiR, DM-NERF, DsRed, DTAF, DY-490, DY-495, DY-505, DY-530, DY-547, DY-548, DY-549, DY-549P1, DY-550, DY-554, DY-555, DY-556, DY-560 ， DY-590, DY-591, DY-594, DY-605, DY-610, DY-615, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, DY-647, DY-648, DY-649, DY-649P1, DY-650, DY-651, DY-652, DY-654, DY-675, DY-676, DY-677, DY-678, DY-679, DY-679P1, DY-680, DY-681, DY-682, DY-700, DY-701, DY-703 ，DY-704, DY-730, DY-731, DY-732, DY-734, DY-749, DY-750, DY-751, DY-752, DY-754, DY-776, DY-777, DY-77 8. DY-780, DY-781, DY-782, DY-800, DY-831, eosin, erythrosine, FITC, Fluo-3, Fluo-4, Fluor-Ruby, FluorX, FM1-43, FM 1-46, iFluor 488, iFluor 555, iFluor594, iFluor 647, iFluor 680, iFluor700, iFluor 750, iFluor 780, Lyso Tracker Green, Lyso Tracker Yellow, Mitotracker Green, Mitotracker Orange, Mitotracker Red, NBD, Oregon Green 488, Oregon Green 514, PKH26, PKH67, Halogen, RH414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine 6G, Rhodamine B, Rhodamine Green, Rhodamine Red, Rose Red, Spectral Green, Spectral Orange, Spectral Red

[0376] SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX blue, SYTOX green, SYTOX orange, Texas red, TideFluor 2 (TF2), Tide Fluor 2WS (TF2WS), Tide Fluor 3(TF3), Tide Fluor 3WS(TF3WS), Tide Fluor 4(TF4), Tide Fluor 5WS(TF5WS), Tide Fluor 6WS(TF6WS), Tide Fluor 7WS(TF7WS), Tide Fluor 8WS(TF8WS), TRITC, and XTRITC.

[0377] In some cases, the fluorophores, chromophores, donor dyes, or acceptor dyes that can be used in this disclosure may include, for example, cyanine dyes, succinate dyes, etc. Dyes, coumarin dyes, thiazine dyes, acridine dyes, FITC, CY3B, Cy55, Alexa488, Texas Red, Cy5, Cy7, Alexa 750, Cy55, Cy3B, Cy3.5, Alexa 750, 800CW, Biotium CF555, diethylcoumarin, DY705 (Dyomics), DY431, DY485XL, DY500XL, DY610, DY640, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY778, DY782, DY800, DY831 and 800CW.

[0378] In some cases, BtCy compounds can function as acceptor dyes, with fluorescent polymer dyes / protein dyes acting as donors. The fluorescent polymer dye can be any suitable fluorescent polymer dye. For example, fluorescent polymer dyes are mentioned in U.S. Patent Nos. 11,208,527; 11,584,825; 11,119,107; 11,485,825; 11,492,493; 9159,465; 11,215,612; 11,209,43; 10,288,620; 8,969,509; 8431,416; 11,099,190; 10,604,657; 10,533,092; 10,920,082.

[0379] And disclosed in U.S. patent applications US2020 / 0048469; US 2022 / 0082568; US2022 / 0276255; US2021 / 0373029; US 2022 / 0340813; US 2021 / 0108083; and US 2022 / 0348770, each of which is incorporated herein by reference in its entirety. The polymer dye may also be BD Horizon Brilliant. TM Polymer dyes (Becton Dickinson Biosciences) or Super Bright polymer dyes (ThermoFisher Scientific).

[0380] The acceptor dye may be a side-group acceptor dye. The tandem dye may comprise a benzothiophene-pyrrole-cyanine compound according to this disclosure, said compound optionally comprising one or more, or two or more, additional fluorophores, chromophores, or acceptor dye moieties. The tandem dye may comprise a fluorophore or chromophore donor dye and a BtCy acceptor dye according to the present invention.

[0381] Fluorescent tandem dyes can be prepared using techniques known to those skilled in the art or by combining methods known in the art with those described herein. The tandem dyes can be water-soluble.

[0382] In some embodiments, the fluorophore, acceptor dye, chromophore, and / or functional moiety and binding partner are not directly linked to the compound, but are instead linked to the BtCy compound of this disclosure via a linker portion using a direct modification method as described in US2020 / 0190253 (which is incorporated herein by reference in its entirety). In some embodiments, this disclosure provides a tandem dye comprising: a fluorescent compound or a labeled specific binding partner according to this disclosure; and a fluorophore, chromophore, or acceptor dye covalently linked to the fluorescent compound or the labeled specific binding partner.

[0383] VI. Methods for detecting analytes

[0384] This disclosure provides a method for detecting a target analyte in a sample, the method comprising: providing a sample suspected of containing the target analyte; and contacting the sample with a specific binding partner conjugated to a fluorescent BtCy or BtCy tandem dye compound according to this disclosure, wherein the binding partner is capable of interacting with the target analyte.

[0385] A light source that can excite a fluorescent compound or donor dye is applied to the sample; and the light emitted from the conjugated fluorescent compound complex or acceptor dye is detected. In a typical assay, the water-soluble fluorescent compound of this disclosure can be excited by light with wavelengths of about 500 nm to about 1200 nm, about 550 nm to about 1150 nm, about 575 nm to about 1100 nm, or about 600 nm to about 1000 nm, and the emitted light is typically about 600 nm to about 1200 nm, about 650 nm to about 1150 nm, or about 700 nm to about 1100 nm.

[0386] Alternatively, the wavelength of the excitation light can be from about 600 nm to about 1100 nm, and the wavelength of the emitted light can be from about 650 nm to about 1200 nm. Depending on the design of the compound, the fluorescent compounds of this disclosure can have excitation spectra tuned to yellow, orange, red, and NIR or another laser.

[0387] In the methods of this disclosure, the fluorescent BtCy compound can be any water-soluble fluorescent BtCy compound or BtCy tandem dye as disclosed herein. The binding partner can be a protein, peptide, affinity ligand, antibody, antibody fragment, carbohydrate, lipid, nucleic acid, or aptamer. When the binding partner is an antibody, the method can be configured for flow cytometry; the water-soluble fluorescent BtCy dye or tandem dye can bind to a substrate; the analyte can be a protein expressed on a cell surface; the method can be configured for immunoassay; or the method may further include providing additional specific binding partners for the simultaneous detection of additional analytes.

[0388] sample

[0389] The sample used in the methods of this disclosure may be, for example, blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate (or any cells obtained from bone marrow), urine (lavage fluid), serum, saliva, cerebrospinal fluid, urine, amniotic fluid, interstitial fluid, feces, mucus, or tissue (e.g., tumor sample, decomposed tissue, decomposed solid tumor). In some embodiments, the sample is a blood sample. In some embodiments, the blood sample is whole blood. Whole blood can be obtained from the subject using standard clinical procedures. In some embodiments, the sample is a subset of one or more cell types from whole blood (e.g., red blood cells, white blood cells, lymphocytes (e.g., T cells, B cells, or NK cells), phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, or any cell type having one or more detectable markers). In some embodiments, the sample may be derived from cell cultures.

[0390] The subject can be a human (e.g., a patient with a disease), a commercially important mammal, including, for example, a monkey, a cow, or a horse. Samples may also be obtained from domestic pets, including, for example, dogs or cats. In some embodiments, the subject is a laboratory animal used as a disease animal model or for drug screening, such as a mouse, rat, rabbit, or guinea pig.

[0391] Analytes

[0392] As used herein, "analyte" or "target analyte" refers to a substance, such as a molecule, whose abundance / concentration is determined by some analytical procedure. For example, in this disclosure, an analyte can be a protein, peptide, nucleic acid, lipid, carbohydrate small molecule, or target-related biomolecule.

[0393] The target analyte can be, for example, nucleic acids (DNA, RNA, mRNA, tRNA, or rRNA), peptides, polypeptides, proteins, lipids, ions, monosaccharides, oligosaccharides, polysaccharides, lipoproteins, glycoproteins, glycolipids, or fragments thereof. In some embodiments, the target analyte is a protein and can be, for example, structural microfilaments, microtubules and intermediate filament proteins, organelle-specific markers, proteasomes, transmembrane proteins, surface receptors, nuclear porins, protein / peptide translocases, protein folding chaperones, signal transduction scaffolds, ion channels, etc. The protein can be an activatable protein or a differentially expressed or activated protein in diseased or abnormal cells, including but not limited to transcription factors, DNA and / or RNA-binding proteins and modified proteins, nuclear import and export acceptors, apoptosis or survival regulators, etc.

[0394] Measurement

[0395] Assay systems that utilize binding couplers and fluorescent labeling to quantify bound molecules are well known. Examples of such systems include flow cytometry, scanning cytometry, imaging cytometry, fluorescence microscopy, and confocal fluorescence microscopy.

[0396] In some implementations, flow cytometry is used to detect fluorescence. Many devices suitable for this purpose are available and are known to those skilled in the art. Some examples include the BCI Navios, Gallios, Aquios, and CytoFLEX flow cytometers.

[0397] In other embodiments, an assay is used. The assay may be an immunoassay. Some examples of immunoassays that can be used in this disclosure include, but are not limited to, fluoroluminescence assays (FLA). Assays may also be performed on protein arrays.

[0398] When the binding partner is an antibody, antibody or multi-antibody sandwich assays can also be used. A sandwich assay refers to the use of successive recognition events to construct a layer of multiple binding partners and reporter elements to signal the presence of a specific analyte. Some examples of sandwich assays are disclosed in U.S. Patent No. 4,486,530 and the references cited therein.

[0399] VII. Examples

[0400] Example 1. Synthesis of intermediate compounds of benzothiophene / pyrrole and indole

[0401] The intermediate and product compounds are characterized by mass spectrometry and / or 1 H-NMR.

[0402] Example 1A. Benzothiophene-pyrrole intermediate 33-(2,3,3-trimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Synthesis of 1-yl)propane-1-sulfonate / ester.

[0403] Benzothiophene-pyrrole intermediate 3 Synthesis such as Figure 2 Scheme 1 is as shown.

[0404] compound 1 1-(benzo[b]thiophene-2-yl)hydrazine-1-carboxylic acid tert-butyl ester

[0405] In a round-bottom flask, 5.7 g of 2-iodobenzo[b]thiophene (21.9 mmol) and 7.24 g of tert-butyl hydrazine carboxylate (54.8 mmol) were dissolved in 10 mL of DMF. Then, 10 g of Cs₂CO₃ (30.7 mmol), 0.39 g of 1,10-phenanthroline (2.19 mmol), and 42 mg of CuI (0.22 mmol) were added. The mixture was purged with nitrogen for 10 min and then reacted at 48 °C for 4 days.

[0406] Next, 15 ml of H2O was added to the reaction mixture and stirred for 10 minutes. The mixture was diluted with a 1 / 1 brine / water mixture and extracted three times with ethyl acetate. The organic fractions were combined, washed with brine, and then concentrated. The residue was purified using an automated chromatographic system using neutral alumina as the stationary phase and hexane / ethyl acetate as the mobile phase. 1.36 g (23%) of the product was collected as an orange solid to provide the compound. 1 . 1 The desired structure was determined by H NMR and MS.

[0407] 1 H NMR D6DMSO (500MHz): 7.75 (1H, d, J = 13.1Hz), 7.61 (1H, d, J = 13.1Hz), 7.75 (1H, t, J = 12.8Hz), 7.15 (1H, t, J = 12.8Hz), 5.59 (2H, bs), 1.55 (9H, s). HRMS (ESI+): m / z measured 248.0465 [M+H-NH2].

[0408] compound 2 2,3,3-Trimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole contains 300 mg of the compound dissolved in 5 mL of ethanol. 1243 μL of 3-methylbutanone (2.27 mmol) and 648 mg of p-toluenesulfonic acid (pTSA) (3.4 mmol) were added to a 1.14 mmol flask. The mixture was heated to 85 °C for 6 hours and then left at room temperature overnight. Afterward, 8 mL of water was added, and the pH of the resulting solution was adjusted to 9 by adding 3 mL of 2 M Na₂CO₃. The mixture was extracted with CHCl₃ to bind and concentrate the organic fraction. The residue was purified using an automated chromatographic system with neutral silica as the stationary phase and hexane / ethyl acetate as the mobile phase. 175 mg g (71%) of the product was collected as an orange oil to provide the compound. 2 HRMS (ESI+): m / z measured value 216.0839 [M+H].

[0409] compound 3 3-(2,3,3-trimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- -1-yl)propane-1-sulfonate

[0410] 175mg of the compound 2 (0.81 mmol) was dissolved in 1 mL of hot 1,2-dichlorobenzene, and 109 mg of 1,3-propanesulfonic acid lactone (0.9 mmol) was added with the aid of 0.25 mL THF. The mixture was heated at 120 °C for 3 hours and then cooled to room temperature. 1 mL of hexane was added, and the liquid was decanted. Then 0.6 g of toluene was added, the mixture was sonicated, and then decanted. The resulting solid was dissolved in 0.5 mL of MeOH and precipitated again by adding diethyl ether. The solid compound was collected. 3 245mg (89%). 1 H-NMR and mass spectrometry identified the compound 3 The expected structure.

[0411] 1 H NMR D6DMSO (500MHz): 8.12 (1H, d, J = 7.4Hz), 8.06 (1H, d, J = 7.4Hz), 7.63-7.55 (2H, m), 4.19 (2H, m), 2.60-2.55 (2H, m), 2.58 (3H, s), 2.16-2.10 (2H, m), 1.59 (6H, s). HRMS (ESI+): m / z measured 360.0699 [M+Na].

[0412] Example 1B. Benzothiophene-pyrrole intermediate 6 3-(2,3-dimethyl-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Synthesis of 1-yl)propane-1-sulfonate / ester

[0413] Benzothiophene-pyrrole intermediate 6 Synthesis such as Figure 2 Scheme 2 is as shown.

[0414] compound 4 4-Methyl-5-oxohexane-1-sulfonic acid

[0415] Add 3.84 g of ethyl 2-methyl-3-oxobutyrate (26.16 mmol) to a solution of 3.56 g potassium tert-butoxide (31.9 mmol) in 52 mL of tert-butanol. While stirring, add 3.25 g of 1,3-propanesulfonic acid lactone (26.6 mmol) in portions. Heat the mixture to 110 °C for 2 hours, then allow it to cool to room temperature. Evaporate the solvent and dissolve the residue in 30 mL of water. Wash the resulting solution with hexane, discard the organic fraction, and acidify the aqueous layer to pH 1 with concentrated HCl. Evaporate the solvent, add 80 mL of MeOH to the residue, and sonicate the mixture for 5 minutes. Filter out the insoluble residue and collect the methanol solution.

[0416] 3.6 g of NaOH (86.3 mmol) was dissolved in 18 mL of water, and the solution was added to the methanol solution above. The resulting mixture was heated to 50 °C for 12 hours. Afterward, the MeOH was evaporated, and the residue was acidified to pH 1 using concentrated HCl. The solvent was removed using a rotary evaporator. 60 mL of acetone was added to the residue, and the mixture was sonicated for 10 minutes. The insoluble substance was filtered off, and the acetone was evaporated to give 2.6 g of the product compound as a pale yellow oil. 4 (50%). 1 H-NMR identified the compound 4 The expected structure.

[0417] 1 H NMR D6DMSO (500MHz): 2.53-2.46 (3H, m), 2.08 (3H, s), 1.65-1.58 (1H, m), 1.55-1.45-(2H, m), 1.36-1.29 (1H, m), 0.97 (3H, d, J=7.1Hz).

[0418] compound 5 3-(2,3-dimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrolo-3-yl)propane-1-sulfonic acid

[0419] 340mg of the compound 4 (1.75 mmol) and 420 mg of benzothiophene hydrazine compound1 (1.5 mmol) was dissolved in 8 mL of acetic acid, and the mixture was heated to 92 °C for 12 hours. Afterward, the solvent was evaporated, and the residue was dissolved in approximately 8 mL of MeOH, followed by the addition of 8 mL of water to induce precipitation. The mixture was centrifuged, and the supernatant was collected and concentrated. The resulting substance was purified by C18 reversed-phase chromatography using a 1 / 1 MeOH / H2O mobile phase to give 440 mg of the compound as a deep orange solid. 5 (91%), which can be used in the next step. 1 H-NMR and mass spectrometry identified the compound 5 The expected structure.

[0420] 1 H NMR D6DMSO (500MHz): 7.84 (1H, d, J = 7.6Hz), 7.64 (1H, d, J = 7.6Hz), 7.37-7.29 (2H, m), 2.29-2.25 (5H, m), 1.96-1.90 (1H, m), 1.77-1.71 (1H, m), 1.24 (3H, s), 1.18-1.06 (2H, m). HRMS (ESI+): m / z measured value 324.1005 [M+H].

[0421] compound 6 3-(2,3-dimethyl-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- -1-yl)propane-1-sulfonate / ester

[0422] 90mg of the compound 5 (0.28 mmol) was dissolved in 1 mL of hot 1,2-dichlorobenzene, and 41 mg of 1,3-propanesulfonic acid lactone (0.33 mmol) was added. The mixture was heated at 120 °C for 1 hour and then cooled to room temperature. 1 mL of hexane was added, and the liquid was decanted. Then 0.6 mL of toluene was added, the mixture was sonicated, and then decanted. The resulting solid was washed with hexane, and the residue was purified by preparative HPLC using a C8 column as the stationary phase and MeOH / H2O as the mobile phase. 80 mg (64%) of the product compound was collected as a deep purple solid. 6 . 1 H-NMR and mass spectrometry identified the compound 6 The expected structure.

[0423] 1H NMR D6DMSO (500MHz): 8.15 (1H, d, J = 8.6Hz), 8.08 (1H, d, J = 8.6Hz), 7.63-7.56 (2H, m), 4.25-4.10 (2H, m), 2.59-2.86 (5H, m), 2.40-2.28 (4H, m), 2.20-2.06 (2H, m), 1.61 (3H, s), 0.99-0.91 (2H, m). HRMS (ESI-): m / z measured 444.1738 [MH].

[0424] Example 1C. Benzothiophene-pyrrole intermediate compound 7 1-(2-Carboxyethyl)-2,3,3-trimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Synthesis of iodides.

[0425] like Figure 3 As shown in scheme 3A, the compound 7 From compounds 2 For use in compounds 3 It is made in a similar way, except that 3-iodopropionic acid is used instead of 1,3-propanesulfonic acid lactone. 1 H-NMR and mass spectrometry identified the compound 7 The expected structure.

[0426] 1 H NMR D6DMSO (500MHz): 8.14-8.05 (2H, m), 7.65-7.58 (2H, m), 4.24 (2H, t, J = 13.6Hz), 2.93-2.85 (2H, m), 2.59 (3H, s), 1.61 (6H, s). HRMS (ESI+): m / z measured value 288.1044 [M+].

[0427] Example 1D. Benzothiophene-pyrrole intermediate compound 8 1-(2-Carboxyethyl)-2,3-dimethyl-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Synthesis of iodides.

[0428] like Figure 3 As shown in scheme 3B, compound 8 With compounds 7 It is made in a similar way, the difference being the use of compounds. 5 Instead of compounds 2 . 1 H-NMR and mass spectrometry identified the compound 8 The expected structure.

[0429] 1 H NMR D6DMSO (500MHz): 8.16-8.06 (2H, m), 7.69-7.51 (2H, m), 4.26-4.21 (2H, m), 2.96-2.86 (2H, m), 2.67-2.57 (5H, m), 2.38-2.33 (2H, m), 1.61 (3H, s), 0.99-0.92 (2H, m). HRMS (ESI-): m / z measured value 394.1062 [M-2H].

[0430] Example 1E. Indole intermediate compound 10 5-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)oxy)-1-(2-carboxyethyl)-2,3,3-trimethyl-3H-indole-1- Synthesis

[0431] Indole intermediate compounds 10 Synthesis such as Figure 3 Scheme 4 is as shown.

[0432] compound 9 5-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)oxy)-2,3,3-trimethyl-3H-indole

[0433] The starting material 2,3,3-trimethyl-3H-indole-5-ol was prepared according to the literature protocol. (Tomasulo et al., 2007, J Org Chem 72, 2, 595-605). 130 mg of 2,3,3-trimethyl-3H-indole-5-ol (0.74 mmol) and 406 mg of m-PEG8-Tos PEG connector (0.82 mmol) containing toluenesulfonyl BP22358 from BroadPharm were dissolved in 10 mL of acetone, and 0.4 g of K2CO3 (2.9 mmol) was added. The mixture was heated to 60 °C for 14 hours, followed by solvent evaporation. The residue was purified using an automated chromatographic system with silica as the stationary phase and CHCl3 / MeOH as the mobile phase. 240 mg of the product compound was collected. 9 (50%). 1 H-NMR identified the compound 9 The expected structure.

[0434] 1H NMR CDC13 (500MHz): 7.51 (1H, d, J = 8.6Hz), 6.92 (1H, d, J = 3.5Hz), 6.87 (1H, dd, J = 8.6Hz, J = 3.5Hz), 4.17 (2H, t, J = 4.5Hz), 3 .89 (2H, t, J = 5.1Hz), 3.76-3.74 (2H, m), 3.71-3.65 (20H, m), 3.57-3.55 (2H, m), 3.39 (3H, s), 2.38 (3H, s), 1.34 (6H, s).

[0435] compound 10 5-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)oxy)-1-(2-carboxyethyl)-2,3,3-trimethyl-3H-indole-1- Typical object.

[0436] 105 mg of the compound 2 0.21 mmol of 3-iodopropionic acid and 47 mg of 3-iodopropionic acid (0.23 mmol) were dissolved in 1 mL of toluene. The mixture was heated to 100 °C for 12 hours, followed by evaporation of the solvent. The residue was washed with hexane, then dissolved in 0.5 mL of EtOAc, and precipitated by adding 20 mL of hexane to give 143 mg (98%) of the compound. 10 . 1 H-NMR and mass spectrometry identified the compound 10 The expected structure.

[0437] HRMS (ESI+): Measured m / z value 570.3019 (M + -I). 1 H NMR CDC13 (500MHz): 7.69 (1H, d, J=8.8Hz), 7.13 (1H, dd, J=9.0Hz, J=2.3Hz), 7.10 (1H, d, J=2.2Hz), 4.84 (2H, t, J=6.4Hz), 4.24 (2H, t, J=4.7Hz), 3 .89 (2H, t, J=4.5Hz), 3.73-3.71 (2H, m), 3.67-3.62 (20H, m), 3.55-3.5 3(2H,m), 3.37(3H,s), 3.23(2H,t,J=5.5Hz), 2.97(3H,s), 1.58(6H,s).

[0438] Example 2. Synthesis of Symmetrical BtCy Dyes

[0439] Symmetrical BtCy dyes such as Figure 4 and Figure 5The preparation method is outlined in the document.

[0440] Example 2A. Compound 11 (BtCy3): 3-(2-((1E,3Z)-3-(3,3-dimethyl-1-(3-sulfonylpropyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-yl)prop-1-en-1-yl)-3,3-dimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Sodium 1-propane-1-sulfonate (e.g.) Figure 4 The preparation method is shown in Scheme 5.

[0441] 9 mg of the compound was placed in a glass vial. 3 (26.7 μmol) was dissolved in 0.2 mL of EtOH, followed by the addition of 2.3 mg of N,N'-diphenylmethanemid (11.6 μmol), 0.6 mL of acetic anhydride, and 30 mg of NaOAc (36 μmol). The mixture was heated to 80 °C for 1 hour. Afterward, the solvent was evaporated, and the residue was purified by automated column chromatography using silica as the stationary phase and chloroform / methanol as the mobile phase to provide the compound. 11 For compounds 11 The UV Vis shows the maximum absorbance at 758 nm (MeOH) and the emission at 769 nm (MeOH).

[0442] Example 2B. Compound 12 (BtCy5): 3-(2-((1E,3E,5Z)-5-(3,3-dimethyl-1-(3-sulfonylpropyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-yl)pent-1,3-dien-1-yl)-3,3-dimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Sodium 1-propane-1-sulfonate (e.g.) Figure 4 Scheme 6 is generated as shown.

[0443] Put 20 mg of the compound in a glass vial. 3 (59 μmol) was dissolved in 0.3 mL of EtOH, then 4.4 mg of N-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline (17 μmol), 0.8 mL of acetic anhydride, and 50 mg of NaOAc (61 μmol) were added. The mixture was heated to 90 °C for 2 hours. Afterward, the solvent was evaporated, and the residue was purified by automated column chromatography using silica as the stationary phase and chloroform / methanol as the mobile phase to give 11 mg of the product compound. 12ES1-TOF mass spectrometry: calculated exact mass: 709.15 g / mol; MW: 709.93; m / z: 709.15 (100%). For compound 12, UV Vis shows the maximum absorbance at 860 nm (MeOH) and the emission at 875 nm (MeOH). 1 H-NMR and mass spectrometry identified the compound 12 The expected structure.

[0444] HRMS (ESI-): Measured m / z value 709.1495 (M - ). 1 H NMRD6DMSO (500MHz): 7.96 (2H, d, J = 7.2Hz), 7.91 (2H, d, J = 8.1Hz), 7.84 (2H, t, J = 13.3Hz), 7.47 (2H, t, J = 7.9Hz), 7.3 1 (2H, t, J=7.7Hz), 3.81-6.74 (3H, m), 3.90 (4H, t, J=8.0Hz), 2.57 (4H, t, J=7.1Hz), 2.09-2.06 (4H, m), 1.71 (s, 12H).

[0445] Example 2C. Compound 13 (BtCy7): 3-(2-((1E,3E,5E,7Z)-7-(3,3-dimethyl-1-(3-sulfonylpropyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-yl)hept-1,3,5-trien-1-yl)-3,3-dimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Sodium 1-propane-1-sulfonate (e.g.) Figure 4 Scheme 7 is generated as shown.

[0446] 9 mg of the compound was placed in a glass vial. 3 (27 μmol) was dissolved in 0.6 mL of EtOH, then 3.5 mg of N-((1E,3E,5Z)-5-(phenylimino)pent-1,3-dien-1-yl)aniline (12 μmol), 0.6 mL of acetic anhydride, and 0.2 mL of pyridine were added. The mixture was heated to 110 °C for 1 hour. Afterward, the solvent was evaporated, and the residue was purified by automated column chromatography using silica as the stationary phase and chloroform / methanol as the mobile phase to give 5 mg of the product compound. 13For compound 13, UV Vis shows a maximum absorbance at 972 nm (MeOH) and emission at 997 nm (MeOH). ESI (negative mode) shows a peak at m / z = 735.22. HRMS (ESI-): measured m / z 735.2250 M. - ).

[0447] Example 2D. Compound 14 3-(3-methyl-2-((1E,3E,5Z)-5-(3-methyl-1-(3-sulfonylpropyl)-3-(3-sulfopropyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-yl)pent-1,3-dien-1-yl)-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- Sodium 1-propane-1-sulfonate (e.g.) Figure 5 The compound was produced as shown in scheme 8. 14 Use with compounds 12 A similar experimental protocol was prepared, but using compounds 6 as starting material rather than compound 3 For compound 14, UV Vis shows the maximum absorbance at 863 nm (MeOH) and the emission at 887 nm (MeOH).

[0448] Example 3. Synthesis of Asymmetric BtCy Dyes

[0449] Asymmetric BtCy dyes such as Figure 6 , Figure 7 , Figure 8 and Figure 9 The preparation method is outlined in the document.

[0450] Example 3A. Compound 15 : 4-((Z)-3,3-dimethyl-2-((2E,4E)-5-(1,3,3-trimethyl-3H-indole-1- -2-yl)pent-2,4-dien-1-ylide)-2,3-dihydro-1H-benzo[4,5]thieno[2,3-b]pyrrolo-1-yl)butane-1-sulfonate / ester Figure 6 Scheme 9 is shown.

[0451] Initiated by 1,2,3,3-tetramethyl-3H-indole-1- The iodide was prepared according to reference EP2289563 and is also commercially available (Aldrich, CAS 5418-63-3).

[0452] In a glass vial, add 30 mg of 1,2,3,3-tetramethyl-3H-indole-1- Iodide (0.1 mmol) was dissolved in 0.6 mL of acetic anhydride, then 25.6 mg of N-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline (0.1 mmol) was added, and the mixture was heated to 90 °C for 40 minutes. Afterwards, 35 mg of the compound dissolved in 0.4 mL of EtOH was added... 3 (0.11 mmol) was added together with 66 mg of NaOAc (0.8 mmol). The mixture was heated to 90 °C for 1 hour. Afterwards, the solvent was evaporated, and the residue was purified by automated column chromatography using silica as the stationary phase and chloroform / methanol as the mobile phase to give 6 mg of the product compound. 15 For compound 15, UV Vis data show a maximum absorbance at 740 nm (MeOH) and an emission at 766 nm (MeOH). ESI MS (positive mode) shows peaks at m / z 547.2 and M+Na at 569.2. HRMS (ESI+): measured m / z values ​​of 547.2089 (M+H) and 569.1891 (M+Na).

[0453] Example 3B. Compound 17 : 1-(2-Carboxyethyl)-2-((1E,3E,5Z)-5-(3,3-dimethyl-1-(4-sulfonylbutyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-ylidene)pent-1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1- Sodium 5-sulfonate, such as Figure 6 Scheme 10 is generated as shown.

[0454] compound 17 Use with compounds 15 A similar experimental protocol was prepared, but using compounds 16 Instead of 1,2,3,3-tetramethyl-3H-indole-1- Iodides. Compounds 16 1-(2-Carboxyethyl)-2,3,3-trimethyl-5-sulfonyl-3H-indole-1- The iodides were prepared according to the literature protocol. Tomasulo et al., 2007, J Org Chem 72, 2, 595-605. For compound 17, UV Vis shows the maximum absorbance at 747 nm (DMF) and the emission at 777 nm (DMF).

[0455] Example 3C. Compound19 1-(2-Carboxyethyl)-3,3-dimethyl-2-((1E,3E,5E,7Z)-7-(3-methyl-1,3-bis(3-sulfopropyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-ylidene)hept-1,3,5-trien-1-yl)-3H-indole-1- Iodides such Figure 7 Scheme 11 is shown.

[0456] compound 19 Use with compounds 15 A similar experimental protocol was prepared, but using compounds 18 Instead of 1,2,3,3-tetramethyl-3H-indole-1- Iodides and compounds used 6 Instead of compounds 3 Compounds 18 1-(2-Carboxyethyl)-2,3,3-trimethyl-3H-indole-1- Iodides were produced according to a literature protocol. Fissi et al., Macromolecules 1995, 28, 302-309. For compound 19, UV Vis shows the maximum absorbance at 842 nm (MeOH) and the emission at 885 nm (MeOH).

[0457] Example 3D. Compound 20 :3-(2-((1E,3E,5E)-7-((Z)-5-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)oxy)-1-(2-carboxyethyl)-3,3-dimethylindoline-2-yl)hept-1,3,5-trien-1-yl)-3-methyl-3-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- -1-yl)propane-1-sulfonate / ester Figure 7 Scheme 12 is shown.

[0458] compound 20 Use with compounds 15 A similar experimental protocol was prepared, but using compounds 10 Instead of 1,2,3,3-tetramethyl-3H-indole-1- Iodides and compounds 6 Instead of compounds 3 For compound 20, UV Vis shows the maximum absorbance at 859 nm (MeOH).

[0459] Example 3E. Compound 21 1-(2-Carboxyethyl)-3,3-dimethyl-2-((1E,3Z)-3-(3-methyl-1,3-bis(3-sulfopropyl)-1,3-dihydro-2H-benzo[4,5]thieno[2,3-b]pyrrole-2-ylidene)prop-1-en-1-yl)-3H-indole-1- Iodides such Figure 8 Scheme 13 is generated as shown.

[0460] compound 21 Use with compounds 19 Similar experimental protocols are derived from compounds 18 and compounds 6 It was prepared, differing in that N,N'-diphenylmethanemidane was used instead of N-((1E,3E)-3-(phenylimino)prop-1-en-1-yl)aniline. For compound 21, UV Vis showed a maximum absorbance at 642 nm (MeOH) and emission at 662 nm (MeOH). ESIMS (negative mode) showed a peak at m / z = 685.25. HRMS (ESI-): measured m / z 685.2531.

[0461] Example 3F. Compound 24 :3-(2-((1E,3E,5E)-7-((Z)-1-(2-carboxyethyl)-3,3-dimethyl-5-(3-sulfonylpropoxy)indoline-2-ylidene)hept-1,3,5-trien-1-yl)-3-methyl-3-(3-sulfonylpropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- -1-yl)propane-1-sulfonate / ester Figure 9 Schemes 14 and 15 are shown.

[0462] intermediate compounds 22 3-((2,3,3-trimethyl-3H-indol-5-yl)oxy)propane-1-sulfonic acid Figure 9 It is made as shown in scheme 14.

[0463] 70 mg of 2,3,3-trimethyl-3H-indole-5-ol (0.4 mmol) was dissolved in acetone (10 mL) to form a solution. 166 mg of K₂CO₃ (1.2 mmol) and 51 mg of 1,3-propanesulfonic acid lactone (0.42 mmol) were added to the solution. The mixture was heated to 70 °C for 12 hours, and then the solvent was evaporated. The residue was dissolved in 2 mL of H₂O and then acidified with 1 M HCl to pH < 2. The resulting precipitate was collected to give 120 mg (99%) of the product compound. 22 . 1H-NMR and mass spectrometry determined the desired structure of compound 22.

[0464] HRMS (ESI-): m / z measured value 296.2177. 1 NMR: D6DMSO (500MHz): 7.27 (1H, d, J = 8.3Hz), 7.02 (1H, d, J = 2.5Hz), 6.78 (1H, dd, J 1 =8.3Hz, J 2 =2.5Hz), 4.04 (2H, t, J = 6.5Hz), 2.55 (2H, t, J = 6.3Hz), 2.15 (3H, s), 2.02-1.96 (2H, m), 1.21 (6H, s).

[0465] intermediate compounds 23 1-(2-Carboxyethyl)-2,3,3-trimethyl-5-(3-sulfonylpropoxy)-3H-indole-1- Iodides such Figure 9 It is made as shown in scheme 14.

[0466] 120mg of the compound 22 (0.4 mmol) was dissolved in 1 mL of dichlorobenzene, and 104 mg of 3-iodopropionic acid (0.48 mmol) was added. The mixture was reacted at 120 °C for 14 hours. Afterwards, the solvent was decanted, and the residue was washed with hexane and finally with CHCl3. The residue was purified by preparative HPLC using a C8 column as the stationary phase and MeOH / H2O as the mobile phase. 200 mg (99%) of the product compound was collected as a dark brown solid. 23 . 1 H-NMR and mass spectrometry determined the desired structure of compound 23.

[0467] HRMS(ESI-): m / z 368.2203(MI). 1 NMR: D6DMSO (500MHz): 7.86 (1H, d, J = 8.8Hz), 7.49 (1H, d, J = 2.5Hz), 7.12 (1H, dd, J 1 =8.9Hz, J 2 =2.5Hz), 4.59 (2H, t, J = 7.0Hz), 4.18 (2H, t, J = 6.6Hz), 2.94 (2H, t, J = 7.0Hz), 2.77 (3H, s), 2.56 (2H, t, J = 7.2Hz), 2.02 (2H, m), 1.50 (6H, s).

[0468] Example 3G. Compound 24:1-((l1-oxoalkyl)dioxo-l6-thioalkyl)-3-(2-((1E,3E,5E)-7-((Z)-1-(2-carboxyethyl)-3,3-dimethyl-5-(3-sulfonylpropoxy)indoline-2-ylidene)hept-1,3,5-trien-1-yl)-3-methyl-3-(3-sulfonylpropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- -1-Base)Pro-1-ide Figure 9 It is made as shown in Scheme 15.

[0469] compound 24 Use with compounds 20 A similar experimental protocol was prepared, but using compounds 23 as starting material rather than compound 10 For compounds 24 The UV Vis shows the maximum absorbance at 869 nm (MeOH) and the emission at 899 nm (MeOH).

[0470] Example 3H. Compound 25 3-(2-((E)-2-((E)-2-chloro-3-(2-((E)-1,3,3-trimethylindoline-2-ethylene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- 1-yl)propane-1-sulfonate / ester

[0471] compound 25 like Figure 15 It is made as shown in Scheme 17.

[0472] In a glass vial, 40 mg of 1,2,3,3-tetramethyl-3H-indole-1- Iodide (0.13 mmol) was dissolved in 1.4 mL of acetic anhydride, and then 55 mg of N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohexyl-2-en-1-yl)methyl)aniline (0.15 mmol) was added. The mixture was heated to 100 °C for 30 min. Afterward, the solvent was evaporated, Et₂O was added to the residue, and the mixture was then vortexed. The purified residue was dissolved in fresh acetic anhydride (1 mL), and 55 mg of compound 3 (0.16 mmol) dissolved in 0.8 mL of EtOH was added together with 65 mg of NaOAc (0.8 mmol). The mixture was heated to 90 °C for 2.5 h. Afterward, the solvent was evaporated, and the residue was purified by automated column chromatography using silica as the stationary phase and chloroform / methanol as the mobile phase to give 28 mg of the product.25 Structure through 1 To determine by H NMR.

[0473] 1 H NMR: D6DMSO (500MHz): 8.3 (1H, s), 8.08 (1H, d), 8.05 (1H, d), 7.58-7.51 (2H, m), 7.44-7.37 (2H, m). 7.26 (1H, t), 7.17 (1H, d), 7.07 (1H, d), 7.00 (1H, t), 6.82 (1H, d), 5.75 (1H, d), 4.12 (2H, t), 3.37 (3H, s), 2.71 (2H, m ), 2.64-2.56 (4H, m), 2.13 (2H, m) 1.85 (2H, m), 1.67 (6H, s), 1.54 (6H, s).

[0474] For compounds 25 The UV Vis shows the maximum absorbance at 875 nm and the emission at 895 nm (MeOH).

[0475] Example 3I. Compound 27 3-(2-((E)-2-((E)-2-chloro-3-(2-((E)-1,3,3-trimethylindoline-2-ethylene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfonyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- 1-yl)propane-1-sulfonate / ester

[0476] Asymmetric benzothiophene-pyrrole-cyanine dye compounds 27 like Figure 16 It is made as shown.

[0477] intermediate compounds 26 2,3,3-Trimethyl-5-sulfono-1-(3-sulfopropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- like Figure 16 The preparation is shown in Scheme 18.

[0478] 90 mg of compound 3 was added to a vial containing 1 mL of fuming sulfuric acid, which had previously been frozen at -20°C for 1 hour. The mixture was stirred in an ice bath and warmed to room temperature overnight. Afterward, the mixture was transferred to a centrifuge vial containing 10 mL of ethyl acetate and centrifuged. The compound was collected as a precipitate. 26 It was washed with diethyl ether and used in the next step. ESI (positive mode) showed a peak at m / z = 418.02 (M+).

[0479] HRMS (ESI+): m / z measured value 418.0243. 1 NMR: D6DMSO (500MHz): 8.11 (1H, d, J = 1.5Hz), 8.01 (1H, d, J = 8.5Hz), 7.81 (1H, d d, J=2Hz, J=8.5Hz), 4.18 (2H, m), 2.62-2.55 (5H, m), 2.13 (2H, m), 1.59 (6H, s).

[0480] compound 27 like Figure 16 It was prepared as shown in Scheme 19. 20 mg of 1,2,3,3-tetramethyl-3H-indole-1- was placed in a glass vial. Iodide (0.066 mmol) was dissolved in 0.6 mL of acetic anhydride, then 27 mg of N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohexyl-2-en-1-yl)methyl)aniline (0.076 mmol) was added, and the mixture was heated to 100 °C for 30 min. Afterward, the solvent was evaporated, Et₂O was added to the residue, and then vortexed. The purified residue was dissolved in fresh acetic anhydride (1 mL), and 33 mg of the compound dissolved in 0.3 mL of EtOH was added. 26 (0.08 mmol) was added together with 33 mg of NaOAc (0.398 mmol). The mixture was heated to 90 °C for 3 hours. Afterwards, the solvent was evaporated, and the residue was purified by automated column chromatography using silica as the stationary phase and chloroform / methanol as the mobile phase to give 7 mg of the product compound. 27 Mass spectrometry identified the compound. 27 The expected structure. ESI (negative mode) shows a peak at m / z = 749.28 (M+Na). HRMS (ESI-): m / z = 749.2884 (M+Na). - +Na).Compound 27 It exhibits a maximum absorbance at 883 nm (MeOH) and an emission at 900 nm (MeOH).

[0481] Example 3J. Compound 29 :3-(2-((E)-2-((E)-3-(2-((E)-1-(5-carboxypentyl)-3,3-dimethylindoline-2-ethylene)ethylene)-2-chlorocyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfonyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- 1-yl)propane-1-sulfonate / ester

[0482] Asymmetric benzothiophene-pyrrole-cyanine dye compounds 29 like Figure 17 It is made as shown in schemes 20 and 21.

[0483] Intermediate compound 281-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole-1- like Figure 17 The compound was prepared as shown in Scheme 20. 1.1 g of compound 2,3,3-trimethylindole (6.9 mmol) was dissolved in 5 mL of dichlorobenzene, and 2.7 g of 6-bromohexanoic acid (13.8 mmol) was added. The mixture was reacted at 120 °C for 14 hours. Afterward, 10 mL of hexane was added, and the solvent was decanted. The residue was washed three times with 6 mL of ethyl acetate and finally dissolved in 4 mL of MeOH. The solution was slowly added to a flask containing 140 mL of isopropyl ether, and the precipitate was collected to give 2.1 g of the compound. 28 . 1 H-NMR and mass spectrometry identified the compound 28 The expected structure.

[0484] 1 H-NMR: D6DMSO (500MHz): 7.97 (1H, m), 7.84 (1H, m), 7.62 (2H, m), 4.46 (2H, t, J = 7.6Hz), 2.84 (3H, s), 2.22 (2H, t, J = 7.0Hz), 1.84 (2H, m), 1.60–1.52 (2H, m), 1.53 (6H, s), 1.43 (2H, m). HRMS (ESI+): Measured value 274.1685.

[0485] Compound 29 was prepared using the same experimental procedure as compound 27, but with the use of an intermediate compound. 28 Instead of 1,2,3,3-tetramethyl-3H-indole-1- Iodides. Compound 29 exhibits a maximum absorbance at 891 nm (MeOH) and emission at 908 nm (MeOH). HRMS (ESI-): Measured value 825.2941.

[0486] Example 3K. Compound 30 3-(2-((E)-2-((E)-3-(2-((E)-1-(5-carboxypentyl)-3,3-dimethylindoline-2-ethylene)ethylene)-2-(phenylthio)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfonyl-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- 1-yl)propane-1-sulfonate / ester

[0487] Asymmetric benzothiophene-pyrrole-cyanine dye compounds 30 like Figure 18 The solution was prepared as shown in Scheme 22. 3 μL of thiophenol (0.029 mmol) and 11 mg of Cs₂CO₃ (0.033 mmol) were dispersed in 100 μL of MeCN in a glass vial, and the solution was stirred for 45 minutes at 0 °C under a nitrogen atmosphere. Subsequently, 17 mg of the compound was added dropwise. 29 A solution of (0.021 mmol) was prepared in 200 μL of DMF, and the reaction mixture was reacted overnight in an ice bath. The mixture was then filtered through a thin reverse-phase C18 filter washed with MeOH. The solvent was evaporated, and the residue was washed with cold diethyl ether and dried to give 18 mg of the compound. 30 Compounds 30 It exhibits a maximum absorbance at 903 nm (MeOH) and an emission at 925 nm (MeOH).

[0488] Example 3L. Compound 33 :2-((E)-2-((E)-3-((E)-2-(1-(5-carboxypentyl)-1-methyl-3-(3-sulfonylpropyl)-1,3-dihydro-2H-benzo[e]indol-2-ethylene)ethylene)-2-chlorocyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-sulfonylpropyl)-3H-benzo[4,5]thieno[2,3-b]pyrrole-1- 5-Sulfonate / Ester

[0489] Asymmetric benzothiophene-pyrrole-cyanine dye compounds 33 like Figure 19 It is made as shown in schemes 23 and 24.

[0490] intermediate compounds 31 6-(1,2-Dimethyl-1H-benzo[e]indol-1-yl)hexanoic acid Figure 19 The product was prepared as shown in Scheme 23. 964 mg of naphthylhydrazine HCl (4.95 mmol) and 967 mg of 7-oxooctanoic acid (5.2 mmol) were dissolved in 10 mL of acetic acid in a sealed flask. The mixture was heated to 120 °C for 14 hours and then cooled to room temperature. The solvent was evaporated, and the residue was purified by automated column chromatography using silica as the stationary phase and hexane / ethyl acetate as the mobile phase to give 1.23 g (87%) of the product.

[0491] 1NMR D6DMSO (500MHz): 8.07 (1H, d, J = 82Hz), 8.00 (1H, d, J = 8.2Hz), 7.90 (1H, d, J = 8.2H z), 7.67 (1H, d, J = 8.2Hz), 7.57 (1H, t, J = 6.9Hz), 7.46 (1H, t, J = 6.9Hz), 2.38-2.32 (1H, m), 2.27 (3H, s), 2.03-1.94 (3H, m), 1.44 (3H, s), 1.22 (2H, t, J = 7.4 Hz), 1.09-0.98 (2H, m), 0.51-0.44 (1H, m), 0.21-0.14 (1H, m). HRMS (ESI-): m / z measured value 308.1948.

[0492] intermediate compounds 32 1-(5-Carboxypentyl)-1,2-Dimethyl-3-(3-sulfopropyl)-1H-benzo[e]indole-3- like Figure 19 The compound was prepared as shown in Scheme 23. 423 mg of compound 31 (1.37 mmol) was dissolved in 2 mL of 1,2-dichlorobenzene, and 217 mg of 1,3-propanesulfonic acid lactone (1.78 mmol) was added. The mixture was heated at 120 °C for 48 hours and then cooled to room temperature. 6 mL of hexane was added, and the liquid was decanted. The residue was washed with hexane (3 times, 6 mL each time), then with toluene (once, 6 mL), and then again with hexane (2 times, 6 mL each time). After drying, 472 mg (80%) of the compound was collected. 32 .

[0493] 1 NMR D6DMSO (500MHz): 8.34 (1H, d, J = 8.5Hz), 8.30 (1H, d, J = 88Hz), 8.25-8.21 (2H, m), 7.78 ( IH, t, J=6.1Hz), 7.73 (1H, t, J=6.1Hz), 4.82 (2H, m), 342 (2H, t,, J=6.5Hz), 2.97 (3H, s), 2.69-2.63 (2H, m), 2.21-2.17 (2H, m), 1.74 (3H, s), 1.69-1.78 (2H, m), 1.27-1.20 (2H, m), 1.09-0.99 (2H, m), 0.61-0.55 (1H, m), 0.20-0.14 (1H, m). HRMS (ESI-): m / z measured value 430.2076

[0494] compound 33 Use with compounds 27Prepared using the same experimental procedure, but using compounds 32 Instead of 1,2,3,3-tetramethyl-3H-indole-1- Iodides. Compound 33 exhibits a maximum absorbance at 907 nm (MeOH) and an emission at 928 nm (MeOH). HRMS (ESI-): (M+CH3COO-) measured value 1043.2855.

[0495] The foregoing description, examples, and data provide a complete description of the manufacture and use of the compounds of the present invention. Since many embodiments of the invention can be carried out without departing from the spirit and scope thereof, the invention is encompassed in the appended terms and claims.

[0496] Terms and Conditions

[0497] Clause 1. A fluorescent compound comprising a structure according to formula (I):

[0498] in

[0499] Selected from substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl, polycyclic aryl, monocyclic heteroaryl and polycyclic heteroaryl;

[0500] Selected from substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl and polycyclic aryl;

[0501] Each T is independently for NR 4’ CR 1 CR 1 R 2 ,O,S,Se,or Te

[0502] V is NR 11 CR 8 CR 8 R 9 O, S, Se, or Te, or each TV together can represent a selection from SO2, -CR 1 -O-,-O-CR 1 -,-CO-O-,-O-CO-,-CO-NR 11 -, or -NR 4’ -CO- structural elements;

[0503] G is either C or N;

[0504] Each R 1 R 2 R 8 and R 9Independently selected from water-soluble moieties, linked water-soluble moieties, linker moieties, E, linked E, reactive groups, linked reactive groups, conjugated mating bodies, linked conjugated mating bodies, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonic acid, sulfonate / ester, alkylsulfonate / ester, alkylsulfonate, alkoxysulfonate / ester, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, alkoxycarboxylate / ester, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylsulfonamide PEG, alkylamide.

[0505]

[0506] Each R 4 R 4’ R 10 and R 11 Independently selected from the following: linker portion, chromophore, linked chromophore, reactive group, linked reactive group, water-soluble moiety, linked water-soluble moiety, conjugated mating body, linked conjugated mating body, E, linked E, H, halogenated alkyl, alkenyl, alkynyl, PEG group, linked PEG group, cycloalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, sulfonic acid, sulfonate / ester, alkyl sulfonate / ester, alkyl sulfonate, carboxylic acid, carboxylate / ester, alkyl carboxylate / ester, alkyl sulfonamide, alkyl sulfonamide PEG, alkylamide, alkylamide-PEG

[0507]

[0508] Or its protected group; or R 4 and R 12 Together, R 14 and R 10 Together, R 4 and R 13 Together, R 13 and R 10 Together, R 13 and R 11 Together, R 12 and R 14 Together, R 4’ and R 13 Together, R 4’ and R 12 Together, R 13 and R 10 Together, R 4R 12 R 14 and R 10 Together, R 4 R 13 and R 10 Together, R 4 R 13 and R 11 Together, R 4 R 12 and R 14 Together, R 4’ R 13 and R 11 Together, and R 12 R 14 and R 10 One, two, three, or four together form unsubstituted or substituted unsaturated or partially unsaturated C3-C atoms. 10 cycloalkyl; unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 10 Heterocyclic alkyl groups; unsubstituted or substituted unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polycyclic alkyl; or unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polyhedraloyl groups;

[0509] Each R 3 Independently selected from H, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, alkyl sulfonate / ester, alkyl carboxylate / ester, water-soluble moiety, linked water-soluble moiety, chromophore, linked chromophore, functional moiety, linked functional moiety, conjugate, linked conjugate, PEG group and linked PEG group;

[0510] Each Q is independently a bond, O, NH, NR 4 C1-C 12 Alkylene, CHR 4 Or CH2;

[0511] Each Z is independently CH2, CHR 4 O, NR 4 Or NH;

[0512] Each W 1 It is a water-soluble component on its own.

[0513] L 1 L 2 and L 3Each connector is selected independently.

[0514] Each E is independently selected from chromophores, functional moieties, substrates, reactive groups, and binding partners;

[0515] Each R 7 Independently selected from H, hydroxyl, C1-C 12 Alkyl, C1-C 12 Heteroalkyl, C2-C 12 Olefins, C2-C 12 Alkynes, C3-C 12 cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy group, C2-C 18 (Miscellaneous) arylamino, carboxylates / esters, carboxylic acids, C2-C 12 Alkyl carboxylic acids, C2-C 12 Alkyl carboxylates / esters, C2-C 12 Alkyl carboxylic acid esters, aryl carboxylic acids, aryl carboxylic acid esters, C1-C 12 Alkyl groups, water-soluble moiety, PEG moiety, protected or unprotected functional groups, chemoselective functional groups, linkers, sulfonic acids, sulfonates / esters, C1-C 12 Alkyl sulfonates / esters, sulfonamides;

[0516] Each R 12 R 13 and R 14 Independently selected from hydrogen, halogen, one or more heteroatoms, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C1-C6 olefin, substituted or unsubstituted C3-C 10 Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C3-C 10 Heterocyclic alkyl groups, CO2R 1 CONR 1 R 2 -O-CH2CH2-PEG-R 7 -S-CH2CH2-PEG-R 7 -N-CH2CH2-PEG-R 7 O-aryl, S-aryl, N-aryl, -O-alkyl, S-alkyl, N-alkyl, wherein each alkyl or aryl group may optionally be etched with one or more R groups. 7 PEG, PEG-R 7 Or, alternatively, a linking group is substituted, wherein each R 12 R 13and R 14 Independently replaced by the following: R 7 Group; or R 1 R 2 R 4 R 4’ R 8 R 9 R 10 R 11 R 12 R 13 , and R 14 At least two of them together, including but not limited to R 1 R 2 R 4 R 4’ R 8 R 9 R 10 R 11 R 12 R 13 and R 14 Two, three, or four of them together, optionally including but not limited to R 1 R 2 R 4 R 4’ R 8 R 9 R 10 R 11 R 12 R 13 and R 14 One, two, three or four together, including but not limited to R 4 and R 12 Together, R 14 and R 10 Together, R 4 and R 13 Together, R 13 and R 10 Together, R 13 and R 11 Together, R 12 and R 14 Together, R 4’ and R 13 Together, R 4’ and R 12 Together, R 13 and R 10 Together, R 4 R 12 R 14 and R 10 Together, R 4 R 13 and R 10 Together, R4 R 13 and R 11 Together, R 4 R 12 and R 14 Together, R 4’ R 13 and R 11 Together, and R 12 R 14 and R 10 Formation of unsaturated or partially unsaturated C3-C, whether substituted or substituted. 10 Cycloalkyl, unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 10 Heterocyclic alkyl groups, unsubstituted or substituted, unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polycyclic alkyl groups, or substituted unsaturated or partially unsaturated C3-C groups, either unsubstituted or optionally O-substituted. 14 C3-C 10 Or C3-C8 polyhedraloyl groups;

[0517] Each K is independently a covalent bond, O, S, Se, P, NR 1 or CR 1 R 2 ;

[0518] Each f is an independent integer from 0 to 50, 1 to 30, or 2 to 20;

[0519] Each m and m' is independently 0, 1, 2, or 3;

[0520] Each n is an independent integer from 1 to 20; from 1 to 10; or 0, 1, 2, or 3.

[0521] Each p is independently 1, 2, 3 or 4;

[0522] Each s is independently 1 or 2;

[0523] Each t is independently 0, 1, 2, 3, or 4; and

[0524] X is a counter ion.

[0525] Clause 2. The compound described in Clause 1, comprising a structure according to any one of formulas (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), (IIg), (IIh), (IIi), (IIj), (IIk), (IIl), (IIm), (IIIn), and (IIo):

[0526]

[0527]

[0528]

[0529]

[0530] in

[0531] T is NR 4’ CR 1 CR 1 R 2 , O or S;

[0532] V is NR 11 CR 8 CR 8 R 9 , O or S;

[0533] Each R 5 and R 6 Independently selected from water-soluble moieties, linked water-soluble moieties, linker moieties, E, linked E, reactive groups, linked reactive groups, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 Independently selected from hydrogen, sulfonates / esters, alkyl sulfonates / esters, alkyl sulfonamides, alkyl sulfonamide-PEG, phosphate groups, carboxylic acids, carboxylates / esters, amides, alkylamides, amide-PEG, and water-soluble groups; and

[0534] R 16 It's KR 13 Optional H, halogen, OC 1-6 Alkyl, SC 1-6 Alkyl, O-aryl, S-aryl, NHC 1-6 Alkyl groups, Ph-NCS, Ph-CO2H, Ph-(CH2) 1-4 CO2H; and

[0535] It is an optional substituted cycloalkenyl or polycycloalkenyl moiety, which may be partially unsaturated.

[0536] Clause 3. The compound described in Clause 1 or 2, comprising a structure according to any one of formulas (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), (IIIj), (IIIk), (IIIl), (IIIm), or (IIIn):

[0537]

[0538]

[0539]

[0540]

[0541] in

[0542] Each R 5 and R 6 Independently selected from water-soluble moieties, linked water-soluble moieties, linker moieties, E, linked E, reactive groups, linked reactive groups, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 Independently selected from hydrogen, sulfonates / esters, alkyl sulfonates / esters, alkyl sulfonamides, alkyl sulfonamide-PEG, phosphate groups, carboxylic acids, carboxylates / esters, amides, alkylamides, amide-PEG, and water-soluble groups; and It is an optional substituted cycloalkenyl or polycycloalkenyl moiety.

[0543] Clause 4. The compound described in Clause 1 or 2, comprising a structure according to any one of the formulas (IVva), (IVb), (IVc), (IVd), (IVe), (IVf), (IVg), (IVh), (IVVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVu), (IVv), (IVw), and (IVx):

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550] in

[0551] Each R 5 and R 6 Independently selected from water-soluble moieties, linked water-soluble moieties, linker moieties, E, linked E, reactive groups, linked reactive groups, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphinamide, phosphinamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 Independently selected from hydrogen, sulfonates / esters, alkyl sulfonates / esters, alkyl sulfonamides, alkyl sulfonamide-PEG, phosphate groups, carboxylic acids, carboxylates / esters, amides, alkylamides, amide-PEG, and water-soluble groups; and It is an optional substituted cycloalkenyl or polycycloalkenyl moiety.

[0552] Clause 5. The compound of any one of Clauses 1 to 4, comprising a structure according to any one of the formulas (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi), (Vj), (Vk), and (Vl):

[0553]

[0554]

[0555]

[0556]

[0557] in

[0558] Y is selected from NR 4 CR 1 CR 1 R 2 O and S.

[0559] Clause 6. The compound of any one of Clauses 1 to 5, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains E or is connected to E.

[0560] Clause 7. The compound of any one of Clauses 1 to 6, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains a combined spouse or a connected combined spouse.

[0561] Clause 8. The compound of any one of Clauses 1 to 7, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R8 R 9 R 10 , and R 11 At least one, at least two, at least three, or at least four of them contain water-soluble portions or connected water-soluble portions.

[0562] Clause 9. The compound of any one of Clauses 1 to 8, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains a reactive group or a linked reactive group.

[0563] Clause 10. The compound of any one of Clauses 1 to 9, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains a chromophore or a connected chromophore.

[0564] Clause 11. The compound of any one of Clauses 1 to 3 or 5 to 10, wherein the compound is symmetrical.

[0565] Clause 12. The compound of any one of Clauses 1, 2, 4 or 5 to 10, wherein the compound is asymmetric.

[0566] Clause 13. The compound of any one of Clauses 1 to 12, wherein the water-soluble portion is selected from carboxylates / esters, carboxylic acids, phosphonates / esters, phosphates / esters, sulfonates / esters, sulfonamides, sulfates / esters, sulfinates / esters, sulfonium, esters, polyoxyalkylene oxides, polyoxyalkylene oxides containing repeating units of the formula -(CH2-CH2-O)n-, polyamide-epoxyalkylene oxides containing repeating units of the formula -(CH2-CH2-O)n-, polyethylene glycol (PEG), modified PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, hydroxyl groups, amines, amino acids, ammonium, guanidine salts, pyridine, polyamines and sulfonium, polyols, linear or cyclic sugars, primary amines, secondary amines, tertiary or quaternary amines and polyamines, glycols, polyethers, -COOX, -SO3X, -PO3X, -NR3+ X, (CH2CH2O) f R 15 and its mixtures, wherein R 15 X is hydrogen, a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted C1-C6 alkoxy group, and X is a counterion.

[0567] Clause 14. The compound according to any one of Clauses 1 to 13, wherein X is an antiion selected from: F - Cl - , Br - I - ClO4 - CF3CO2 - CH3CO2 - PO4 3- SO4 2- BF4 - Na + K + Mg ++ , and Ca ++ .

[0568] Clause 15. The compound of any one of Clauses 1 to 14, wherein the reactive group is selected from thiols, maleimides, halomaleimides, iodoacetamides, amines, alkyl carboxylates / esters, alkyl sulfonates / esters, carboxylic amines, carbamates, carboxylic esters, N-hydroxysuccinimides, halogens, borate esters, boric acids, hydrazones, carboxylic acids or their active esters, azides, alkynes, cyclooctyne, tetrazines, trans-cyclooctene, dienes, dienophiles, sulfo(VI) fluorine (SuFEX), sulfonyl fluoride, hydroxyl groups, hydrazines, hydrazines, aldehydes, ketones, azides, alkynes, phosphines, epoxides, and their protected groups.

[0569] Clause 16. The compound according to any one of the preceding clauses, wherein when m is 0, each T is independently CR. 1 or CR 1 R 2 .

[0570] Clause 17. A compound according to any one of the preceding clauses, wherein when m is 0, T is not NR. 4’ S or O.

[0571] Clause 18. The compound according to any one of the preceding clauses, wherein when m is 1, each T is independently NR. 4’ , O, S, Se or Te.

[0572] Clause 19. A compound according to any one of the preceding clauses, wherein when m is 1, T is not CR. 1or CR 1 R 2 .

[0573] Clause 20. The compound according to any one of the preceding clauses comprises a structure selected from:

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580] Clause 21. A tandem dye comprising a fluorescent compound as described in any one of Clauses 1 to 20; and an acceptor or donor chromophore covalently linked to said fluorescent compound.

[0581] Clause 22. A labeled specific binding partner comprising a fluorescent compound according to any one of Clauses 1 to 20 or a tandem dye according to Clause 21; and a specific binding partner covalently linked to said fluorescent compound or tandem dye.

[0582] Clause 23. The labeled specific binding partner as described in Clause 22, wherein the specific binding partner is selected from proteins, peptides, affinity ligands, antibodies, antibody fragments, carbohydrates, lipids, nucleic acids, and aptamers.

[0583] Clause 24. The labeled specific binding partner as described in Clause 22 or 23, wherein the specific binding partner is an antibody.

[0584] Clause 25. The compounds of any one of Clauses 1 to 20, the tandem dyes of Clause 21, or the labeled specific binding couplers of any one of Clauses 22 to 24, wherein the compounds exhibit solubility in water at ambient room temperature selected from the following: >1 mg / mL, >2 mg / mL, >3 mg / mL, >4 mg / mL, >5 mg / mL, >6 mg / mL, >7 mg / mL, >8 mg / mL, >9 mg / mL, >10 mg / mL, >20 mg / mL, >30 mg / mL, >30 mg / mL, and >50 mg / mL.

[0585] Clause 26. The compound of any one of Clauses 1 to 20, the tandem dye of Clause 21, or the labeled specific binding partner of any one of Clauses 22 to 24, wherein the compound exhibits a maximum absorbance of >500 nm, >600 nm, >700 nm, >800 nm, >850 nm, or 500 nm to 1200 nm, 500 nm to 1000 nm, or 600 nm to 950 nm.

[0586] Clause 27. The compound of any one of Clauses 1 to 20, the tandem dye of Clause 21, or the labeled specific binding partner of any one of Clauses 22 to 24, wherein the compound exhibits a maximum emission value of >550 nm, >650 nm, >750 nm, >850 nm, or >900 nm, or 550 nm to 1300 nm, 550 nm to 1050 nm, or 650 nm to 1000 nm.

[0587] Clause 28. A method for detecting a target analyte in a sample, comprising: providing a sample suspected of containing the analyte; and contacting the sample with a specific binding partner of a fluorescent compound as described in any one of Clauses 1 to 20 or a tandem dye conjugated as described in Clause 21, wherein the specific binding partner is capable of interacting with the target analyte.

[0588] Clause 29. The method of Clause 28, wherein the binding partner is an antibody, optionally wherein: (a) the method is configured for flow cytometry; (b) a water-soluble fluorescent compound binds to a substrate; (c) the analyte is a protein expressed on a cell surface; (d) the method is configured for immunoassay; or (e) the method further includes providing an additional binding partner for simultaneous detection of an additional analyte.

[0589] Clause 30. A kit comprising at least one fluorescent compound as described in any one of Clauses 1 to 20, a tandem dye as described in Clause 21, or a labeled specific binding partner as described in any one of Clauses 22 to 24, wherein the compound or tandem dye optionally comprises a conjugation tag.

Claims

1. A fluorescent compound comprising the structure according to formula (I): in Selected from substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl, polycyclic aryl, monocyclic heteroaryl and polycyclic heteroaryl; Selected from substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl and polycyclic aryl; Each T is independently for NR 4’ CR 1 CR 1 R 2 , O, S, Se, or Te; V is NR 11 CR 8 CR 8 R 9 O, S, Se, or Te, or each TV together can represent a selection from SO2, -CR 1 -O-,-O-CR 1 -,-CO-O-,-O-CO-,-CO-NR 11 -, or -NR 4’ -CO- structural elements; G is either C or N; Each R 1 R 2 R 8 and R 9 Independently selected from water-soluble portion, linked water-soluble portion, linker portion, E, linked E, reactive group, linked reactive group, conjugated tag, linked conjugated tag, conjugated partner, linked conjugated partner, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonic acid, sulfonate / ester, alkylsulfonate / ester, alkylsulfonate, alkoxysulfonate / ester, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, alkoxycarboxylate / ester, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphorinamide, phosphineamide, alkoxysulfonamide PEG, alkylsulfonamide PEG, alkylamide Each R 4 R 4’ R 10 and R 11 Independently selected from the following: linker portion, chromophore, linked chromophore, reactive group, linked reactive group, conjugated tag, linked conjugated tag, water-soluble portion, linked water-soluble portion, conjugated mating body, linked conjugated mating body, E, linked E, H, halogen, alkyl, alkenyl, alkynyl, PEG group, linked PEG group, cycloalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, sulfonic acid, sulfonate / ester, alkyl sulfonate / ester, alkyl sulfonate, carboxylic acid, carboxylate / ester, alkyl carboxylate / ester, alkyl sulfonamide, alkyl sulfonamide PEG, alkylamide, alkylamide-PEG Or its protected group; or R 4 and R 12 Together, R 14 and R 10 Together, R 4 and R 13 Together, R 13 and R 10 Together, R 13 and R 11 Together, R 12 and R 14 Together, R 4’ and R 13 Together, R 4’ and R 12 Together, R 13 and R 10 Together, R 4 R 12 R 14 and R 10 Together, R 4 R 13 and R 10 Together, R 4 R 13 and R 11 Together, R 4 R 12 and R 14 Together, R 4’ R 13 and R 11 Together, and R 12 R 14 and R 10 One, two, three, or four together form unsubstituted or substituted unsaturated or partially unsaturated C3-C atoms. 10 Cycloalkyl, unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 10 Heterocyclic alkyl groups, unsubstituted or substituted, unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polycyclic alkyl groups, or substituted unsaturated or partially unsaturated C3-C groups, either unsubstituted or optionally O-substituted. 14 C3-C 10 Or C3-C8 polyhexanediols; Each R 3 Independently selected from H, alkyl, olefin, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, alkyl sulfonate / ester, alkyl carboxylate / ester, water-soluble moiety, linked water-soluble moiety, chromophore, linked chromophore, functional moiety, linked functional moiety, conjugate, linked conjugate, PEG group and linked PEG group; Each Q is independently a bond, O, NH, NR 4 C1-C 12 Alkylene, CHR 4 Or CH2; Each Z is independently CH2, CHR 4 O, NR 4 Or NH; Each W 1 It is a water-soluble component on its own. L 1 L 2 and L 3 Each connector is selected independently. Each E is independently selected from chromophores, functional moieties, substrates, reactive groups, conjugated tags, and binding partners; Each R 7 Independently selected from H, hydroxyl, C1-C 12 Alkyl, C1-C 12 Heteroalkyl, C2-C 12 Olefins, C2-C 12 Alkynes, C3-C 12 cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy group, C2-C 18 (Miscellaneous) arylamino, carboxylates / esters, carboxylic acids, C2-C 12 Alkyl carboxylic acids, C2-C 12 Alkyl carboxylates / esters, C2-C 12 Alkyl carboxylic acid esters, aryl carboxylic acids, aryl carboxylic acid esters, C1-C 12 Alkyl groups, water-soluble moiety, PEG moiety, functional groups, chemoselective functional groups, conjugated labels, linked conjugated labels, connectors, sulfonic acids, sulfonates / esters, C1-C 12 Alkyl sulfonates / esters, sulfonamides; Each R 12 R 13 and R 14 Independently selected from hydrogen, halogen, one or more heteroatoms, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C1-C6 olefin, substituted or unsubstituted C3-C 10 Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C3-C 10 Heterocyclic alkyl groups, CO2R 1 CONR 1 R 2 O-aryl, S-aryl, N-aryl, -O-alkyl, S-alkyl, N-alkyl, wherein each alkyl or aryl group may optionally be represented by one or more R groups. 7 PEG or PEG-R 7 Replace, optionally, each of R 12 R 13 and R 14 Independently replaced by the following: R 7 Group; or R 1 R 2 R 4 R 4’ R 8 R 9 R 10 R 11 R 12 R 13 and R 14 At least two of them together, including but not limited to R 1 R 2 R 4 R 4’ R 8 R 9 R 10 R 11 R 12 R 13 and R 14 Two, three, or four of them together, optionally including but not limited to R 1 R 2 R 4 R 4’ R 8 R 9 R 10 R 11 R 12 R 13 and R 14 One, two, three or four together, including but not limited to R 4 and R 12 Together, R 14 and R 10 Together, R 4 and R 13 Together, R 13 and R 10 Together, R 13 and R 11 Together, R 12 and R 14 Together, R 4’ and R 13 Together, R 4’ and R 12 Together, R 13 and R 10 Together, R 4 R 12 R 14 and R 10 Together, R 4 R 13 and R 10 Together, R 4 R 13 and R 11 Together, R 4 R 12 and R 14 Together, R 4’ R 13 and R 11 Together, and R 12 R 14 and R 10 Formation of unsaturated or partially unsaturated C3-C, whether substituted or substituted. 10 Cycloalkyl, unsubstituted or optionally O-substituted substituted unsaturated or partially unsaturated C3-C 10 Heterocyclic alkyl groups, unsubstituted or substituted, unsaturated or partially unsaturated C3-C 14 C3-C 10 Or C3-C8 polycyclic alkyl groups, or substituted unsaturated or partially unsaturated C3-C groups, either unsubstituted or optionally O-substituted. 14 C3-C 10 Or C3-C8 polyhexanediols; Each K is independently a covalent bond, O, S, Se, P, NR 1 or CR 1 R 2 ; Each f is an independent integer from 0 to 50, 1 to 30, or 2 to 20; Each m and m' is independently 0, 1, 2, or 3; Each n is an independent integer from 1 to 20; from 1 to 10; or 0, 1, 2, or 3. Each p is independently 1, 2, 3 or 4; Each s is independently 1 or 2; Each t is independently 0, 1, 2, 3, or 4; and X is a counter ion.

2. The compound of claim 1, comprising a structure according to any one of formulas (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), (IIg), (IIh), (IIi), (IIj), (IIk), (IIl), (IIm), (IIIn), and (IIo): in T is NR 4’ CR 1 CR 1 R 2 , O or S; V is NR 11 CR 8 CR 8 R 9 , O or S; Each R 5 and R 6 Independently selected from water-soluble portion, linked water-soluble portion, linker portion, E, linked E, reactive group, linked reactive group, conjugation tag, linked conjugation tag, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphorinamide, phosphineamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 Independently selected from hydrogen, sulfonate / ester, alkyl sulfonate / ester, alkyl sulfonamide, alkyl sulfonamide-PEG, phosphate group, carboxylic acid, carboxylate / ester, amide, alkylamide, amide-PEG and water-soluble group; Each R 16 Independently selected from KR 13 H, halogens, OC 1-6 Alkyl, SC 1-6 Alkyl, O-aryl, S-aryl, NHC 1-6 Alkyl, Ph-NCS, Ph-CO2H, or Ph-(CH2) 1-4 CO2H; and It is an optional substituted cycloalkenyl or polycycloalkenyl moiety.

3. The compound of claim 1, comprising a structure according to any one of formulas (IIIa), (IIIb), (IIHc), (IId), (IIIe), (IIIf), (IIIg), (IIIh), (IIi), (IIIj), (IIIk), (IIIl), (IIIm), and (IIIn): in Each R 5 and R 6 Independently selected from water-soluble portion, linked water-soluble portion, linker portion, E, linked E, reactive group, linked reactive group, conjugation tag, linked conjugation tag, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 Independently selected from hydrogen, sulfonate / ester, alkyl sulfonate / ester, alkyl sulfonamide, alkyl sulfonamide-PEG, phosphate group, carboxylic acid, carboxylate / ester, amide, alkylamide, amide-PEG and water-soluble group; Each R 16 Independently selected from KR 13 H, halogens, OC 1-6 Alkyl, SC 1-6 Alkyl, O-aryl, S-aryl, NHC 1-6 Alkyl, Ph-NCS, Ph-CO2H, or Ph-(CH2) 1-4 CO2H; and It is an optional substituted cycloalkenyl or polycycloalkenyl moiety.

4. The compound of claim 1, comprising a structure according to any one of formulas (IVa), (IVb), (IVc), (IVd), (IVe), (IVf), (IVg), (IVh), (IVVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVu), (IVv), (IVw), and (IVx): in Each R 5 and R 6 Independently selected from water-soluble portion, linked water-soluble portion, linker portion, E, linked E, reactive group, linked reactive group, conjugation tag, linked conjugation tag, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 Independently selected from hydrogen, sulfonate / ester, alkyl sulfonate / ester, alkyl sulfonamide, alkyl sulfonamide-PEG, phosphate group, carboxylic acid, carboxylate / ester, amide, alkylamide, amide-PEG and water-soluble group; Each R 16 Independently selected from KR 13 H, halogens, OC 1-6 Alkyl, SC 1-6 Alkyl, O-aryl, S-aryl, NHC 1-6 Alkyl, Ph-NCS, Ph-CO2H, or Ph-(CH2) 1-4 CO2H, and It is an optional substituted cycloalkenyl or polycycloalkenyl moiety.

5. The compound of claim 1, comprising a structure according to any one of formulas (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi), (Vj), (Vk), and (Vl): in Y is selected from NR 4 CR 1 CR 1 R 2 O and S; and each R 5 and R 6 Independently selected from water-soluble portion, linked water-soluble portion, linker portion, E, linked E, reactive group, linked reactive group, conjugation tag, linked conjugation tag, hydrogen, hydroxyl, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoether ammonium salt, carbonyl, acyl, sulfonate / ester, alkylsulfonate / ester, alkylsulfonamide, phosphate group, carboxylic acid, carboxylate / ester, alkylcarboxylate / ester, sulfonic acid, sulfonic acid alkyl salt, sulfonic acid alkoxy salt, sulfonic acid oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide ester, hypophosphamide, alkoxysulfonamide PEG, alkylamide, alkoxysulfonate / ester, alkylsulfonate, optionally wherein R 5 and R 6 It is independently selected from hydrogen, sulfonate / ester, alkyl sulfonate / ester, alkyl sulfonamide, alkyl sulfonamide-PEG, phosphate group, carboxylic acid, carboxylate / ester, amide, alkylamide, amide-PEG and water-soluble group.

6. The compound according to any one of claims 1 to 5, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains E or is connected to E.

7. The compound according to any one of claims 1 to 6, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains a combined spouse or a connected combined spouse.

8. The compound according to any one of claims 1 to 7, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one, at least two, at least three, or at least four of them contain water-soluble portions or connected water-soluble portions.

9. The compound according to any one of claims 1 to 8, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains a reactive group or a linked reactive group.

10. The compound according to any one of claims 1 to 9, wherein R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 , and R 11 At least one of them contains a chromophore or a connected chromophore.

11. The compound of claim 1, wherein the compound is symmetrical.

12. The compound of claim 1, wherein the compound is asymmetric.

13. The compound of any one of claims 1 to 12, wherein the water-soluble portion is selected from carboxylates / esters, carboxylic acids, phosphonates / esters, phosphates / esters, sulfonates / esters, sulfonamides, sulfates / esters, sulfinates / esters, etc. Esters, polyepoxides, polyepoxides containing repeating ethylene oxide units of the formula -(CH2-CH2-O)n-, polyamide epoxides containing repeating ethylene oxide units of the formula -(CH2-CH2-O)n-, polyethylene glycol (PEG), modified PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, hydroxyl groups, amines, amino acids, ammonium, guanidine salts, pyridine, polyamines, and Polyols, linear or cyclic sugars, primary amines, secondary amines, tertiary or quaternary amines and polyamines, glycols, polyethers, -COOX, -SO3X, -PO3X, -NR 3+ X, (CH2CH2O) f R 15 and its mixtures, wherein R 15 X is hydrogen, a substituted or unsubstituted C1-C6 alkyl group, or a substituted or unsubstituted C1-C6 alkoxy group, and X is a counterion.

14. The compound of claim 1, wherein X is a counterion selected from: F - , Cl, Br - , I - , ClO4 - , CF3CO2 - , CH3CO2 - , PO4 3- , SO4 2- , BF4 - , Na + , K, Mg ++ , and Ca ++ .

15. The compound of any one of claims 1 to 14, wherein the reactive group is selected from thiols, maleimides, halomaleimides, iodoacetamides, amines, alkyl carboxylates / esters, alkyl sulfonates / esters, carboxylic amines, carbamates, carboxylic esters, N-hydroxysuccinimides, halogens, borate esters, boric acids, hydrazones, carboxylic acids or their active esters, azides, alkynes, cyclooctyne, tetrazines, trans-cyclooctene, dienes, dienophiles, sulfo(VI) fluorine (SuFEX), sulfonyl fluoride, hydroxyl groups, hydrazines, hydrazines, aldehydes, ketones, azides, alkynes, phosphine, epoxides, and their protected groups.

16. The compound according to any one of claims 1 to 15, wherein when m is 0, each T is independently CR. 1 or CR 1 R 2 .

17. The compound according to any one of claims 1 to 15, wherein when m is 0, T is not NR. 4’ S or O.

18. The compound according to any one of claims 1 to 15, wherein when m is 1, each T is independently NR. 4’ , O, S, Se or Te.

19. The compound according to any one of claims 1 to 15, wherein when m is 1, T is not CR. 1 or CR 1 R 2 .

20. The compound of claim 1, comprising a structure selected from:

21. Tandem dyes, comprising: The fluorescent compound according to any one of claims 1 to 20; and The acceptor chromophore or donor chromophore is covalently linked to the fluorescent compound or a labeled specific binding partner.

22. A labeled specific binding partner comprising: The fluorescent compound according to any one of claims 1 to 20 or the tandem dye according to claim 21; and A specific binding partner that is covalently linked to the fluorescent compound or tandem dye.

23. The labeled specific binding partner of claim 22, wherein the specific binding partner is selected from proteins, peptides, affinity ligands, antibodies, antibody fragments, carbohydrates, lipids, nucleic acids, and aptamers.

24. The labeled specific binding partner of claim 23, wherein the specific binding partner is an antibody.

25. The compound of any one of claims 1 to 20, the tandem dye of claim 21, or the labeled specific binding pair of any one of claims 22 to 24, wherein the compound exhibits solubility in water at ambient room temperature of >1 mg / mL, >2 mg / mL, >3 mg / mL, >4 mg / mL, >5 mg / mL, >6 mg / mL, >7 mg / mL, >8 mg / mL, >9 mg / mL, >10 mg / mL, >20 mg / mL, >30 mg / mL, >30 mg / mL, or >50 mg / mL.

26. The compound of any one of claims 1 to 20, the tandem dye of claim 21, or the labeled specific binding pair of any one of claims 22 to 24, wherein the compound exhibits a maximum absorbance of >500 nm, >600 nm, >700 nm, >800 nm, >850 nm, >900 nm, >1000 nm, >1100 nm, or >1200 nm.

27. The compound of any one of claims 1 to 20, the tandem dye of claim 21, or the labeled specific binding pair of any one of claims 22 to 24, wherein the compound exhibits a maximum emission value of >550 nm, >650 nm, >750 nm, >850 nm, >900 nm, >1000 nm, >1100 nm, >1200 nm, or >1300 nm.

28. A method for detecting a target analyte in a sample, comprising: Provide a sample suspected of containing the analyte; as well as The sample is brought into contact with a specific binding partner of the fluorescent compound of any one of claims 1 to 20 or the tandem dye conjugate of claim 21, wherein the specific binding partner is capable of interacting with the target analyte.

29. The method of claim 28, wherein the binding partner is an antibody, optionally wherein: a. The method is configured for use in flow cytometry; b. Water-soluble fluorescent compounds bind to the substrate; c. The analyte is a protein expressed on the cell surface; d. The method is configured for immunoassay; or e. The method further includes providing an additional binding pair for the simultaneous detection of another analyte.

30. A kit comprising at least one fluorescent compound of any one of claims 1 to 20, a tandem dye of claim 21, or a labeled specific binding pair of any one of claims 22 to 24, wherein the compound or tandem dye optionally comprises a conjugation tag.

Images