WO2025064842A1 - Dihydrophenanthrene (dhp) bridged dyes for use in flow cytometry - Google Patents

Abstract

The present disclosure provides novel dihydrophenanthrene (DHP) bridged small molecule fluorescent dye compounds. The DHP bridged dye compounds can be excited and/or exhibit emission in UV, violet, blue, yellow, green, red, and near infrared (NIR) wavelengths. Tandem dyes and kits comprising the DHP bridged compounds, are also provided. The dye compounds and tandem dyes may be conjugated to antibodies for detection of target analytes in biological samples and are suitable for use in flow cytometry and other analyses. An exemplary compound has formula 20 below: (I).

Description

NOVEL FLUORESCENT COMPOUNDS

[0001] This application is being filed on 20 September 2024, as a PCT International Application and claims the benefit of priority to U.S. Provisional Application No.

63/584,412, filed 21 September 2023, which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] There is an increasing demand for a variety of fluorescent dyes for use in current flow cytometers as well as in spectral flow instruments. Fluorescent compounds and their conjugates can be used in a variety of biological applications by generating signals which can be monitored in real time and provide simple and rapid methods for the detection of biological targets and events, e.g., in diagnostic kits, in microscopy, in cytometry, or in drug screening.

[0003] Molecular recognition involves the specific binding of two molecules.

Molecules which have binding specificity for a target biomolecule find use in a variety of research and diagnostic applications, such as the labelling and separation of analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separations and chromatography. Target biomolecules may be detected by labelling with a fluorescent compound.

[0004] Current dihydrophenanthrene (DHP) based polymer dyes have strong excitation in the ultraviolet and violet regions of 350-450 nm. US Pat. No. 11,208,527 describes water soluble DHP based fluorescent polymer dyes, for example, exhibiting excitation maxima between 395-415 nm with emitted light between about 415-475 nm. US Pat. No. 11,584,825 describes water soluble dihydrophenanthrene (DHP) based violet excitable polymers and tandem dyes.

[0005] Demands have increased for multicolor panels for both conventional and spectral flow cytometry that require additional fluorescent dyes excitable with other lasers (e.g., 355, 488, 563, 638, and 808 nm). Parameters considered by a user in choosing a fluorescent dye may include excitation wavelength maximum, the emission wavelength maximum, brightness of the dye, and the fluorescence lifetime. Brightness of a dye is an overall contribution from the extinction coefficient (ε, measure of the amount of light absorbed at a particular wavelength) and fluorescence quantum yield (Φ, measure of the light emitted in the form of radiation from its singlet excited state).

[0006] Organic fluorescent compounds that can be excited using UV, violet, blue, yellow, green, red, and near infrared (NIR) wavelengths are desirable.

SUMMARY OF THE INVENTION

[0007] The present disclosure generally provides dihydrophenanthrene (DHP) bridged compound dyes, optionally conjugated to a specific binding partner, their complexes, and methods for detecting analytes in a sample using the complexes comprising the water-soluble DHP bridged compound dyes conjugated to a binding partner. The DHP bridged compound dye may be a DHP bridged small molecule dye. Tandem dyes are also provided comprising the DHP bridged small molecule dyes or labeled specific binding partners according to the disclosure. The DHP bridged small molecule dyes, labeled specific binding partners, and tandem dyes according to the present disclosure are usefill in biological applications, including for the detection of target analytes and use in diagnostic kits, etc. The kits may comprise DHP bridged small molecule compounds, labeled specific binding partners, and/or tandem dyes according to the present disclosure, optionally having a conjugation tag.

[0008] The disclosure provides a compound comprising a structure according to Formula (I):

wherein each

and

is independently selected from the group consisting of a substituted or unsubstituted benzene, benzene derivative, monocyclic aryl group, polycyclic aryl group, monocyclic heteroaryl group, and polycyclic heteroaryl group; each T is independently selected from the group consisting of C, C(R¹), N, N(R¹), P, O, S, and Si(R¹); each X is independently C, Si, O, N, or P; each Y is independently C, Si, O, N, or P; each R¹ is independently selected from the group consisting of a water- solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, hydroxy, halogen, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfmamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

,

each R² is independently selected from the group consisting of a water- solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

each R³ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a water- solubilizing moiety, a chromophore, functional moiety, binding partner, E, a PEG group, a PEG ester, and a PEG carboxylic acid; each optional R^3' is independently selected from the group consisting of R¹, R², unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, halogen, a water- solubilizing moiety, functional moiety, a PEG group, PEG carboxylic acid, alkyl amido PEG ester, alkyl amido PEG carboxylic acid, -B(R³)₂, O-alkyl, O-aryl, arylalkoxy, alkylamide, alkylamidoPEG, aryl-CN, NR'R', NHR', NH₂, -S-R', SO₃H, - SO₂R', - SO₂NR', -PR'₃, POR'₃, -SiR'₃, -ammonium, alkylammonium, and arylammonium; each R' is independently selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted aryl, optionally substituted with one or more independently selected PEG groups, water solubilizing moiety, linked water- solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group; each R⁴ is independently selected from the group consisting of H, alkyl, PEG, a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a chromophore, a functional group, a conjugation tag, a binding partner, a linked E, carboxylic amine, amine, carbamate, carboxylic acid, carboxylate ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, alkene, tetrazine, aldehyde, and thiol, or protected groups thereof; each optional R⁴' is selected from the group consisting of R¹, R², R³ and R³', optionally wherein two R⁴' together form an unsubstituted or substituted cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, cycloalkoxy, aryl, or heteroaryl having 3 to 9 ring members; each R⁷ is independently selected from the group consisting of H, hydroxyl, C₁- C₁₂ alkyl, C₂- C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁- C₁₂haloalkyl, C₁- C₁₂ alkoxy, C₂-C₁₈ (hetero)aryloxy, C₂-C₁₈ (hetero)arylamino, C₂-C₁₂ carboxylic acid, C₂- C₁₂ carboxylate ester, and C₁-C₁₂ alkoxy, a functional group, a chemoselective functional group, conjugation tag, linked conjugation tag, E, a linker, sulfonic acid, sulfonate, C₁- C₁₂ alkyl sulfonate, sulfonamide; at least one of R¹, R², R³, R³', R⁴, or R⁴' comprises an independently selected water-solubilizing moiety or linked water-solubilizing moiety; each Q is independently a bond, NH, NR⁴, C₁-C₁₂ alkylene, CHR⁴, and CH₂; each Z is independently CH₂, CHR⁴, O, NR⁴, or NH; each W¹ is independently a water-solubilizing moiety;

L¹, L², and L³ are each independently selected linker moieties; each E is independently selected from the group consisting of a chromophore, a functional moiety, a substrate, a conjugation tag, and a binding partner; each f is independently an integer from 0 to 50; each m is independently 0 or 1; each n is independently 0, 1, 2, 3, or 4; each s is independently 1 or 2; and each t is independently 0, 1, 2, or 3.

[0009] In some cases, R¹ and R² are each independently selected from the group consisting of a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, hydroxy, halogen, hydrogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, (hetero)aryloxy, (hetero)arylamino, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, alkoxy sulfonic acid, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfmamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

[0010] In some cases, each X is independently C or Si. In some cases, each Y is independently C or Si. In some cases, both X and Y are C. In some cases, each T is independently selected from the group consisting of C, C(R¹), N, and N(R¹). In some cases, each X and Y are independently C or Si; and each T is independently selected from the group consisting of C, C(R¹), N, and N(R¹). In some cases, each X and Y are C; and each T is independently C or C(R¹).

[0011] In some cases, R¹ and R² are different. In some cases, R¹ and R² are the same.

[0012] The disclosure provides a fluorescent compound comprising a structure according to any one of Formulas (IIa), (IIb), (IIc), (IId), or (IIe):

[0013] In some cases, in the structure according to any one of Formulas (IIa), (IIb), (IIc), (IId), or (IIe), X and Y are both C or Si. In some cases, in the structure according to any one of Formulas (IIa), (IIb), (IIc), (IId), or (IIe), X and Y are both C.

[0014] The disclosure provides a fluorescent compound comprising a structure according to any one of Formulas (IIIa), (IIIb), (IIIc), or (IIId):

[0015] In some cases, in the structure according to any one of Formulas (IIIa), (IIIb), (IIIc), or (IIId), X and Y are both C or Si. In some cases, in the structure according to any one of Formulas (IIIa), (IIIb), (IIIc), or (IIId), X and Y are both C.

[0016] The disclosure provides a fluorescent compound comprising a structure according to any one of Formulas (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf):

[0017] In some cases, in the structure according to any one of Formulas (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf), X and Y are both C or Si. In some cases, in the structure according to any one of Formulas (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf), X and Y are both C.

[0018] The disclosure provides a fluorescent compound comprising a structure according to any one of Formulas (Va), (Vb), (Vc), or (Vd):

[0019] In some cases, in the structure according to any one of Formulas (Va), (Vb), (Vc), or (Vd), X and Y are both C or Si. In some cases, in the structure according to any one of Formulas (Va), (Vb), (Vc), or (Vd), X and Y are both C.

[0020] In some cases, in the structures according to any one of (IIa), (IIb), (IIc), (IId), (IIe), (IIIa), (IIIb), (IIIc), (IId), (IVa), (IVb), (IVc), (IVd), (IVe), (IVf), (Va), (Vb), (Vc), X and Y are both Si or C; and R³' is independently selected from the group consisting of hydrogen, halogen, C_1-6 alkyl, C_1-6 alkoxy, NH₂, unsubstituted or substituted aryl, a water- solubilizing moiety, a functional moiety, a PEG group, PEG carboxylic acid, alkyl amido PEG ester, alkyl amido PEG carboxylic acid, arylalkoxy, alkylamide, alkylamidoPEG, aryl-CN,

[0021] In some cases, each of and

is independently selected

from the group consisting of

, wherein each of

and is independently optionally substituted with (R^3')_n; and

p=1, 2, 3, 4, 5, 6, or 7. In some cases,

an

d are each independently selected from the group consisting of:

[0022] In some cases, R² is C₁-C₆ alkyl, phenyl, or phenyl substituted with halogen, C₁-C₆ alkoxy, or CN.

[0023] In some cases,

and

are the same.

In some cases,

and

are different. In some cases, each of

and

are not optionally substituted DHP groups. In some cases, each of

and

are not optionally substituted fluorenyl groups. In some cases, each of

and

are independently selected from optionally substituted DHP groups or optionally substituted fluorenyl groups.

[0024] The disclosure provides a fluorescent compound comprising a structure selected from the group consisting of:

[0025] In some cases, each G is independently propyl, phenyl, 4-methoxyphenyl, or 4-cy anophenyl. [0026] In some cases, the fluorescent compound of the disclosure further comprises a binding partner bonded to said compound.

[0027] The disclosure provides a labeled specific binding partner, comprising a compound according to the present disclosure; and a specific binding partner covalently linked to the fluorescent compound. The specific binding partner may be selected from the group consisting of a protein, peptide, affinity ligand, antibody, antibody fragment, carbohydrate, lipid, nucleic acid, and an aptamer. The specific binding partner may be an antibody. The specific binding partner may be an antibody specific for a target analyte.

[0028] The disclosure provides a tandem dye, comprising: a fluorescent compound or labeled specific binding partner according to the present disclosure; and a donor or acceptor chromophore covalently linked to the fluorescent compound. In some cases, the fluorescent compound of the invention is an acceptor dye. In some cases, the fluorescent compound of the invention is a donor dye.

[0029] A fluorescent dye bonded to one or more, two or more, three or more, four or more, or from 1-30, 2-20, 3-15, or 4-10 DHP bridged compound dyes of the disclosure is provided. A binding partner bonded to one or more, two or more, three or more, four or more, or from 1-30, 2-20, 3-15, or 4-10 DHP bridged compound dyes of the disclosure is provided. The bond may be a covalent bond. The bond may be a non-covalent bond.

[0030] The compound, labeled specific binding partner, or tandem dye may be water- soluble. The compound, labeled specific binding partner, or tandem dye may be fluorescent. The compound, labeled specific binding partner, or tandem dye may be a water-soluble fluorescent dye.

[0031] A method is provided for detecting a target analyte in a sample comprising: providing a sample that is suspected of containing the analyte; and contacting the sample with a specific binding partner conjugated to a water-soluble fluorescent compound or tandem dye according to the present disclosure, wherein the binding partner is capable of interacting with the target analyte.

[0032] The method may include one or more of wherein the method is configured for flow cytometry; the water-soluble fluorescent DHP bridged compound according to the disclosure is bound to a substrate; the analyte is a protein expressed on a cell surface; the method is configured as an immunoassay; and/or the method further comprises providing additional binding partners for detecting additional analytes simultaneously. [0033] The disclosure provides a kit comprising at least one fluorescent compound, labeled specific binding partner, or tandem dye according to the present disclosure. The kit can also comprise a container, buffer, and/or instructions. The compound, labeled specific binding partner, or tandem dye according to the disclosure may include a conjugation tag. The conjugation tag can be used for attachment of an acceptor dye, donor dye, support, or binding partner to the compound or tandem dye.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 shows Scheme 1 illustrating a representative synthetic route to provide inventive DHP bridged compound 4: a 3,3'-((2,7-bis(4-(diphenylamino)phenyl)-9,10- dihydrophenanthrene-9, 10-diyl)bis(oxy))bis(N-(PEG-OMe)propane-1-sulfonamide) from reaction of intermediate compound A with an aryl boronic acid pinacol ester intermediate. [0035] FIG. 2 shows Scheme 2 illustrating a representative synthetic route to provide PEGylated aryl boronic acid pinacol ester intermediates such as intermediate compound 16: 3-(2-(2-(2-(2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2 yl)phenoxy)ethoxy) ethoxy)ethoxy)propanoic acid, from a bromo-phenolic starting material.

[0036] FIG. 3 shows Scheme 3 illustrating a representative synthetic route to provide inventive DHP bridged compound 18: 3,3'-(((((((((9,10-bis(3-sulfopropoxy)-9,10- dihydrophenanthrene-2,7-diyl)bis(2-fluoro-4, 1 -phenylene))bis(oxy))bis(ethane-2, 1 - diyl))bis(oxy))bis(ethane-2, 1 -diyl))bis(oxy))bis(ethane-2, 1 -diyl))bis(oxy))dipropionic acid from reaction of compound 17: 3,3'-((2,7-dibromo-9,10-dihydrophenanthrene-9,10- diyl)bis(oxy))bis(propane-1 -sulfonic acid) and PEGylated aryl boronic acid pinacol ester 16.

[0037] FIG. 4 shows Scheme 4 illustrating a representative synthetic route to provide inventive DHP bridged compound 7: 3,3'-((2,7-bis(4-aminophenyl)-9,10- dihydrophenanthrene-9,10-diyl)bis(oxy))bis(N-(2,5,8,11,14,17,20,23,26,29,32- undecaoxatetratriacontan-34-yl)propane-1-sulfonamide) from intermediate compound B and a p-Boc-aminophenyl boronic acid pinacol ester intermediate followed by deprotection of the Boc amino groups.

[0038] FIG. 5 shows Scheme 5 illustrating a representative synthetic route to prepare inventive DHP bridged compounds 8a-8e from 2-alkyl- or 2-aryl-6-bromo-lH- benzo[de]isoquinoline-1,3(2H)-diones compounds 23a-23d. [0039] FIG. 6 shows Scheme 6 illustrating a representative synthetic route to provide a DHP bridged compound-antibody conjugate from a carboxylate-derivitized DHP bridged compound.

[0040] FIG. 7 shows Scheme 7 illustrating a representative synthetic route to provide mono-bromo-DHP-sulfonamide PEG monomer compound 36 from phenanthrene, 9, 10- dione compound 31.

[0041] FIG. 8 shows Scheme 8 illustrating a representative synthetic route to provide inventive DHP bridged-sulfonamide PEG compound 20 from mono-bromo-DHP- sulfonamide PEG monomer compound 36 and DHP-sulfonamide PEG, diboronic ester compound 28 via Suzuki coupling.

[0042] FIG. 9 shows an absorption spectrum of the DHP bridged -sulfonamide PEG compound 20 exhibiting an absorption maxima at 359 nm (left panel) and fluorescence excitation/emission spectra of the DHP-sulfonamide PEG trimer compound 20 exhibiting an emission max at 419 nm (right panel).

[0043] FIG. 10 shows Scheme 10 illustrating a representative synthetic route to provide inventive amino-derivitized DHP trimer compound 22 from DHP-sulfonamide PEG trimer compound 20 to intermediate protected NHBoc amino DHP trimer compound 21 and deprotection of Boc protecting groups.

[0044] FIG. 11 shows absorption spectrum of DHP trimer-Dy495 tandem dye compound 25 exhibiting two absorption maxima at 370 nm and 500 nm (left panel). A fluorescence emission spectrum of DHP timer-Dy495 tandem dye compound 25 after excitation at 355 nm shows an emission max at 532 nm (right panel).

[0045] FIG. 12 shows Scheme 11 illustrating a representative synthetic route to inventive amino-derivitized DHP bridged fluorenyl sulfonamide PEG compound 30 from mono-bromo-fluorenyl compound 27 and DHP-sulfonamide PEG, diboronic ester compound 28.

[0046] FIG. 13 shows a chart showing normalized emission spectra of UV excited DHP bridged fluorenyl conjugate compound 37 and UV DHP bridged fluorenyl tandem conjugates 38-46 upon excitation at 355 nm in PBS lx. A single excitation wavelength results in multiple emission wavelengths from 415 nm to 925 nm.

[0047] FIG. 14 shows an overlay of Absorption spectra of UV DHP bridged fluorenyl CF850 tandem (A) conjugate 45 and of reference dye CF850 (B) in PBS lx. [0048] FIG. 15 shows emission spectra of UV DHP bridged fluorenyl Compound 30 donor molecule before (A) and after (B) attachment of acceptor dye CF850 to form UV DHP bridged fluorenyl tandem conjugate 45 showing the donor emission quenching (PBS lx ex at 355nm).

[0049] FIG. 16 shows emission spectra of UV DHP bridged fluorenyl CF850 tandem conjugate 45 after ex at 355nm (A) with an em max at 887 run and blank (B) in PBS lx. [0050] FIG. 17 shows flow cytometry results from CD8 UV DHP bridged fluorenyl CF850 tandem conjugate 45 in whole blood sample (Cytoflex LX bp 885/40). The circled area corresponds to CDS positive cells.

DETAILED DESCRIPTION OF THE INVENTION

I. General

[0051] The present disclosure provides novel 9, 10-dihydrophenanthrene (DHP) bridged compounds (“DHP bridged compounds”). The DHP bridged compounds can be DHP bridged small molecule dyes (“DHP bridged dyes”). In some embodiments, DHP bridged small molecule dyes have been designed to be water soluble. The DHP bridged small molecule dyes can be water soluble. The present disclosure also provides labeled specific binding partners comprising DHP bridged compounds or dyes and a binding partner. The disclosure also provides tandem dyes comprising the DHP bridged compounds or labeled specific binding partners and an acceptor dye or donor dye. The DHP bridged dyes or labeled specific binding partners can be an acceptor dye. The DHP bridged dyes or labeled specific binding partners can be a donor dye. The DHP bridged dyes can be conjugated to a binding partner.

[0052] Methods are provided for detecting target analytes in a sample comprising the DHP bridged compounds or dyes of the disclosure conjugated to binding partners (labeled specific binding partners). The DHP bridged dyes of the present disclosure demonstrate water solubility and can be excited using UV, violet, blue, yellow, green, red, and near infrared (NIR) wavelengths. In some cases, the DHP bridged dyes of the present disclosure demonstrate water solubility are excited/absorb at a UV wavelength. In some cases, the DHP bridged dyes of the present disclosure demonstrate water solubility and emit at a UV, violet, blue, yellow, green, red, and near infrared (NIR) wavelengths. In some cases, the DHP bridged dyes of the present disclosure demonstrate water solubility and are excited/absorb at a UV wavelength and emit at a UV, violet, blue, yellow, green, red, and near infrared (NIR) wavelengths. There is an increasing demand for a variety of fluorescent dyes for use in current flow cytometers as well as spectral flow instruments. [0053] The DHP bridged compounds of the disclosure may be produced using any appropriate cross-coupling reaction. In the present disclosure, a DHP intermediate compound can be modified to include covalently bonded, optionally substituted,

aryl groups to form the DHP bridged dye compounds (“DHP bridged compounds” or “DHP bridged dyes”). For example, a DHP intermediate can be covalently bonded to optionally substituted

and

aryl groups to form electron rich DHP bridged compounds of the disclosure in a single synthetic step, for example, using Suzuki coupling of di-bromo dihydrophenanthrene and commercially available

and

- functionalized with a mono boronic ester group as illustrated in Schemes 1, 3, and 4. Conversely, the DHP intermediate can be functionalized with di-boronic esters and mono Br-functionalized

and

intermediates may be employed in the Suzuki coupling, as illustrated in

Scheme 5. The DHP moiety and

and

groups may include optional substitutions. The DHP bridged compounds can be excited using UV, violet, blue, green, red, or NIR light, depending on the

and

groups attached to the DHP moiety. The DHP bridged compounds comprising an acceptor or donor dye can emit at a UV, violet, blue, green, red, or NIR wavelength, depending on the

and

groups attached to the DHP moiety.

[0054] Various chemical modifications of the core DHP molecule allowed preparation of compounds which can be excited using different lasers. Small organic dyes were prepared using this approach. Symmetric as well as unsymmetric dyes were made using this approach which allows fine tuning of the absorption and emission of the dyes. DHP bridged compounds according to the present disclosure exhibit excitation maxima in a range of from about 300 to about 900 nm, 300 to about 810 nm, or about 300 to about 650 nm, or about 300 to about 565 nm, or about 300 to about 500 nm, or about 320 to about 420 nm. In some cases, DHP bridged compounds according to the present disclosure exhibit emission in a range of from about 350 to about 925 nm, or about 350 to about 810, or about 350 to about 650 nm, or about 350 to about 565 nm, or about 350 to about 500 nm, or about 350 to about 420 nm. In some cases, the tandem dyes comprising DHP bridged compounds and acceptor or donor dyes according to the present disclosure exhibit emission in a range of from 400 to about 925 nm, or about 400 to about 810, or about 400 to about 650 nm, or about 400 to about 565 nm, or about 400 to about 500 nm, or about 400 to about 420 nm.

[0055] Kits comprising at least one fluorescent DHP bridged compound, labeled specific binding partner, or tandem dye according to the present disclosure are also provided. The kits can optionally also include a container, buffer, and/or instructions. The fluorescent DHP bridged compounds, labeled specific binding partners, and/or tandem dyes may include a conjugation tag.

II. Definitions

[0056] The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0057] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings.

While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0058] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub- ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0059] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “of” is used to refer to a nonexclusive “of' unless otherwise indicated. The statement “at least one of A and B” or

“at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section.

[0060] All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0061] In the methods described herein, the acts can be carried out in any order without departing from the principles of the present disclosure, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately.

For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0062] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0063] The term “substantially” as used herein refers to a majority of, or mostly, as in 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 more, or 100%.

[0064] The term “substantially free of' as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than or equal to about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0065] The term “weight average molecular weight” or “mass average molar mass”, M_w, refers to the weight fraction distribution of different sized molecules in a sample. M_w can be determined by any appropriate technique known in the art. In some cases, M_w can be determined by, for example, static light scattering or small angle laser light scattering.

[0066] The term “number average molecular weight” of “number average molar mass”, M_n, refers to the mole fraction distribution of different sized molecules in a sample. M_n is the ordinary arithmetic mean or average of the molecular masses of the individual molecules. M_n can be determined by any appropriate means known in the art. The M_n can be determined, for example, by gel permeation chromatography, viscometry via Mark-Houwink equation, vapor pressure osmometry, or proton NMR.

[0067] The term “covalent bond” refers to a region of relatively high electron density between nuclei which arises at least partly from sharing of electrons and gives rise to an attractive force and characteristic intemuclear distance. IUPAC Glossary of Terms used in Physical Organic Chemistry, Pure & Appl. Chem. Vol. 66, no. 5, pp. 1077-1184, p. 1101.

[0068] The bonds that hold the antigen to the combining site of an antibody are noncovalent, and hence, they are reversible in nature. These noncovalent bonds may be hydrogen bonds, electrostatic bonds, or van der Waals forces. [0069] The term “reactive group” refers to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases, after optional activation of one of the functional groups. Chemoselective functional groups of interest include, but are not limited to, thiols, maleimides, halogenated maleimides, iodoacetamides, amines, amide, sulfonamide, alkyl carboxylates, activated esters, alkyl sulfonates, carboxylic amines, carbamate, carboxylate esters, N- hydroxysuccinimidyl (NHS), imido ester, halogen, boronic esters, boronic acids, hydrazonyl, carboxylic acids or active esters thereof, as well as groups that can react with one another via Click chemistry, e.g., azide and alkyne groups (e.g., cyclooctyne groups), tetrazine and alkene groups (e.g., cyclooctene groups), dienes and dienophiles, sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, as well as hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, alkyne, phosphine, epoxide, urea, thiourea, thioester, imine, disulfides, and protected groups thereof and the like, or protected groups thereof. The reactive group may be a conjugation tag. The chemoselective functional group may be protected or unprotected. Additionally, orthogonal “functional group(s)” can be included that can be used for either bioconjugation of a binding partner to or the attachment of acceptor signaling chromophores in donor acceptor tandem dyes.

[0070] 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, isocyanates, acyl azides, NHS esters, imidoesters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. The amine-reactive group can be a NHS ester or imidoesters.

[0071] In some cases, non-covalent linking may involve specific binding between two moieties of interest (e.g., two affinity moieties such as a hapten and an antibody or a biotin moiety and a streptavidin, etc.). In certain cases, non-covalent linking may involve absorption to a substrate.

[0072] The term “symmetric” in reference to a compound of the present disclosure refers to wherein each terminal heterocyclic ring system

and

is the same, and substituents may be the same or different. [0073] The term “asymmetric” in reference to a compound of the present disclosure . refers to wherein each terminal heterocyclic ring system

and

is different.

[0074] The term “counterion” refers to an ion that is charge balancing to the fluorescent compound according to the disclosure. The counter ion may be a cation. The counterion may be an anion. In some cases, the counterion may be a halogen ion, perchlorate ion, PF^6-, phosphate ion, sulfate ion, and the like. The counterion may be, F-, Cl-, Br, I-, ClO₄-, CF₃CO₂-, CH₃CO₂-, PO₄^3-, SO₄^2-, BF₄-, and the like. In some cases, the counterion may be Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺, and the like.

[0075] The term “organic group” as used herein refers to any carbon-containing functional moiety. Examples can include an oxygen-containing group such as an alkoxy group; aryloxy group; aralkyloxy group; oxo(carbonyl) group; an amine group, including alkyl amine amine esters, and sulfonamide groups; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group, thiol, thiol reactive group, and sulfone group; maleimide; iodoacetamide; azide group; alkyne group; and other heteroatom-containing groups. 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, 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)O-₂N(R)C(O)R, (CH₂)O-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- C₁₀₀)hydrocarbyl, wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

[0076] The term “elevated temperature” refers to a temperature above room temperature (25 deg C). In some cases, the term elevated temperature may refer to a temperature >50 deg C, >60 deg C, > 70 deg C, or >75 deg C. [0077] The term “heteroatom” as used herein refers to any appropriate atom that is not carbon, such as, for example, N, O, S, Se, P, B, Al, Si, and Ge, inserted between adjacent carbon atoms in an organic group. The organic group may be a cyclic, aryl, or straight or branched chain group (e.g., alkyl or alkene). More than one heteroatom (e.g., 1, 2, 3, 4 or 5heteroatoms) may be inserted between adjacent carbon atoms. The heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- and S(O)₂-, sulfinate, sulfonamide.

[0078] The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms, such as, for example an alkyl, aryl, or a functional group. The “substituted” group may include one or more groups selected from halogen, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, alkoxy, SO₃H, -SO₂R', -SO₂NR', -PR'₃, POR'₃, and -SiR'₃. In some instances, the “substituted” group is selected from R¹, R², R³, R³'and/or R⁴ groups as defined herein, In some instances the “substituted group, is selected from R¹, R², aryl, heteroaryl, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, halogen, a water-solubilizing moiety, functional moiety, a PEG group, -B(R³)₂, O-alkyl, O-aryl, NR'R', NHR', NH₂, -S-R', SO₃H, -SO₂R', -SO₂NR', -PR'₃, POR'₃, -SiR'₃, -ammonium, alkylammonium, and arylammonium, where each R' is independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted aryl, optionally substituted with one or more PEG groups, water solubilizing moieties, linking moieties, linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group.

[0079] The term “functional group,” “functional moiety,” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); alkene; cycloalkene; alkyne; cycloalkyne; an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, imides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)₂, CN, NO, NO₂, O NO₂, azido, 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)O-₂N(R)C(O)R, (CH₂)O-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, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C₁- C₁₀₀)hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl ring system.

[0080] The subject fluorescent compounds can include one or more “chemoselective functional group(s)” (also referred to as a “conjugation tag”) that provide for bioconjugation to a dye having the corresponding “chemoselective functional group” or “conjugation tag”. In some cases, such functionality may be used to covalently attach an acceptor or donor dye, or a biomolecule or binding partner such as a protein, peptide, affinity ligand, antibody, antibody fragment, polynucleotide, or aptamer. In some cases the functional group or conjugation tag may be selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N- hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, isothiocyanate, azide, alkyne, cycloalkyne (e.g., , alkene, cycloalkene (e.g., cyclooctene), tetrazine, aldehyde, thiol, and protected groups thereof for conjugation to a substrate, acceptor dye, functional moiety, or binding partner. The functional group may be protected or unprotected. The functional group may be a reactive or chemoselective functional group that can react with another group via copper-free click chemistry, including strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse-electron-demand Diels-Alder (iEDDA) reactions that enable fast and specific chemical conjugation. See Kim et al., Chem. Sci., 2019, 10, 7835; and Davis et al., J. Org. Chem. 2016, 81, 6816-6819, both incorporated herein by reference in their entireties. The functional group or conjugation tag can be, for example, cycloalkene (e.g., cyclooctene); alkyne; cycloalkyne (e.g., cyclooctyne group, such as, for example, bicyclo[6.1.0] nonyne (BCN)), Dibenzocyclooctyne (DBCO)); cycloalkene (e.g., cyclooctene group, such as, for example, trans-cyclooctene (TCO)); an azide group; or a tetrazine group.

[0081] As used herein, the term “activated ester” or “active esters” by itself or as part of another substituent refers to carboxyl-activating groups employed in peptide chemistry to promote facile condensation of a carboxyl group with a free amino group of an amino acid derivative. Descriptions of these carboxyl-activating groups are found in general textbooks of peptide chemistry, for example 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.

[0082] As used herein, the term “ammonium” by itself or as part of another substituent refers to a cation having the formula NHR3⁺ where each R group, independently, is hydrogen or a substituted or unsubstituted alkyl, aryl, aralkyl, or alkoxy group. Preferably, each of the R groups is hydrogen.

[0083] The term “hydrocarbon” or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein some or all the hydrogen atoms are substituted with other functional groups. The term “hydrocarbyl” 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. Hydrocarbyl groups can be shown as (C_a-C_b)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (C₁-C₄)hydrocarbyl means the hydrocarbyl group can be methyl (C₁), ethyl (C₂), propyl (C₃), or butyl (C₄), and (C₀-C_b)hydrocarbyl means in certain embodiments there is no hydrocarbyl group. A hydrocarbylene group is a diradical hydrocarbon, e.g., a hydrocarbon that is bonded at two locations.

[0084] As used herein, the term “alkyl” by itself or as part of another substituent refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl groups can be optionally substituted alkyl groups. For example, C₁-C₆ alkyl includes, but is 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 can include any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. Alkyl can also refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted alkyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy. The alkyl group is typically monovalent, but can be divalent, such as when the alkyl group links two moieties together.

[0085] As used herein, the term “alkylene” refers to an alkyl group, as defined above, linking at least two other groups (i.e., a divalent alkyl radical). The two moieties linked to the alkylene group can be linked to the same carbon atom or different carbon atoms of the alkylene group.

[0086] As used herein, the term “alkoxy” by itself or as part of another substituent refers to an alkyl group, as defined above, having an oxygen atom that connects the alkyl group to the point of attachment. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can be further substituted with a variety of substituents described within. For example, the alkoxy groups can be substituted with halogens to form a “halo-alkoxy” group.

[0087] As used herein, the term “alkene” or “alkenyl” by itself or as part of another substituent refers to either a straight chain, branched chain, or cyclic hydrocarbon, having at least one double bond between two carbon atoms. Examples of alkene 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-hexatrienyl. The alkene group is typically monovalent, but can be divalent, such as when the alkenyl group links two moieties together.

[0088] As used herein, the term “alkyne” or “alkynyl” by itself or as part of another substituent refers to either a straight chain or branched hydrocarbon, having at least one triple bond between two carbon atoms. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1 -pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1- hexynyl, 2-hexynyl, 3-hexynyl, 1,3 -hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4- hexadiynyl, or 1,3,5-hexatriynyl. The alkynyl group is typically monovalent, but can be divalent, such as when the alkynyl group links two moieties together. [0089] The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a “formyl” group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acyl group can optionally also include heteroatoms within the meaning herein. Examples of acyl groups include, but are not limited to, a nicotinoyl group (pyridyl-3- carbonyl) acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.

[0090] As used herein, the term “aldehyde” by itself or as part of another substituent refers to a chemical compound that has a — CHO group.

[0091] As used herein, the term “aryl” by itself or as part of another substituent refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the aromatic ring assembly. “Aryl” groups can be a monocyclic or fused bicyclic, tricyclic, tetracyclic, pentacyclic, or greater, aromatic ring assembly containing 6 to 22, 14 to 22, 17 to 22, or 6 to 16 ring carbon atoms. For example, aryl may be, but is not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, benzyl, benzoxazole, dihydrophenanthrenyl, 9, 10-dihydrophenanthrenyl, 2-R²- 1H-benzo[de]isoquinoline- 1,3(2H)-dionyl, or naphthyl, and the like. Aryl groups can include substituted aryl groups. Substituted aryl groups may be mono- or disubstituted by amino, alkoxy, optionally substituted phenyl, halogen, alkyl or trifluoromethyl, hydroxyl, C₁-C₁₂ alkyl, C₂- C₁₂ alkene, C₂- C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂ haloalkyl, C₁-C₁₂ alkoxy, C₂- C₁₈ (hetero)aryloxy, C₂-C₁₈ (hetero)arylamino, carboxylate, carboxylic acid, C₂-C₁₂ alkyl carboxylic acid, C₂-C₁₂ alkyl carboxylate, C₂-C₁₂ alkyl carboxylate ester, C₁-C₁₂ alkoxy, a water-solubilizing group (WSG), a functional group, sulfonic acid, sulfonate, or C₁-C₁₂ alkyl sulfonate. In some cases, the substituted aryl group may be mono- or disubstituted by a functional group, a WSG, optionally a WSG comprising a functional group, alkoxy, halogen or trifluoromethyl. The WSG can be a branched WSG, optionally comprising a functional group, such as, for example, a WSG comprising PEG and a functional group. In some cases, WSG comprises a PEG group.

[0092] Preferred aryl groups may include 2-R²-1H-benzo[de]isoquinoline-1,3(2H)- dionyl substituted by alkyl, phenyl, p-methoxyphenyl, or p-CN-phenyl; alkyl substituted fluorenyl; benzoxazole naphthyl;; phenyl or phenyl mono- or disubstituted by amino, alkoxy, phenyl, phenyl amino, diphenyl amino, PEG carboxylic acid, halogen, alkyl or trifluoromethyl, especially phenyl or phenyl-mono- or disubstituted by alkoxy, halogen, or trifluoromethyl, and in particular, phenyl.

[0093] The term “monocyclic heteroaryl” refers to an unsubstituted or substituted heteroaryl ring system comprising 1 aryl ring, and wherein the monocyclic ring system comprises one or more, two or more, three or more, or four or more heteroatoms. Nonlimiting examples of a monocyclic heteroaryl group are substituted or unsubstituted pyridinyl, pyranyl, thiophenyl, furanyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl.

[0094] The term “polycyclic aryl” refers to an unsubstituted or substituted polycyclic ring system comprising 2 to 9, 2 to 8, or 2 to 6 aryl rings with or without fused cycloalkyl or cycloalkenyl rings. Examples of polycyclic aryl groups may include fluorene, 9H- fluorene, phenanthrene, dihydrophenanthrene, 9,10-dihydrophenanthrene, naphthalene, anthracene, tetracene, pentacene, and the like.

[0095] 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 with or without fused cycloalkyl or cycloalkenyl rings, and wherein the polycyclic ring system comprises one or more, two or more, three or more, or four or more heteroatoms. Non-limiting examples of polycyclic heteroaryl systems may include quinoline, benzoxazole, benzothiazole, benzimidazole, indole, benzindole, pyridinium, benzopyrylium, thiopyrylium, 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-dihydrophenanthro[3,2-d]thiazole, 4,5-dihydrophenanthro[2, 1 - d]thiazole, 6,7-dihydrophenanthro[4,3-d]thiazole, 5,6-dihydrophenanthro[3,2-d]oxazole, 4,5-dihydrophenanthro[2, 1-d] oxazole, 6,7-dihydrophenanthro[4,3-d]oxazole, 5,6- dihydronaphtho[2, 1-g] quinoline, 7,8-dihydronaphtho[2, 1-h] quinoline, 5,6- dihydronaphtho[2, 1-f] quinoline, 5,6-dihydro-8X2-phenanthro[3,2-d]imidazole, 4,5- dihydro-3A2-phenanthro [2, 1-d] imidazole, 6,7-dihydro-3X2-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-dihydropentapheno[3,2, 1-cd: 10, 11,12- c'd']diindole, 3,8-dihydrophenanthro[2,3-e:7,6-e']diindole, 3, 5,6,8- tetrahydrophenanthro[2,3-e:7,6-e']diindole, 1,2,3,5,6,8-hexahydrophenanthro[2,3-e:7,6- e']diindole-1, 10-diium salt, 3,5,6,8-tetrahydrophenanthro[2,3-e:7,6-e']diindole— ethane (1/1), 5,6-dihydropentapheno[3,4-d: 10,9-d']bis(oxazole), 1,2,5,6,9,10- hexahydropentapheno[3,4-d: 10, 9-d']bis(thiazole)-1, 10-diium, pentapheno[3,4-d: 10,9- d']bis(thiazole), 1 ,2,9, 10-tetrahydropentapheno[3,4-d: 10,9-d']bis(thiazole)-1,10-diium,

6.7-dihydrophenanthro[2,3-f:7,6-f]diquinoline-1,12-diium, 6,7-dihydrophenanthro[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), and the like.

[0096] The term “Arylene” refers to a divalent radical derived from an aryl group. Aryl groups can be mono-, di- or tri-substituted by one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C₂-C₃-alkylene; all of which are optionally further substituted, for instance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl.

Alkylenedioxy is a divalent substitute attached to two adjacent carbon atoms of phenyl, e.g., methylenedioxy or ethylenedioxy. Oxy-C₂-C₃-alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, e.g., oxyethylene or oxypropylene. An example for oxy-C₂-C₃-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl. The polycyclic heteroaryl may be substituted or unsubstituted.

[0097] As used herein, the term “aryloxy” by itself or as part of another substituent refers to a O-aryl group, wherein aryl is as defined above. An aryloxy group can be unsubstituted or substituted with one or two suitable substituents. The term “phenoxy” refers to an aryloxy group wherein the aryl moiety is a phenyl ring. The term “(hetero)aryloxy” as used herein means an — O-heteroaryl group, wherein heteroaryl is as defined below. The term “(hetero)aryloxy” is used to indicate the moiety is either an aryloxy or (hetero)aryloxy group.

[0098] The term “aralkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

[0099] As used herein, the term “amine” by itself or as part of another substituent as used herein refers to an alkyl groups as defined within, having one or more amino groups. The amino groups can be primary, secondary or tertiary. The alkyl amine can be further substituted with a hydroxy group. Amines useful in the present disclosure include, but are not limited to, ethyl amine, propyl amine, isopropyl amine, ethylene diamine and ethanolamine. The amino group can link the alkyl amine to the point of attachment with the rest of the compound, be at the omega position of the alkyl group, or link together at least two carbon atoms of the alkyl group. One of skill in the art will appreciate that other alkyl amines are usefill in the present disclosure.

[00100] The term “amino group” as used herein refers to a substituent of the form - NH₂, -NHR, -NR₂, -NR₃⁺, wherein each R is independently selected, and protonated forms of each, except for -NR₃⁺, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group may include a monoalkylamino, dialkylamino, or trialkylamino group.

[00101] 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 “amido” refers to a substituent containing an amide group.

[00102] As used herein, the term “carbamate” by itself or as part of another substituent refers to the functional group having the structure — NR"CO₂R', where 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. Examples of carbamates include t-Boc, Fmoc, benzyloxy-carbonyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7- dibromo)fluorenylmethyl carbamate, Tbfmoc, Climoc, Bimoc, DBD-Tmoc, Bsmoc, Troc, Teoc, 2-phenyl ethyl carbamate, Adpoc, 2-chloroethyl carbamate, 1,1-dimethyl-2- haloethyl carbamate, DB-t-BOC, TCBOC, Bpoc, t-Bumeoc, Pyoc, Bnpeoc, V-(2- pivaloylamino)- 1,1 -dimethylethyl carbamate, NpSSPeoc. [00103] As used herein, the term “carboxylic acid” by itself or as part of another substituent refers to a structure R-COOH where R is a carbon-containing group of atoms. [00104] As used herein, the term “carboxylate” by itself or as part of another substituent refers to the conjugate base of a carboxylic acid, which generally can be represented by the formula RCOO-. For example, the term “magnesium carboxylate” refers to the magnesium salt of the carboxylic acid The term “carboxylate ester” as used herein by itself or as part of another substituent refers to a compound derived from a carboxylic acid, which generally can be 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 group of atoms. R' can optionally contain functional groups.

[00105] As used herein, the term “cycloalkyl” by itself or as part of another substituent refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms or the number of atoms indicated. Cycloalkyl can include any number of carbons, such as C_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, norbomane, [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 cycloalkyl groups that are partially unsaturated 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), norbomene, and norbomadiene. When cycloalkyl is a saturated monocyclic C_3-8 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C_3-6 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted cycloalkyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy. The term “lower cycloalkyl” refers to a cycloalkyl radical having from 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, norbomane, decahydronaphthalene and adamantane. For example, C_3-8cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbomane. Polycyclic ring systems may be substituted or unsubstituted.

[00106] As used herein, the term “cycloalkylene” refers to a cycloalkyl group, as defined above, linking at least two other groups (i.e., a divalent cycloalkyl radical). The two moieties linked to the cycloalkylene group can be linked to the same atom or different atoms of the cycloalkylene group.

[00107] As used herein, the term “haloalkyl” by itself or as part of another substituent refers to alkyl as defined above where some or all of the hydrogen atoms are substituted with halogen atoms. Halogen (halo) preferably represents chloro or fluoro, but may also be bromo or iodo. For example, haloalkyl includes trifluoromethyl, flouromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term “perfluoro” defines a compound or radical which has at least two available hydrogens substituted with 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.

[00108] As used herein, the term “halogen” by itself or as part of another substituent refers to fluorine, chlorine, bromine, and iodine.

[00109] As used herein, the term “heteroaryl” by itself or as part of another substituent refers to a monocyclic or fused polycyclic, such as bicyclic, tricyclic, tetracyclic, or pentacylic aromatic ring assembly, for example, containing 5 to 22, 14 to 22, 17 to 22, 6 to 16, or 5 to 16 ring atoms, where from 1 to 4 of the ring atoms may be a heteroatom, such as N, O, or S. Additional heteroatoms can also be usefill, including, but not limited to, B, Al, Si, or P. The heteroaryl may be substituted or unsubstituted. Substituted heteroaryl may include one or more K-R¹³, halogen, O-C_1-6 alkyl, S-C_1-6 alkyl, O-aryl, S- aryl, NHC_1-6alkyl, Ph-NCS, Ph-CO₂H, Ph-(CH₂)_1-4CO₂H substituents. The heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)₂-. For example, heteroaryl may include pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radicals substituted, especially mono- or di-substituted, by, e.g., alkyl, nitro or halogen. Pyridyl may represent 2-, 3- or 4-pyridyl, advantageously 2- or 3 -pyridyl. Thienyl may represent 2- or 3 -thienyl. Quinolinyl may represent preferably 2-, 3- or 4-quinolinyl. Isoquinolinyl may represent preferably 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranyl represents preferably 3 -benzopyranyl or 3 -benzothiopyranyl, respectively. Thiazolyl may represent preferably 2- or 4-thiazolyl, and most preferred, 4- thiazolyl. Triazolyl may represent preferably 1-, 2- or 5-(l,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl. The heteroaryl may include aryloxy or arylamino groups. In some embodiments, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted, especially mono- or di-substituted.

[00110] As used herein, the terms “heteroalkyl” or “heteroalkoxy” by itself or as part of another substituent refers to an alkyl or alkoxy group, preferably a C₁-C₁₂ alkyl group or C₁-C₁₂ alkoxy group where a C is substituted by a heteroatom such as N, O or S. For example, heteroalkyl or heteroalkoxy can include ethers, thioethers and alkyl-amines. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si, or P. The heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- , -S(O)₂-, sulfinate, sulfonamide. The heteroatom portion of the heteroalkyl can replace a hydrogen atom of the alkyl group to form a hydroxy, thio or amino group. Alternatively, the heteroatom portion can be the connecting atom, or be inserted between two carbon atoms.

[00111] As used herein, the term “heteroalkylene” refers to a heteroalkyl group, as defined above, linking at least two other groups (i.e., a divalent heteroalkyl radical). The two moieties linked to the heteroalkylene group can be linked to the same atom or different atoms of the heteroalkylene group.

[00112] As used herein, the term “(hetero)arylamino” by itself or as part of another substituent refers an amine radical substituted with an aryl group (e.g., — NH-aryl). An arylamino may also be an aryl radical substituted with an amine group (e.g., -aryl-NH₂). Arylaminos may be substituted or unsubstituted.

[00113] In some embodiments, substituents for the aryl, heteroaryl, and heteroalkylene groups are varied and are 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, perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where 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.

[00114] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O) — ( CH₂)_q — U — , wherein T and U are independently — NH — , — O — , — CH₂ — or a single bond, and q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A- (CH₂)_r — B — , wherein A and B are independently — CH₂ — , — O — , — NH — , — S — , — S(O) — , — S(O)₂ — , — S(O)₂NR' — or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula — (CH₂)s — X' — (CH₂)t — , where s and t are independently integers of 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.

[00115] As used herein, the term “oligoether” is understood to mean an oligomer containing structural repeat units having an ether functionality. As used herein, an “oligomer” is understood to mean a molecule that contains one or more identifiable structural repeat units of the same or different formula.

[00116] As used herein, the terms “polyethylene glycol”, “PEG”, “PEG group”, “polyethylene oxide” or “PEG” refer to the family of biocompatible water-solubilizing linear polymers based on the ethylene glycol monomer unit described by the formula — (CH₂ — CH₂ — O — )n — or a derivative thereof. 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 may be a monodispersed PEG or a polydispersed PEG. Monodispersed PEGs are discreet compounds having a single molecular weight (MW). Polydispersed PEG compounds are mixtures of PEG compounds having an average molecular weight. The molecular weights of polydisperse PEGs can be described in terms of distribution ranges. Molecular weight distribution can be confirmed by the polydispersity index (PDI) also known as dispersion coefficient, or dispersity. PDI of a polymer is calculated as the ratio of weight average molecular weight by number average molecular weight. PDI = M_w/M_n. PEG shapes include linear, branches, star, with a combination of different PEG chain lengths. It is understood that the PEG polymeric group may be of any convenient length and may include a variety of terminal groups and/or further substituent groups, including but not limited to, alkyl, alkoxy, aryl, hydroxyl, amino, acyl, carboxylic acid, carboxylate ester, acyloxy, and amido terminal and/or substituent groups. As used herein, PEG groups include, but are not limited to, PEG, modified PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, alkyl sulfonamide-PEG and alkoxy sulfonamide-PEG. It is understood that the PEG polymeric moiety may be of any convenient length and may include a variety of terminal groups and/or further substituent groups, including but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amido terminal and/or substituent groups. PEG groups that may be adapted for use with the subject compounds include those PEGs 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, N.Y. (1992); and by J.M. Harris and S Zalipsky in “Poly(ethylene glycol) Chemistry and Biological Applications”, ACS (1997). In some instances, PEG and modified PEG moieties can be, for example, those taught in International Patent Applications: 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. Pat. 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 each of which are incorporated herein by reference).

[00117] As used herein, the term “PEG ester” by itself or as part of another substituent refers to a group of formula — PEG — C(O) — OR where R is a carbon-containing group of atoms.

[00118] As used herein, the term “PEG carboxylic acid” by itself or as part of another substituent refers to a group of formula — PEG — C(O) — OH.

[00119] As used herein, the term “sulfonate functional group” or “sulfonate” either by itself or as part of another substituent refers to both the free sulfonate anion ( — S(=O)₂O — ) and salts thereof. Therefore, the term sulfonate encompasses sulfonate salts such as sodium, lithium, potassium and ammonium sulfonate.

[00120] As used herein, the term “sulfonamide” by itself or as part of another substituent refers to a group of formula — SO₂NR2 where each R can independently be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or functional group and can contain carboxylic groups. R can be a water- solubilizing polymer including, but not limited to, a polymer comprising 6 or more monomeric units, a non-ionic water-soluble polymer, such as PEG, modified PEG terminated with, for example, a carboxylic acid, a carboxylic ester, or an alkoxy group (e.g., OMethyl or OEthyl). The “sulfonamide” attached to another molecule by a linker or bond. The “sulfonamide” can be, for example, sulfonamide-PEG, alkyl sulfonamide, alkoxy sulfonamide, alkyl sulfonamide PEG, alkoxy sulfonamide PEG, alkyl sulfonamide PEG carboxylate, alkoxy sulfonamide PEG carboxylate.

[00121] As used herein, the term “sulfonamido” by itself or as part of another substituent refers to a group of formula — SO₂NR — where R can be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or functional group and can contain carboxylic groups. R can be a water-solubilizing polymer including, but not limited to, a polymer comprising 6 or more monomeric units, a non-ionic water-soluble polymer, such as PEG, modified PEG terminated with a carboxylic acid or a carboxylic ester. The “sulfonamido” can be attached to another molecule by a linker or bond. The “sulfonamido” can be, for example, sulfonamido-PEG, alkyl sulfonamido, alkoxy sulfonamido, alkyl sulfonamido PEG, alkoxy sulfonamido PEG, alkyl sulfonamido PEG carboxylate, alkoxy sulfonamido PEG carboxylate. [00122] As used herein, the term “sulfinamide” by itself or as part of another substituent refers to a group of formula — SONR₂ where each R can independently be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or functional group and can contain carboxylic groups. R can be a water- solubilizing polymer including, but not limited to, a polymer comprising 6 or more monomeric units, a non-ionic water-soluble polymer, such as PEG, modified PEG terminated with a carboxylic acid or a carboxylic ester. The “sulfinamide” can be attached to another molecule by a linker or bond. The “sulfinamide” can be, for example, sulfinamide-PEG, alkyl sulfinamide, alkoxy sulfinamide, alkyl sulfinamide PEG, alkoxy sulfinamide PEG, alkyl sulfinamide PEG carboxylate, alkoxy sulfinamide PEG carboxylate.

[00123] As used herein, the term “sulfonic acid” by itself or as part of another substituent refers to a group of formula — R — S(O)₂ — OH where R is a carbon- containing group of atoms.

[00124] As used herein, the terms “hydrazine” and “hydrazide” by themselves or as part of another substituent refer to compounds that contain singly bonded nitrogens, one of which is a primary amine functional group. For example, the term “hydrazine” refers to a moiety having the structure -NHNH₂.

[00125] As used herein, the term “thiol” by itself or as part of another substituent refers to a compound that contains the functional group composed 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 group of atoms.

[00126] As used herein, the term “silyl” by itself or as part of another substituent refers to Si(R^z)₃ wherein each R^z independently is alkyl, aryl or other carbon-containing group of atoms.

[00127] As used herein, the term “diazonium salt” by itself or as part of another substituent refers to a group of organic compounds with a structure of R — N₂⁺X'- wherein R can be any organic group (e.g., alkyl or aryl) and X' is an inorganic or organic anion (e.g., halogen).

[00128] As used herein, the term “triflate” by itself or as part of another substituent also referred to as trifluoromethanesulfonate, is a group with the formula CF₃SO₃.

[00129] As used herein, the term “boronic acid” by itself or as part of another substituent refers to a structure -B(OH)₂. It is recognized by those skilled in the art that a boronic acid may be present as a boronate ester at various stages in the synthesis of the quenchers. Boronic acid is meant to include such esters. The term “boronic ester” or “boronate ester” as used herein refers to a chemical compound containing a — B(Z¹)(Z²) moiety, wherein Z¹ and Z² together form a moiety where the atom attached to boron in each case is an oxygen atom. In some embodiments, the boronic ester moiety is a 5- membered ring. In some other embodiments, the boronic ester moiety is a 6-membered ring. In some other embodiments, the boronic ester moiety is a mixture of a 5-membered ring and a 6-membered ring.

[00130] As used herein, the term “maleimide” by itself or as part of another substituent refers a structure

where R can be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other group and can contain carboxylic groups. R can be a water-solubilizing polymer including, but not limited to, a polymer comprising 6 or more monomeric units, a non-ionic water-soluble polymer, PEG, modified PEG terminated with a carboxylic acid or a carboxylic ester. [00131] As used herein, the term “hydrazone” by itself or as part of another substituent refers to a structure where R can be, for example, a water solubilizing

moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other group and can contain carboxylic groups. R can be a water-solubilizing polymer including, but not limited to, a polymer comprising 6 or more monomeric units, a non-ionic water-soluble polymer, PEG, modified PEG terminated with a carboxylic acid or a carboxylic ester. [00132] As used herein, the term “azide” by itself or as part of another substituent refers to a structure-N₃.

[00133] As used herein, the term “tetrazine” by itself or as part of another substituent refers to a compound having the molecular formula C₂H₂N₄ that consists of a six- membered heteroaromatic ring structure having 4 nitrogen atoms. The term “tetrazine” includes all of its isomers, namely 1,2,3,4-tetrazines, 1,2,3,5-tetrazines, and 1, 2,4,5- tetrazines. [00134] As used herein, the term “N-hydroxysuccinimidyl” by itself or as part of another substituent refers to a structure

where R can be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other group and can contain carboxylic groups. R can be a water-solubilizing polymer including, but not limited to, a polymer comprising 6 or more monomeric units, a non- ionic water-soluble polymer, PEG, modified PEG terminated with a carboxylic acid or a carboxylic ester.

[00135] As used herein, the term “phosphoramide” by itself or as part of another substituent refers to a structure

where R can be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other group and can contain carboxylic groups. R can be a water-solubilizing polymer including, but not limited to, a polymer comprising 6 or more monomeric units, a non- ionic water-soluble polymer, PEG, modified PEG terminated with a carboxylic acid or a carboxylic ester.

[00136] As used herein, the term “phosphonamidate” by itself or as part of another substituent refers to a structure

where R can be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other group and can contain carboxylic groups. R can be a water-solubilizing polymer including, but not limited to, a polymer comprising 6 or more monomeric units, a non- ionic water-soluble polymer, PEG, modified PEG terminated with a carboxylic acid or a carboxylic ester. The “phosphonamidite” can be attached to another molecule by a linker or bond. The “phosphonamidite”can be, for example, phosphonamidite-PEG, alkyl phosphonamidite, alkoxy phosphonamidite, alkyl phosphonamidite PEG, alkoxy phosphonamidite PEG, alkyl phosphonamidite PEG carboxylate, alkoxy phosphonamidite PEG carboxylate.

[00137] As used herein, the term “phosphinamide” by itself or as part of another substituent refers to a structure

where R can be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other group and can contain carboxylic groups. R can be a water-solubilizing polymer including, but not limited to, a polymer comprising 6 or more monomeric units, a non- ionic water-soluble polymer, PEG, modified PEG terminated with a carboxylic acid or a carboxylic ester. The “phosphinamide” can be attached to another molecule by a linker or bond. The “phosphinamide” can be, for example, phosphinamide-PEG, alkyl phosphinamide, alkoxy phosphinamide, alkyl phosphinamide PEG, alkoxy phosphinamide PEG, alkyl phosphinamide PEG carboxylate, alkoxy phosphinamide PEG carboxylate.

[00138] The term “fluorescent” as used herein refers to a compound which, when irradiated by light of a wavelength that the compound absorbs, emits light of a (typically) different wavelength. Fluorescence is the emission of light 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.

[00139] The term “absorption maxima” or “Abs λ max” or “max λ abs” refer to wavelength of maximum absorption measured by UV Vis spectroscopy.

[00140] The term “excitation wavelength” or “λ ex” refers to the wavelength where the compound can be excited to induce fluorescence emission, it does not necessarily have to be at the Abs λ max.

[00141] The term “chromophore” refers to a molecular entity or a portion thereof consisting of an atom or a group of atoms in which the electronic transition responsible for a given spectral band is approximately localized. In some instances, the “chromophore” may itself be fluorescent. In some cases, the chromophore may be a fluorophore. In some cases, the fluorophore may be an acceptor dye. As used herein, the terms “fluorescent chromophore” and “fluorescent dye” are used interchangeably and refer to a compound which has a structure capable of harvesting light with a particular absorption maximum wavelength and converting it to emitted light at a longer emission maximum wavelength. A chromophore may have a reactive group (e.g., a carboxylate moiety, an amino moiety, a haloalkyl moiety, or the like) that can be covalently bonded. Examples of suitable chromophores include, but are not limited to, those described in U.S. Pat. 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; 6,005,113; 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, which patents are incorporated herein by reference in their entirety.

[00142] The term “moiety” refers to a group as a portion of a molecule, which may be a functional group, or a portion of a molecule with multiple groups which share common structural and/or functional aspects. Examples of group or moiety include but are not limited to a linker moiety, a functional group, a water-solubilizing moiety, a PEG moiety, according to the present disclosure.

[00143] The term “small molecule dye” as used herein refers to a bridged DHP compound wherein the fluorophore portion of the molecule has a discrete molecular weight. In other words, the fluorophore portion of the molecule, i.e., the individual units of the bridged DHP compound without attached PEG groups or other water solubilizing groups that in some instances may be polydisperse, has a discrete molecular weight. The bridged DHP small molecule dye comprises a fluorophore that is non-polymeric.

[00144] The bridged DHP small molecule dye comprises a fluorophore that is non- polymeric.

[00145] The term “polymer” or “polymeric” refers to a molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass . (See IUPAC Glossary of Basic Terms in Polymer Science, Pure & Appl. Chem., vol. 68, no. 12, pp. 2287-2311, 1996; and What are polymers?-IUPAC International Union of Pure and Applied Chemistry). In contrast to a bridged “small molecule dye” of the disclosure that has a discrete molecular weight derived from an exact number of individual molecules, a polymer has a distribution of monomer units, not one exact number. Polymers can be made via Suzuki coupling reaction between a diboronic monomer with a dibromide monomer. In contrast, the bridged small molecule dyes of the disclosure can be made via Suzuki coupling between a molecule containing a diboronic group and monobromide molecules (or vice versa) (See, for example Schemes 1, 3-5 and 8).

[00146] The term “linker,” “linked” or “linkage” refers to a linking moiety that connects two groups and has a backbone of 100 atoms or less in length. A linker or linkage may be a covalent bond that connects two groups or a chain of between 1 and 100 atoms in length, for example a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom. In some embodiments, the linker is a branching linker that refers to a linking moiety that connects three or more groups. In some embodiments, the linker backbone includes a linking functional group, such as an ether, thioether, amino, amide, carbonyl, acyl, sulfonamide, a sulfinamide, a disulfonamide, a disulfinamide, a sultam, an amide, a secondary amine, a phosphonamide, a phosphinamide, a phosphonamidate, a selenonamide, a seleninamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. In some embodiments, the linker backbone includes a linking functional group, such as an amino, amide, carbonyl, sulfonamide, sulfinamide, a disulfonamide, a disulfinamide, a sultam, an amide, a secondary amine, a phosphonamide, a phosphinamide, a phosphonamidate, a selenonamide, or a seleninamide. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. In some embodiments, the linker backbone includes a linking functional group, such as an ether, thioether, amino, amide, carbonyl, acyl, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. The bonds between backbone atoms may be saturated or unsaturated, and in some cases not more than one, two, or three unsaturated bonds are present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, polyethylene glycol, ethers, thioethers, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1 -methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1- dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3, or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable.

[00147] A linker moiety can be attached to a DHP bridged small molecule dye compound according to the disclosure. A linker moiety can comprise covalent bond, an alkoxy, sulfonamide, disulfonamide, a selenomide, a sulfinamide, a sultam, a disulfinamide, an amide, carbonyl, a seleninamide, a phosphonamide, a phosphinamide, a phosphonamidate, or a secondary amine.

[00148] As described therein, and as each pertains to a linker moiety, the term “sulfonamide,” refers to a moiety -S(O)₂NR-; the term “disulfonamide,” refers to a moiety -S(O)₂NRS(O)₂-; the term “selenonamide,” refers to a moiety -Se(O)₂NR-; the term “sulfinamide,” refers to a moiety -S(O)NR₂; the term “disulfinamide,” refers to a moiety -S(O)NRS(O)-; the term “seleninamide,” refers to a moiety -Se(O)NR-; the term “phosphonamide,” refers to a moiety -NR-PR(O)NR-; the term “phosphinamide,” refers to a moiety -PR(O)NR-; and the term “phosphonamidate,” refers to a moiety - O-PR(O)NR-; and the term “sultam” refers to a cyclic sulfonamide (e.g., wherein the R group is bonded to the sulfur atom via an alkylene moiety); wherein for each term the R group is independently H, alkyl, haloalkyl, or aryl.

[00149] The subject water-soluble fluorescent DHP bridged compounds according to the disclosure may include a linear moiety that can include a functional group that provides for bioconjugation. A covalent bond can be formed to attach a biomolecule such as a protein, peptide, affinity ligand, antibody, antibody fragment, polynucleotide, or aptamer. For example, small molecule dye-labeled antibodies find use in flow cytometry as reagents exhibiting high brightness. Additionally, orthogonal functional groups can be installed that can be used for either bioconjugation or the attachment of the DHP bridged compounds in donor acceptor dyes.

[00150] The phrase “conjugated compound” or “labeled specific binding partner” refers to a compound according to the disclosure having a binding partner conjugated thereto.

[00151] In chemical structures,

represents either a single or double bond.

[00152] The phrase “binding partner” or “binding member” refers to any molecule or complex of molecules capable of specifically binding to a target analyte. A binding partner of the present disclosure includes for example, a protein (e.g., an antibody or an antibody fragment), a carbohydrate (e.g., a polysaccharide), an oligonucleotide, a polynucleotide, a lipid, an affinity ligand, an aptamer, or the like. In some embodiments, the binding partner is an antibody or fragment thereof. Specific binding in the context of the present disclosure refers to a binding reaction which is determinative of the presence of a target analyte in the presence of a heterogeneous population. Thus, under certain assay conditions, the specified binding partners bind preferentially to a particular protein or isoform of the particular protein and do not bind in a significant amount to other proteins or other isoforms present in the sample.

[00153] In some cases, the antibody includes intravenous immunoglobulin (IVIG) and/or antibodies from (e.g., enriched from, purified from, e.g., affinity purified from) IVIG. IVIG is a blood product that contains IgG (immunoglobulin G) pooled from the plasma (e.g., in some cases without any other proteins) from many (e.g., sometimes over 1,000 to 60,000) normal and healthy blood donors. IVIG is commercially available. Aspects of IVIG are described, for example, in US. Pat. Appl. Pub. Nos. 2010/0150942; 2004/0101909; 2013/0177574; 2013/0108619; and 2013/0011388.

[00154] In some cases, the antibody is a monoclonal antibody of a defined sub-class (e.g., IgG1, IgG2, IgG3, or IgG4). If combinations of antibodies are used, the antibodies can be from the same subclass or from different subclasses. For example, the antibodies can be IgG1 antibodies. In some cases, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is humanized.

[00155] The phrase “water-soluble compound complex” refers to a water-soluble fluorescent compound of the present disclosure conjugated with a binding partner. [00156] The phrase “protected group” (also referred to as "protecting group") refers to a reversibly formed derivative of an existing functional group in a molecule attached to decrease reactivity so that the protected functional group does not react under synthetic conditions to which the molecule is subjected. Examples of amine protecting groups include, but are not limited to, benzyloxycarbonyl; 9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl (Boc); allyloxycarbonyl (Alloc); p-toluene sulfonyl (Tos); 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc); 2,2,4,6,7-pentamethyl-2,3- dihydrobenzofuran-5-sulfonyl (Pbf); mesityl-2-sulfonyl (Mts); 4-methoxy-2,3,6- trimethylphenylsulfonyl (Mtr); acetamido; phthalimide; and the like. These and other protecting groups for amines, carboxylic acids, alcohols, and further functional groups can be added to and removed from compounds of the present disclosure using known techniques as described, for example, by Green and Wuts (Protective Groups in Organic Synthesis, 4^th Ed. 2007, Wiley-Interscience, New York).

[00157] The term “sample” refers to a material or mixture of materials, in some cases in liquid form, containing one or more analytes of interest. In some embodiments, the term as used in its broadest sense, refers to any plant, animal or bacterial material containing cells or producing cellular metabolites, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment. The term “sample” may also refer to a “biological sample”. As used herein, the term “a biological sample” refers to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including, but not limited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A “biological sample” can also refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, and organs. In certain embodiments, the sample has been removed from an animal or plant. Biological samples may include cells. The term “cells” is used in its conventional sense to refer to the basic structural unit of living organisms, both eukaryotic and prokaryotic, having at least a nucleus and a cell membrane. In certain embodiments, cells include prokaryotic cells, such as from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples from animals, plants or fungi.

[00158] The term “substrate” refers to a solid material having a variety of configurations. The substrate can be, for example, a sheet, bead, or other structure, such as a plate with wells, a polymer, a particle, a semiconductor surface, nanotubes, a fibrous mesh, a hydrogel, a porous matrix, a pin, a microarray surface, a chromatography support, and the like. In some instances, the substrate is selected from the group consisting of a particle, a planar solid substrate, a fibrous mesh, a hydrogel, a porous matrix, a pin, a microarray surface and a chromatography support.

[00159] The term “water” as used herein refers to any aqueous solution that is primarily water and is compatible with physiological conditions. In some instances, the aqueous solution contains more than 50% water, such as more than 60% water, more than 70% water, more than 80% water, more than 90% water, 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, for example, sugars, proteins, amino acids, and nucleotides. In some instances, “water” may 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 solvent combinations different from water, such as pure alcohols, for example pure methanol or ethanol, pure ethers, for example pure diethyl ether or tetrahydrofuran, or any other pure solvent either miscible or not miscible with water.

[00160] The term “water solubilizing moiety” or “water-solubilizing group” (WSG or W¹) as used herein by itself or part of another group refers to any hydrophilic group that is well solvated in aqueous environments, for example such as under physiological conditions, and is capable of increasing the water solubility of the molecule to which it is attached. The increase in water solubility of the molecule can vary depending upon the moiety attached. In some instances, the increase in water solubility (as compared to the solubility of the molecule without the moiety attached) is 2 fold or more, 5 fold or more, 10 fold or more, 25 fold or more, 50 fold or more, or 100 fold or more. Any convenient WSG may be included in the dyes described herein to provide for increased water- solubility. A water-solubilizing moiety can increase the solubility of a compound in a predominantly aqueous solution, as compared to a control compound which lacks the water-solubilizing moiety. The water-solubilizing moiety may be any convenient hydrophilic moiety that is well solvated in aqueous environments. The “water- solubilizing moiety” includes moieties, such as, but not limited to, PEG groups, carboxy groups including but not limited to carboxylic acids and carboxylates, polyvinyl alcohol, glycols, peptides, polyphosphates, polyalcohols, sulfonates, phosphonates, boronates, amines, ammoniums, sulfoniums, phosphonium, alcohols, polyols, oxazolines, zwitterionic derivatives, carbohydrates, nucleotides, polynucleotides, substituted PEG groups, substituted carboxy groups including but not limited to substituted carboxylic acids and substituted carboxylates, substituted glycols, substituted peptides, substituted polyphosphates, substituted polyalcohols, substituted sulfonates, substituted phosphonates, substituted boronates, substituted amines, substituted ammoniums, substituted sulfoniums, substituted phosphonium, alcohols, substituted zwitterionic derivatives, substituted carbohydrates, substituted nucleotides, substituted polynucleotides, and combinations thereof. In some cases, WSG comprises a PEG group.

[00161] In some instances, the water-solubilizing moiety can be capable of imparting solubility in water (e.g., aqueous buffer) > 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 instances, the water-solubilizing moiety can be capable of imparting 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.

[00162] The increase in water solubility of the molecule can vary depending upon the moiety attached. In some instances, the increase in water solubility (as compared to the solubility of the molecule without the moiety attached) is 2 fold or more, 5 fold or more, 10 fold or more, 25 fold or more, 50 fold or more, or 100 fold or more. In some cases, the water-solubilizing moiety is charged, e.g., a positively or negatively charged hydrophilic moiety. In some instances, the water-solubilizing moiety is a neutral hydrophilic moiety. In some instances, the water-solubilizing moiety is branched (e.g., as described herein). In some instances, the water-solubilizing moiety is linear. Water-solubilizing moieties include, but are not limited to, those taught in US Patent Publication No. 2022/0348770 which is incorporated herein by referenced in its entirety.

[00163] The term “water soluble” when referring to a compound as used herein refers to a compound having solubility in “water” as used herein of 1 mg/mL or more, such as 3 mg/mL or more, 10 mg/mL or more, 20 mg/mL or more, 30 mg/mL or more, 40 mg/mL or more, 50 mg/mL or more, 60 mg/mL or more, 70 mg/mL or more, 80 mg/mL or more, 90 mg/mL or more, 100 mg/mL or more, or even more at ambient room temperature. A “water-soluble compound” may exhibit solubility in water (e.g., 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 at ambient room temperature. In some instances, the water-soluble compound can 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 at ambient room temperature. It is understood that water soluble compounds may, under certain conditions, form discrete water- solvated nanoparticles in aqueous systems and can be resistant to aggregation. The fluorescent compounds of the disclosure can be water soluble.

[00164] Any convenient water-solubilizing moiety or WSG may be included in the dyes described herein to provide for increased water-solubility. WSGs may be, but are not limited to, carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate, sulfonium, ester, polyethylene glycols (PEG) and modified PEGs, linear PEG groups, branched PEG groups, hydroxyl, amine, amino acid, ammonium, guanidinium, pyridinium, polyamine and sulfonium, polyalcohols, straight chain or cyclic saccharides, primary, secondary, tertiary, or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols, including, polyethers, a zwitterionic derivative, a peptide sequence, nucleotides (DNA and RNA), a peptoid, a carbohydrate, an oxazoline, a polyol, a dendron, a dendritic polyglycerol, a cellulose, a chitosan, — COOM', — SO₃M', — PO₃M', — NR³+, Y', (CH₂CH₂O)_pR and mixtures thereof, where Y' can be any halogen, sulfate, sulfonate, or oxygen containing anion, p can be 1 to 500, each R can be independently H or an alkyl (such as methyl) and M' can be a cationic counterion or hydrogen, — (CH₂CH₂O)_yyCH₂CH₂XR^yy, — (CH₂CH₂O)_yyCH₂CH₂X— , — X(CH₂CH₂O)_yyCH₂CH₂ — , glycol, and polyethylene glycol, wherein yy is selected from 1 to 1000, X is selected from O, S, and NR^ZZ, and R^ZZ and R^YY are independently selected from H and C_1-3 alkyl, and combinations or derivatives thereof. In some instances, WSGs include, but are not limited to, PEG, a modified PEG, a peptide sequence, a peptoid, a carbohydrate, an oxazoline, a polyol, a dendron, a dendritic polyglycerol, a cellulose, a chitosan, or a derivative thereof. WSGs may be unsubstituted or substituted. In some cases, WSG comprises a PEG group.

[00165] In some instances, the water-solubilizing moiety or WSGs may be a hydrophilic polymer. For example, hydrophilic polymers that can be utilized in the WSG include, but are not limited to, polyalkylene oxide based polymers comprising an ethylene oxide repeat unit of the formula — (CH₂ — CH₂ — O)_n — or — (O — CH₂ — CH₂)_n — , such as, for example, PEG, polyamide alkylene oxide, or derivatives thereof. Further examples of polymers of interest include a polyamide having a molecular weight greater than 1,000 Daltons of the formula — [C(O)— X— C(O)— NH— Y— NH]_n- or — [NH— Y— NH— C(O) — X — C(O)]_n — , where X and Y are divalent radicals that may be the same or different and may be branched or linear, and n is a discrete integer from 2-100, such as from 2 to 50, and where either or both of X and Y comprises a biocompatible, substantially non-antigenic water-soluble repeat unit that may be linear or branched. The number of such water-soluble repeat units can vary significantly, with the number of such units being from 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6-100, for example from 2 to 50 or 6 to 50. An example of an embodiment is one in which one or both of X and Y is selected from: — ((CH₂)_n1 — (CH₂ — CH₂ — O)_n2 — (CH₂) — or — ((CH₂)_n1 — (O — CH₂ — CH₂)_n2 — (CH₂)_n1 — ), where nl 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 instances, the water-soluble polymer is a group of 1-50 monomeric units, such as 1-40, 1-30, 1-20, 2-24, 2-20, 2-10 or 2-6 monomeric units. A further example of an embodiment is one in which X is — (CH₂ — CH₂)— , and where Y is — (CH₂— (CH₂— CH₂— O)₃— CH₂— CH₂— CH₂)— or — (CH₂— CH₂ — CH₂ — (O — CH₂ — CH₂)₃ — CH₂) — . In certain instances, any one of the formulae described herein may be substituted with a water-soluble moiety that is a dendron, as known in art. [00166] In some instances, hydrophilic polymers can be, for example, PEG, a peptide sequence, a peptoid, a carbohydrate, an oxazoline, a polyol, a dendron, a dendritic polyglycerol, a cellulose, a chitosan, or a derivative thereof.

[00167] In some cases, a water-solubilizing moiety or WSG is (CH₂)_x(OCH₂CH₂)_yOCH₃ where each x is independently an integer from 0-20, each y is independently an integer from 0 to 50. In some instances, the water-soluble polymer is a PEG group or modified PEG polymer of 6-24 monomeric units, such as 10-30, 10-24, 10- 20, 12-24, 12-20, 12-16 or 16-20 monomeric units.

[00168] In some cases, the water-solubilizing moiety or WSG includes a non-ionic polymer (e.g., a PEG polymer) substituted at the terminal with an ionic group (e.g., a sulfonate). In some embodiments of the formulae, the WSG includes a substituent selected from (CH₂)_x(OCH₂CH₂)_yOCH₃ where each x is independently an integer from 0- 20, each y is independently an integer from 0 to 50; and a benzyl optionally substituted with one or more halogen, hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_zOCH₃ where each z is independently an integer from 0 to 50. In some instances, the WSG is (CH₂)₃(OCH₂CH₂)₁₁OCH₃. In some embodiments, one or more of the substituents is a benzyl substituted with at least one WSG groups (e.g., one or two WSG groups) selected from (CH₂)_x(OCH₂CH₂)_yOCH₃ where each x is independently an integer from 0-20 and each y is independently an integer from 0 to 50. It is understood that hydroxy-terminated polymer chains (e.g., PEG chains) instead of methoxy-terminated polymer chains (e.g., PEG chains) may be utilized in any of the water-solubilizing moieties.

[00169] The term modified polymer, such as a modified PEG, refers to water soluble polymers that have been modified or derivatized at either or both terminals, e.g., to include a terminal substituent (e.g., a terminal alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc.) and/or a terminal linking functional group (e.g., an amino or carboxylic acid group suitable for attachment via amide bond formation) suitable for attached of the polymer to a molecule of interest (e.g., to a light harvesting chromophore via a branching group). The subject water-soluble polymers can be adapted to include any convenient linking groups. It is understood that in some cases, the water-soluble polymer can include some dispersity with respect to polymer length, depending on the method of preparation and/or purification of the polymeric starting materials. In some instances, the water-soluble polymers are monodisperse. [00170] The water-soluble polymer can include one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties comprising one or more repeat units employed in a water-soluble polymer, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, and the like, which can contain, for example, up to 18 carbon atoms or even an additional polymer chain.

[00171] The water-soluble polymer moiety, or one or more of the spacers or linkers of the polymer moiety when present, may include polymer chains or units that are biostable or biodegradable. For example, polymers with repeat linkages have varying degrees of stability under physiological conditions depending on bond lability. Polymers with such bonds can be categorized by their relative rates of hydrolysis under physiological conditions based on known hydrolysis rates of low molecular weight analogs, e.g., from less stable to more stable, e.g., polyurethanes ( — NH — C(O) — O — )>polyorthoesters ( — O — C((OR)(R')) — O — )>polyamides ( — C(O) — NH — ). Similarly, the linkage systems attaching a water-soluble polymer to a target molecule may be biostable or biodegradable, e.g., from less stable to more stable: carbonate ( — O — C(O) — O — )>ester ( — C(O) — O — )>urethane ( — NH — C(O) — O — )>orthoester ( — O — C((OR)(R')) — O — )>amide ( — C(O) — NH — ). In general, it may be desirable to avoid use of a sulfated polysaccharide, depending on the lability of the sulfate group. In addition, it may be less desirable to use polycarbonates and polyesters. These bonds are provided by way of example, and are not intended to limit the types of bonds employable in the polymer chains or linkage systems of the water-soluble polymers useful in the WSGs disclosed herein.

[00172] In some instances, the water-solubilizing moieties include, but are not limited to, hydroxy, alkoxy, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate, alkyl sulfonate, alkyl carboxylate, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, alkoxy sulfonamide PEG, alkylcarboxylate, alkylamide, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

[00173] In some instances, the subject compounds may comprise multiple water- solubilizing moieties or WSG attached at a single location in the subject compounds, for example, via a branching linker, such as, for example, an aralkyl substituent further di- substituted with water solubilizing groups. As such, in some cases, the branching linker group is a substituent of the dye that connects the dye to two or more water solubilizing groups. In some instances, multiple water-solubilizing moieties may be attached to the subject compounds via groups having, for example, the following formulas:

, or

, wherein X¹, X² are branching points, L¹, L², L³ are linkers, m' is an integer from 1, 2, or 3; W¹ is a water-solubilizing moiety.

[00174] In some instances, one or more water-solubilizing moieties may be attached to the subject compounds via a group comprising linkers according to the disclosure, for example, as taught in US Published Application No. 2020/0190253 Al, which is incorporated herein by reference in its entirety. A linker moiety can be attached to the

DHP,

aanndd ,//,oorr

groups of the fluorescent compounds of the instant disclosure. A linker may be cleavable or non-cleavable. [00175] One or more water-solubilizing moieties can also be attached to the subject compounds via a group comprising linkers, such as, for example, but not limited to, the following linker formula (Vie):

wherein: each optional L¹ and L³ is an independently selected linker moiety;

X¹, optionally present, is a branching point;

W¹ is a water-soluble moiety, including, but not limited to, a water-soluble polymer comprising 2-50, 4-30, or 6-24 monomeric units; each m is independently 0 or 1; each m' is independently 0 or 1; each m” is independently 0 or 1; each s is independently 1 or 2; each t is independently 0, 1, 2, or 3; and R³ is as defined herein.

[00176] In some instances, L¹, L³, and X¹ are absent and W¹ is a water-solubilizing moiety, for example, a water-soluble polymer such as a PEG group comprising 2-50, 4- 30, or 6-24 monomeric units, such as 10-30, 10-24, 10-20, 12-24, 12-20, 12-16 or 16-20 monomeric units. In some instances, the water-solubilizing moiety can be an alkoxy sulfonic acid. In some cases, the water-solubilizing moiety may be a linear water- solubilizing moiety. For example, L¹ and X may be absent, L³ is a linker (e.g., as disclosed herein), and W¹ is a water-solubilizing moiety.

[00177] In some cases, at least one of, at least two of, or all three of L¹, L² and/or L³ may be selected from an alkyl or substituted alkyl linker, an alkenyl or substituted alkenyl linker, an alkynyl or substituted alkynyl linker, an alkoxy or substituted alkoxy linker, a PEG linker, a sulfonamido-alkyl or substituted sulfonamido-alkyl linker, an amido-alkyl or substituted amido-alkyl linker and an alkyl-amido-alkyl or substituted alkyl-amido- alkyl linker. In certain cases, the linker comprises a carbonyl group. A linker moiety can be a covalent bond, an alkoxy, sulfonamide, disulfonamide, a selenomide, a sulfinamide, a sultam, a disulfinamide, an amide, carbonyl, a seleninamide, a phosphonamide, a phosphinamide, a phosphonamidate, or a secondary amine.

[00178] In some instances, L² and L³ may be linker moieties each independently selected from the group consisting of a covalent bond, C_1-8 alkylene, 2- to 8-membered heteroalkylene, and a chain of between 2 and 200 backbone atoms in length, wherein the chain comprises a linear chain, a branched chain, and/or a cyclic moiety.

[00179] In some instances, L¹ can be a sulfonamide, a sulfinamide, a disulfonamide, a disulfinamide, a sultam, an amide, a secondary amine, a phosphonamide, a phosphinamide, a phosphonamidate, a selenonamide, or a seleninamide.

[00180] In some instances, L³ can be a linker having a backbone of 20 atoms or less in length and W¹ is a water-solubilizing moiety (e.g., as described herein). In some instances, L³ can be selected from an alkyl or substituted alkyl linker, an alkenyl or substituted alkenyl linker, an alkynyl or substituted alkynyl linker, an acyl or substituted acyl, an alkoxy or substituted alkoxy linker, a PEG linker, a sulfonamido-alkyl or substituted sulfonamido-alkyl linker, an amido-alkyl or substituted amido-alkyl linker and an alkyl-amido-alkyl or substituted alkyl-amido-alkyl linker. In some instances, L³ can be a bond. In some instances, L³ can be an alkyl or substituted alkyl linker, an alkenyl or substituted alkenyl linker, an alkynyl or substituted alkynyl linker, an alkoxy or substituted alkoxy linker and X can be an aryl group.

[00181] In some instances, L¹ and L³ are each independently selected from a C₁- C₁₂ alkyl or substituted alkyl linker, a C₁-C₁₂ alkenyl or substituted alkenyl linker, a C₁- C₁₂ alkynyl or substituted alkynyl linker, a C₁-C₁₂ acyl or substituted acyl linker, a C₁- C₁₂ alkoxy or substituted alkoxy linker, a C₁-C₁₂ amido-alkyl or substituted amido-alkyl linker, a C₁-C₁₂ alkyl-amido-alkyl or substituted alkyl-amido-alkyl linker, a sulfonamide, a sulfinamide, a disulfonamide, a disulfinamide, a sultam, an amide, a secondary amine, a phosphonamide, a phosphinamide, a phosphonamidate, a selenonamide, and a seleninamide. In certain cases, L³ comprises a carbonyl group or alkoxy group, and L¹ is a C₁-C₁₂ alkyl or substituted alkyl, a sulfonamide, a sulfinamide, a disulfonamide, a disulfinamide, a sultam, an amide, a secondary amine, a phosphonamide, a phosphinamide, a phosphonamidate, a selenonamide, and a seleninamide. In some instances, L³ can be an alkoxy or substituted alkoxy linker, X can be absent, and L¹ can be a sulfonamide, a sulfinamide, a disulfonamide, a disulfinamide, a sultam, an amide, a secondary amine, a phosphonamide, a phosphinamide, a phosphonamidate, a selenonamide, or a seleninamide.

[00182] In some instances, the branching point X¹ is selected from N, CR', C(=O)N, SO₂N, a tri-substituted aryl moiety (e.g., a 1,3,5-phenyl), a tetra-substituted aryl moiety (e.g., a 1, 3, 4, 5-phenyl), and a tri-substituted heteroaryl group. In certain instances, the branching point X¹ is a nitrogen atom. In other instances, the branching point X¹ is CR', where R' is selected from hydrogen, alkyl, substituted alkyl, or -L³-W¹ (e.g., as described herein). in. DHP Bridged Compounds

[00183] The disclosure provides DHP bridged compounds and methods for making. The DHP bridged dyes exhibit excitation maxima in a range of from about 300 to 900 nm. In addition to the specific structures disclosed herein, structural isomers of the disclosed structures are also included.

[00184] The DHP bridged compounds of the present disclosure each comprise at least one 9,10-dihydrophenanthrene (DHP) moiety or derivative thereof. The DHP bridged compounds according to the disclosure may comprise a structure according to Formula CD:

wherein * each

and

/ is independently selected from the group consisting of a substituted or unsubstituted benzene, benzene derivative, monocyclic aryl group, polycyclic aryl group, monocyclic heteroaryl group, and polycyclic heteroaryl group. [00185] In some cases, each of

and

may be independently selected from the group consisting of

, wherein each of

and

is independently optionally substituted with (R³ )_n; and p=1, 2, 3, 4, 5, 6, or 7. In some cases,

and

may each be independently selected from the group consisting of:

optionally wherein R² is C₁-C₆ alkyl, phenyl, or phenyl substituted with halogen, C₁-C₆ alkoxy, or CN.

[00186] Each T may independently be selected from the group consisting of C, C(R¹), N, N(R¹), P, O, S, and Si(R¹). Each T may independently be C, C(R¹), N(R¹), or N. In some cases, each T is C or C(R¹). In some cases, each T is C.

[00187] Each X may independently be C, O, N, P, or Si. In some cases, each X may be C or Si. In some cases, each X may be C.

[00188] Each Y may independently be C, O, N, P, or Si. In some cases, each Y may be

C or Si. In some cases, each X may be C.

[00189] In some cases, each X and Y are C and each T is C(R¹) or C. In some cases, each X, Y and T are C.

[00190] Each R¹ may independently be selected from the group consisting of a water- solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, hydroxy, halogen, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfmamide, phosphonamidate, phosphinamide, phosphorate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic add, alkyl sulfonate, alkyl sulfonate salt,

[00191] In some instances, each R¹ may independently be selected from the group consisting of a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

[00192] In some cases, each R¹ may be independently selected from the group consisting of a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, sulfonamide-PEG, phosphoramide-PEG, alkoxy sulfonic acid,

[00193] In some cases, each R¹ may be independently selected from the group consisting of sulfonamide-PEG, alkoxy sulfonic acid,

[00194] Each R² may independently be selected from the group consisting of a water- solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

[00195] In some cases, each R² is hydrogen. [00196] Each R³ may independently be selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a water-solubilizing moiety, a chromophore, functional moiety, binding partner, E, and a PEG group.

[00197] Each optional R^3' may independently be selected from the group consisting of R¹, R², R³, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, halogen, a water-solubilizing moiety, functional moiety, a PEG group, PEG carboxylic acid, alkyl amido PEG ester, alkyl amido PEG carboxylic acid, -B(R³)₂, O-alkyl, O-aryl, NR'R', NUR', NH₂, -S-R', SO₃H, -SO₂R', -SO₂NR', -PR'₃, POR'₃, -SiR'₃, -ammonium, alkylammonium, and arylammonium.

[00198] In some cases, each optional R^3' may independently be selected from the group consisting of halogen, NH₂, C₁-C₆ alkyl, C₁-C₆ alkoxy, a PEG group, PEG carboxylic acid, alkyl amido PEG ester, alkyl amido PEG carboxylic acid, phenyl, or phenyl substituted with halogen, C₁-C₆ alkoxy, or CN,

[00199] Each R' may independently be selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted aryl, optionally substituted with one or more independently selected PEG groups, water solubilizing moiety, linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, and a linked functional group.

[00200] Each R⁴ may independently be selected from the group consisting of H, alkyl, a PEG group, a linked PEG group, a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a chromophore, a functional group, a conjugation tag, a binding partner, a linked E, carboxylic amine, amine, carbamate, carboxylic acid, carboxylate ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, alkene, tetrazine, aldehyde, and thiol, or protected groups thereof.

[00201] Each optional R⁴' may independently be selected from the group consisting of R¹, R², R³ and R³', optionally wherein two R⁴' together form an unsubstituted or substituted cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, cycloalkoxy, aryl, or heteroaryl having 3 to 9 ring members.

[00202] Each R⁷ may independently be selected from the group consisting of H, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂haloalkyl, C₁-C₁₂ alkoxy, C₂-C₁₈ (hetero)aryloxy, C₂-C₁₈ (hetero)arylamino, C₂-C₁₂ carboxylic acid, C₂-C₁₂ carboxylate ester, and C₁-C₁₂ alkoxy, a functional group, a chemoselective functional group, conjugation tag, linked conjugation tag, E, a linker, sulfonic acid, sulfonate, C₁-C₁₂ alkyl sulfonate, and sulfonamide. In some cases, each R⁷ may independently be selected from the group consisting of H, hydroxyl, C₁-C₁₂ alkoxy, C₂- C₁₂ carboxylic acid, C₂-C₁₂ carboxylate ester, a functional group, a chemoselective functional group, conjugation tag, linked conjugation tag, and linked E.

[00203] At least one of R¹, R², R³, R³', R⁴, or R⁴' comprises an independently selected water-solubilizing moiety or linked water-solubilizing moiety. In some cases two or more, three or more, four or more, five or more, or six or more R¹, R², R³, R³', R⁴, or R⁴' comprise an independently selected water-solubilizing moiety or linked water- solubilizing moiety

[00204] Each Q may independently be selected from the group consisting of a bond, NH, NR⁴, C₁-C₁₂ alkylene, CHR⁴, and CH₂. In some cases, Q is NH or NR⁴.

[00205] Each Z may independently be CH₂, CHR⁴, O, NR⁴, or NH. In some cases, Z is O. In some cases, Z is CH₂ or CHR⁴. In some cases, each Q is independently NH or NR⁴ and each Z is independently O, CH₂, or CHR⁴.

[00206] Each W¹ may independently be a water-solubilizing moiety.

[00207] L¹, L², and L³ may each independently be selected linker moieties.

[00208] Each E may independently be selected from the group consisting of a chromophore, a functional moiety, a substrate, a conjugation tag, and a binding partner. [00209] Each f may independently be an integer from 0 to 50. In some cases, f is an integer in a range of 1 to 25, 3 to 20, 8 to 18, 10 to 15, or 10 to 11.

[00210] Each m may independently be 0 or 1.

[00211] Each n may independently be 0, 1, 2, 3, or 4.

[00212] Each s may independently be 1 or 2.

[00213] Each t may independently be 0, 1, 2, or 3.

[00214] In some cases, each X is independently C or Si. In some cases, each Y is independently C or Si. In some cases, both X and Y are C. [00215] In some cases, where the compound comprises a structure according to

Formula (I), each

and

is independently selected from the group consisting of a substituted or unsubstituted benzene, benzene derivative, monocyclic aryl group, polycyclic aryl group, monocyclic heteroaryl group, and polycyclic heteroaryl group; each R¹ is independently selected from the group consisting of sulfonamide-PEG, alkoxy sulfonic acid,

optionally each R² is hydrogen; optionally each X, Y and T is C; optionally each optional R³' is independently be selected from the group consisting of halogen, NH₂, C₁-C₆ alkyl, C₁-C₆ alkoxy, a PEG group, PEG carboxylic acid, alkyl amido PEG ester, alkyl amido PEG carboxylic acid, phenyl, or phenyl substituted with halogen, C₁-C₆ alkoxy, or CN,

optionally each R⁷ is independently be selected from the group consisting of H, hydroxyl, C₁-C₁₂ alkoxy, C₂-C₁₂ carboxylic acid, C₂-C₁₂ carboxylate ester, a functional group, a chemoselective functional group, conjugation tag, linked conjugation tag, and linked E; optionally each Q is NH or NR⁴; optionally each Z is O, CH₂ or CHR⁴; and each R³, R', R⁴, R⁴', W¹, L¹, L², L³, E, f, m, n, s, and t are as previously defined.

[00216] In some cases, where the compound comprises a structure according to

Formula (I), each

and

is independently selected from the group consisting of

wherein each

and

is independently optionally substituted with (R³ )n; optionally wherein each R³' is independently be selected from the group consisting of halogen, NH₂, C₁-C₆ alkyl, C₁-C₆ alkoxy, a PEG group, PEG carboxylic acid, alkyl amido PEG ester, alkyl amido PEG carboxylic acid, phenyl, or phenyl substituted with halogen, C₁-C₆ alkoxy, or CN,

, ,

, and p=1, 2,

3, 4, 5, 6, or 7; each R¹ is independently selected from the group consisting of sulfonamide-PEG, alkoxy sulfonic acid,

optionally each R² is hydrogen; optionally each X, Y and T is C; optionally each R⁷ is independently be selected from the group consisting of H, hydroxyl, C₁-C₁₂ alkoxy, C₂-C₁₂ carboxylic acid, C₂-C₁₂ carboxylate ester, a functional group, a chemoselective functional group, conjugation tag, linked conjugation tag, and linked E; optionally Q is NH or NR⁴; optionally Z is O; and each R³, R', R⁴, R⁴', W¹, L¹, L², L³, E, f , m, n, s, and t are as previously defined. In some cases,

and

are each independently selected from the group consisting of:

optionally wherein R² is C₁-C₆ alkyl, phenyl, or phenyl substituted with halogen, C₁-C₆ alkoxy, or CN.

[00217] The DHP bridged compounds of the present disclosure may be water-soluble. The DHP bridged compounds of the present disclosure may be fluorescent. The DHP bridged compounds of the present disclosure may be a fluorescent water-soluble dye. [00218] Methods of making DHP bridged compounds of the present disclosure are also provided.

[00219] An exemplary route of synthesis to obtain DHP bridged compounds of the disclosure is shown in FIG. 1, Scheme 1. DHP intermediate compound A may be prepared as follows. 2,7-dibromo-trans-9,10-dihydrophenanthrene-9,10-diol (DHP-OH) can be prepared by adding 2,7-dibromophenanthrene-9, 10-dione to a stirred mixture of water-ethanol and sodium borohydride, then acidification with dilute HC1 to obtain DHP- OH as a white precipitate. DHP-OH can be dissolved in THF with 18-crown-6 and NaH added under nitrogen. Then 1,3-propanesultone can be dissolved in THF and added dropwise to the DHP-OH solution and stirred for several hours. Solvents can be evaporated, and the precipitate dissolved in water. Acetone can be added to obtain a white precipitate of DHP-OSO₃H disodium salt which can be neutralized with HC1 and precipitated from acetone to obtain DHP-OSO₃H as a white solid. DHP-OSO₃H can be dissolved in DMF and thionyl chloride added and the mixture stirred overnight then poured into water to obtain DHP-OSO₂CI as a precipitate. DHP-OSO₂CI can be mixed with 2.2 equiv. of a PEG amine (e.g., f=l 5) in dichloromethane triethylamine mixture followed by column chromatography to obtain compound A. Intermediate compound B can be prepared in a similar fashion, except DHP-OSO₂CI can be mixed with 2.2 equiv. of a PEG amine (e.g., f=10).

[00220] Inventive fluorescent DHP bridged compounds such as compound 4 can be prepared as shown in Scheme 1 by dissolving DHP intermediate compound A and aryl boronic acid pinacol ester compound N,N-diphenyl-4-(4, 4,5, 5-tetramethyl-1, 3,2- dioxaborolan-2-yl)aniline in DMF, adding CsF dissolved in water and then Pd(OAc)₂, and stirring at elevated temperature overnight. The mixture may be filtered over celite and purified with column chromatography to obtain inventive DHP bridged compound 4. Inventive bridged DHP compounds 1, 2, 3, 5, 9, 10, 11, 12a may be prepared in an analogous fashion using either DHP intermediate compound A or DHP intermediate compound B and an alternative aryl boronic acid pinacol ester.

[00221] To improve water solubility of the DHP bridged compounds, PEGylated aryl boronic acid pinacol ester intermediates may be prepared as shown in FIG. 2, Scheme 2. A brominated hydroxyl aryl compound such as 4-bromo-2-fluorophenol can be treated with a tosylated-PEG-t-butyl ester to obtain brominated aryl PEG t-butyl ester compound 14 or analogous intermediate. In some cases, the brominated hydroxyl aryl compounds are commercially available, for example, from, e.g., SIGMA-ALDRICH. Tosylated- PEG-t-butyl ester are commercially available, for example, from, e.g., BROADPHARM®. The brominated aryl PEG t-butyl ester compound 14 or analogous intermediate may be treated with an acid such as TFA to cleave the t-butyl ester to obtain compound 15, or analogous compound which is then treated with bis(pinacolato)diboron to obtain PEGylated aryl boronic acid pinacol ester intermediates such as compound 16, or analogous intermediate compounds.

[00222] Inventive DHP bridged compounds such as compound 18 can be prepared as shown in FIG. 3, Scheme 3. DHP-OSO₃H (compound 17) can be treated with PEGylated aryl boronic acid pinacol ester intermediates such as compound 16 to obtain fluorescent compound 18. DHP bridged compound 19 may be prepared in an analogous fashion.

[00223] Inventive DHP bridged compounds such as compound 7 can be prepared as shown in FIG. 4, Scheme 4 by dissolving intermediate compound B and tert-butyl (4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate in DMF and adding CsF dissolved in water and then Pd(OAc)₂ and stirring at elevated temperature overnight. The mixture may be filtered over celite and purified with column chromatography to obtain Boc-protected amino aryl fluorescent compound 7b. The Boc protecting group may be cleaved with HC1 to provide amino aryl fluorescent compound 7.

[00224] Inventive DHP bridged compounds 8a-e can be prepared as shown in FIG. 5, Scheme 5 via a Suzuki coupling reaction between PEGylated DHP boronic acid ester 24 and Naphthalimide derivatives 23a-23d by stirring in DMF under N₂ and adding CsF dissolved in water and then Pd(OAc)₂ and stirring at elevated temperature overnight. The bridged DHP compounds 8a-e can be extracted from reaction mixture and purified by column chromatography.

Synthesis of DHP bridged dihydrophenanthrenyl compounds

[00225] Synthesis of inventive water-soluble small molecule DHP bridged dihydrophenanthrenyl (DHP) dyes can employ a Suzuki coupling reaction between a DHP-sulfonamide PEG, diboronic ester and a mono-bromo DHP-sulfonamide PEG, for example, as shown in FIG. 8, Scheme 8. A DHP-sulfonamide PEG, diboronic ester, such as compound 28 and a mono-bromo DHP-sulfonamide PEG such as compound 36 can be employed in the preparation of small molecule DHP bridged-sulfonamide PEG compound 20. DHP-sulfonamide PEG, diboronic esters, such as compound 28, can be prepared was prepared as shown in Scheme 9 from DHP-sulfonamide PEG compounds such as compound B. Briefly, dibromo-functionalized DHP-sulfonamide compound B can be mixed with DMSO under nitrogen with 3 equivalents of bispinacolatodiboron. Then potassium acetate and Pd(dppf)Cl₂ catalyst can be added and reaction mixture stirred for several hours at about 80 °C. The reaction mixture can then be cooled, extracted, and purified to provide the DHP-sulfonamide PEG, diboronic ester. Mono-bromo-DHP- sulfonamide PEG compounds, such as for example compound 36, can be prepared as illustrated in FIG. 7, Scheme 7.

[00226] Briefly, as shown in FIG. 7, commercially available phenanthrene, 9, 10-dione compound 31 can be reacted with N-bromosuccinimide using methane sulfonic acid to provide bromophenanthrene, 9, 10-dione compound 32. 2-bromo-trans-9,10- dihydrophenanthrene-9,10-diol (DHP-OH) compound 33 can be prepared by addition of NaBH4 in a water-ethanol mixture to 2 -bromophenanthrene, 9, 10-dione compound 32. 2- bromo-DHP-O(CH₂)₃-SO₃H compound 34 can be prepared from DHP-OH compound 33 using 18-crown-6 in THF and NaH followed by 1,3 propane sulfone to obtain 2-bromo 9,10 bis-oxypropane sulfonyl compound 34 in the form of disodium salt. 2-bromo-DHP- O(CH₂)₃-SO₂Cl compound 35 can be prepared from 2-bromo-DHP-SO₃H compound 34 in DMF with thionyl chloride SOCl₂. DHP-OSO₂CI compound 35 can be prepared by mixing for several hours with PEG amine in dichloromethane/TEA, extraction, and column chromatography to provide 2-bromo-DHP-sulfonamide PEG compound 36, as shown in FIG. 7.

[00227] DHP bridged-sulfonamide PEG compound 20 can be prepared as shown in FIG. 8, Scheme 8. Briefly, mono-bromo-DHP-sulfonamide PEG compound 36 and DHP- sulfonamide PEG, diboronic ester compound 28 can be dissolved in a DMF -water mixture under an inert gas, then treated with CsF and Pd(OAc)₂, mixed and heated. After the reaction, solvents can be removed via evaporation and the crude material purified by, e.g., column chromatography to provide DHP bridged-sulfonamide PEG compounds such as compound 20.

[00228] Amino-derivatives of DHP bridged-sulfonamide PEG compounds, such as for example, compounds 21 and 22, can be prepared as illustrated in FIG. 10, Scheme 10. Briefly, DHP bridged -sulfonamide PEG DHP compound, e.g., compound 20 can be dissolved in anhydrous DMF, and treated with cesium carbonate and tert-Butyl-3- iodopropyl-carbamate in a sealed flask and heated, cooled, worked-up in the usual fashion, and dried to provide an NHBoc amino DHP bridged compound, such as compound 21. The NHBoc amino DHP bridged compound 21 can be deprotected in a solvent using trifluoroacetic acid, TFA to provide an amino DHP bridged DHP compound such as compound 22, as shown in Scheme 10.

[00229] Tandem DHP bridged DHP dyes can be prepared by acceptor dye manufacturer's protocol, for example, as follows. An amino DHP bridged compound, such as compound 22, can be dissolved in anhydrous DMSO, treated with, e.g., acetonitrile and diisopropylethylamine followed by addition of solution of ~2 eq. acceptor dye NHS ester (e.g., a commercially available acceptor dye NHS ester, e.g., Dy495NHS; Dyomics GmbH) in anhydrous DMSO. After stirring at room temperature, and purification, to provide the tandem small molecule DHP bridged-acceptor dye.

Synthesis of DHP bridged fluorenyl compounds [00230] Inventive small molecule DHP bridged fluorenyl dyes can be prepared from one or more mono-bromo fluorenyl compounds and a DHP-sulfonamide PEG, diboronic ester compound via Suzuki coupling, for example, as shown in FIG. 12, Scheme 11. For example, inventive DHP bridged fluorenyl compound 30 can be prepared from a mono- bromo fluorenyl compound (e.g., compound 27) and a DHP-sulfonamide PEG, diboronic ester compound (e.g., compound 28). An optionally substituted 2-bromo-fluorene can be obtained commercially or prepared according to a literature protocol. For example, 3,3'- (2-bromo-9H-fluorene-9,9-diyl)dipropionic acid, compound 26, can be prepared as described from Chem. Comm. 2019, Vol. 55, 95, 14287-14290, incorporated herein in its entirety. In this case, the available carboxylic acid moieties of compound 26 can be exploited to obtain additional 2-bromo-fluorene compounds. For example, compound 26 can be solubilized, for example, in DMF/acetonitrile and treated with DIPEA, TSTU and an amino-PEG3-t-butyl ester compound to obtain for example, mono-bromo fluorenyl compound 27. The mono-bromo fluorenyl compound can be reacted with a DHP- sulfonamide PEG, diboronic ester via Suzuki coupling to provide DHP bridged fluorenyl compounds, such as compound 29, as shown in FIG. 12, Scheme 11.

[00231] The DHP bridged fluorenyl compounds, such as compound 29, can be dissolved in a solvent, e.g., DMF, treated with CS2CO3 and tert-butyl (3 -iodopropyl) carbamate, and the tert-butyl ester and tert-butyl carbamate groups deprotected to provide an amino-DHP bridged fluorenyl product, such as Compound 30, as shown in Scheme 11. [00232] Tandem small molecule DHP bridged fluorenyl dyes can be made from Compound 30.

[00233] Table 1 shows exemplary symmetric and unsymmetric DHP bridged compounds of the present disclosure along with their fluorescence properties including maximum excitation wavelength (Xex) and emission wavelengths (Xem).

[00234] Table 1. Exemplary DHP Bridged Compounds with Fluorescence Properties

IV. Binding Partners

[00235] A “binding partner” or “specific binding partner” of the present disclosure can be any molecule or complex of molecules capable of specifically binding to a target analyte. A binding partner of this disclosure includes, for example, proteins, small organic molecules, carbohydrates (including polysaccharides), oligonucleotides, polynucleotides, lipids, affinity ligand, antibody, antibody fragment, an aptamer and the like. In some cases, the binding partner is an antibody, antigen-binding fragment of an antibody, protein, peptide, affinity ligand, sugar, lipid, nucleic acid, or aptamer. In some embodiments, the binding partner is an antibody or fragment thereof. Specific binding in the context of the present disclosure refers to a binding reaction which is determinative of the presence of a target analyte in the presence of a heterogeneous population. Thus, under designated assay conditions, the specified binding partners bind preferentially to a particular protein or isoform of the particular protein and do not bind in a significant amount to other proteins or other isoforms present in the sample.

[00236] When the binding partners are antibodies, they may be monoclonal or polyclonal antibodies. The term antibody as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules. Such antibodies include, but are not limited to, polyclonal, monoclonal, mono-specific polyclonal antibodies, antibody mimics, chimeric, single chain, Fab, Fab' and F(ab')₂ fragments, Fv, and a Fab expression library.

[00237] In general, water-soluble fluorescent compounds of the present disclosure can be conjugated to binding partners to form a conjugated water-soluble fluorescent compound (also referred to as a “labeled specific binding partner”) using techniques known to those of skill in the art or using methods known in the art in combination with methods described herein.

[00238] A labeled specific binding partner is provided comprising a fluorescent compound according to the present disclosure; and a specific binding partner covalently linked to the fluorescent compound. The specific binding partner may be an antibody. The specific binding partner may be specific for a target analyte.

[00239] In some embodiments, fluorescent compounds of the present disclosure can be conjugated to binding partners, for example, a small molecule DHP bridged dye-antibody conjugate can be prepared according to the general scheme as shown in Scheme 6 (FIG. 6).

[00240] For example, preparation of DHP bridged compound NHS ester can proceed as follows. Using a clean vial, dissolve 5 mg of compound 19 in 1 mL dry CH₃CN. To this, add 15 mg N,N,N',N'- tetramethyl-O-(N-succinimidyl)uranium tetrafluoroborate (TSTU) and stir for 2 more minutes. To this, add 100 uL N,N-diisopropylethylamine (DIPEA) and continue stirring overnight with the cap sealed with parafilm. Later evaporate off the organic solvents in the reaction mixture. The residue is purified by adding 2 mL diethyl ether, vortex the mix and decant the solvent. This procedure is repeated 3-4 times. The purified NHS product is dried and dissolved in dry DMSO.

[00241] Conjugation of compound NHS with an antibody, e.g., anti-CD4 antibody can proceed as follows. To 0.6 mg of CD4, add 230 uL PBS lx and 51 uL NaHCO₃ buffer (pH=8.01) and the NHS compound as DMSO solution. Vortex quickly for 30 seconds and allow to mix for 3-4 hours in the coulter mix.

[00242] Purification of compound-antibody conjugate through Histrap HP column can proceed as follows. Approach 1 : After the crude reaction purify the conjugate using a Histrap HP column. Load the sample using 1×PBS buffer and collect the unbound fraction. This can be done using 20 CV of buffer. Later change the buffer to wash the bound fraction which has both conjugate and free antibody. This can be done using 1×PBS with 0.25M imidazole running for 10 CV.

[00243] Purification of compound-antibody conjugate through SEC column can proceed as follows. Load the crude conjugate containing free antibody to the Size Exclusion Column, using 1×PBS. Pool the tubes after checking the absorption spectra and concentrate in an Amicon Ultra-15 having a 30 KDa MWCO centrifugal concentrator. [00244] Purification of conjugate through an Anti-mouse anti-H+L antibody-agarose bead can proceed as follows. Mix crude compound-antibody conjugate mixture with anti- mouse anti-H+L antibody-agarose bead in a biological buffer having a pH between about 6 to about 8 for about 30 minutes at room temperature. The anti-mouse anti H+L antibody-agarose bead will bind to the compound antibody conjugate. Remove unreacted compound by washing with the above-mentioned biological buffer using a benchtop centrifuge with a speed of 300 g for 3 minutes. Repeat the washing process at least three times. To elute the compound-antibody conjugate, apply an IgG elution buffer with a pH ranging from about 2 to about 4 to the washed antibody-agarose bead and incubate for about 10 to 15 min. Centrifuge to collect the flow through that contains the compound- antibody conjugate.

[00245] Purification of the antibody conjugate can be achieved by spinning down the conjugation mix on a Zeba column using manufacturer procedure and by collecting the filtrate.

V. Tandem Dyes

[00246] The small molecule dyes of the present disclosure can be either a donor dye or an acceptor dye. The small molecule dyes and labeled specific binding partners of the present disclosure are capable of transferring energy to a linked acceptor chromophore or accepting energy from a linked donor chromophore. When the small molecule dyes and labeled specific binding partners of the disclosure are donor dyes, an acceptor chromophore can be covalently linked to a small molecule DHP bridged dye or labeled specific binding partner according to the disclosure in energy-receiving proximity such that excitation of the donor DHP bridged compound or labeled specific binding partner leads to energy transfer to, and emission from, the covalently attached acceptor signaling chromophore. When the small molecule dyes and labeled specific binding partners of the disclosure are acceptor dyes, they can be covalently linked to a donor chromophore in energy-receiving proximity such that excitation of the donor leads to energy transfer to, and emission from, the covalently attached acceptor signaling chromophore. Mechanisms for energy transfer between the small molecule dyes and labeled specific binding partners of the present disclosure and a linked donor or acceptor chromophore include, for example, resonant energy transfer (e.g., Forster (or fluorescence) resonant energy transfer, FRET), quantum charge exchange (Dexter energy transfer) and the like. In some cases, the “chromophore” may be a “fhiorophore”. In some cases, the “chromophore” may be an acceptor dye. In some cases, the “chromophore” may be a donor dye.

[00247] As such, the present disclosure provides tandem dyes, comprising a fluorescent compound or labeled specific binding partner according to the disclosure, and an acceptor chromophore or a donor chromophore covalently linked to the fluorescent compound or labeled specific binding partner. In some embodiments, the fluorescent compounds of the disclosure, and conjugates thereof, are donor dyes. In some cases, the fluorescent compounds of the disclosure, and conjugates thereof, are acceptor dyes. In some cases, the fluorescent compounds of the disclosure, and conjugates thereof, include additional acceptor dye(s) (e.g., fluorophores or chromophores) attached to a donor dye having a structure of the disclosure, or additional donor dye(s) (e.g., fluorophores or chromophores) attached to an acceptor dye having a structure of the disclosure.

[00248] When a light source excites the donor compound, the fluorophores (FP), acceptor dyes or chromophores can absorb energy of an appropriate wavelength and emit or transfer energy. When the small molecule dye of the disclosure is a donor, the acceptor dye linked to the fluorescent dyes of the disclosure may have an absorption profile with a degree of overlap with the emission profile of the small molecule DHP bridged compounds of the disclosure. When the small molecule dye of the invention is an acceptor dye, the fluorescent dyes of the invention have an absorption profile with a degree of overlap with the emission profile of the donor dye. The compounds of the disclosure may have an absorption maximum longer than 300 nm, and emission maximum longer than 350 nm, and optionally may exhibit with fluorescence quantum yield larger than 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 10%.

[00249] Any convenient fluorescent dyes may be utilized in the tandem dyes as an acceptor chromophore or donor chromophore. The chromophores and fluorophores may be selected from coumarins, fluoresceins, rhodamines, cyanines (Cy), bodipys, or other polycyclic aromatics. Many fluorophores are commercially available and may be selected from but are not limited to, for example, any dye available from Beckman Coulter, Inc., including, but not limited to, SuperNova polymer dyes, Chrome Orange; any dye available from Becton Dickinson Biosciences, including, but not limited to, BD Horizon Brilliant™ polymer dyes; any dye available from Thermo Fisher Scientific or Invitrogen™, a part of Thermo Fisher Scientific, including, but not limited to, Super Bright™ polymer dyes, eFluor® dyes, Pacific dyes, NovaFluor dyes, Brilliant dyes, Alexa Fluor (AF) dyes, including, but not limited to, 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, Phycoerythrin (PE), Allophycocyanin (APC), Alexa Fluor 430, Fluorescein (FITC), DDAO, 1,1'-Dioctadecyl-3,3,3',3'- Tetramethylindodicarbocyanine (DiD), DyLight™ 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 755, DyLight 800, DsRed, 5- (4,6-Dichlorotriazinyl) Aminofluorescein (DTAF), SYTOX ™Blue, SYTOX Green, SYTOX Orange, 5-carboxy-2,7-dichlorofluorescein, 5-Carboxyfluorescein (5- FAM), 5-Carboxynapthofluorescein, 5-Carboxytetramethylrhodamine (5-TAMRA), 5- FAM (5-Carboxyfluorescein), 5-Carboxy-X-Rhodamine (5-ROX), 5- Carboxytetramethylrhodamine (6-TAMRA), 6-Carboxyrhodamine 6G, 6-Carboxy-X- Rhodamine (6-CR 6G), 6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein (6-JOE), 6- Carboxyfluorescein (6-FAM), 6-Carboxy-X-Rhodamine (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, Bodipy 665/676, Bodipy Fl, Bodipy R6G, Bodipy TMR, Bodipy TR, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, 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, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, Erythrosin, Fluo-3, Fluo-4; any dye available from ATTO-Tec, including, but not limited to ATTO 390, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 532, ATTO 550, ATTO 565, ATTO 590, ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740; any dye available from Biotium, including, but not limited to CF® 488A, CF 555, CF 568, CF 594ST, CF 633, CF 640R, CF 647, CF 660C, CF 680, CF680R, CF 750, CF 770, CF 790, CF 820, CF 850, CF 870; CL- NERF, CMFDA, 4-(4-Dihexadecylaminostyryl)-N-methylpyridinium iodide) (DiA), 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine (Dil), 3,3'- Dioctadecyloxacarbocyanine Perchlorate (DiO), 1,1'- Dioctadecyl-3,3,3',3'-

T etram ethyl indotri carbocyanine Iodide (DiR); any dye available from Millipore Sigma, including, but not limited to Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7; any dye available from AAT Bioquest, including, but not limited to DM-NERF, Tide Fluor 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), iFluor® 488, iFluor 555, iFluor 594, iFluor 647, iFluor 680, iFluor 700, iFluor 750, iFluor 780; any dye available from Dyomics, including, but not limited to DY-490, DY-495, DY-505, DY-526XL, 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-778, DY-780, DY-781, DY- 782, DY-800, DY-831; any dye available from Miltenyi Biotec, including, but not limited to all Vio® dyes; any dye available from BioRad, including, but not limited to all StarBright dyes; any dye available from AAT Bioquest, including, but not limited to all iFluor® dyes; any dye available from Ray Biotech, including but not limited to, all RayBright dyes; any dye available from BioLegend, including, but not limited to all Spark dyes; any dye available from Sony Biotechnology Inc, including, but not limited to all Kiravia™ dyes; all dyes available from Cytek, including, but not limited to all cFluor™ dyes and StarBright™ dyes; Eosin, Fluor-Ruby, FluorX, FM 1-43, FM 1- 46, Lyso Tracker Green, Lyso Tracker Yellow, Mitotracker Green, Mitotracker Orange, Mitotracker Red, NBD, Oregon Green 488, Oregon Green 514, PKH26, PKH67, Resorufin, RH 414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine 6G, Rhodamine B, Rhodamine Green, Rhodamine Red, Rose Bengal, Spectrum Green, Spectrum Orange, Spectrum Red, Texas Red, TRITC, and XTRITC. Acceptor dyes useful in the disclosure may include, for example, any of the previously listed dyes, a cyanine (Cy) dye, a xanthene dye, a coumarin dye, a thiazine dye, an acridine dye, FITC, CY3B, Cy55, Alexa 488, Texas red, Cy5, Cy7, Alexa 750, Cy55, Cy3B, Cy3.5, Alexa 750, 800 CW, Biotium CF 555, diethyl coumarin, DY705 (Dyomics), DY431, DY485XL, DY500XL, DY610, DY640, DY654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831 and 800CW. The acceptor dye may be a pendant acceptor dye. The tandem dye may comprise one or more, two or more, three or more, or four or more small molecule DHP bridged dyes according to the present disclosure comprising one or more, two or more, acceptor dye moieties. The tandem dye may comprise a donor dye and one or more, two or more, three or more, 1-30, 2-20, or 2.5-10 small molecule DHP bridged acceptor dye compounds according to the present disclosure.

[00250] Fluorescent tandem dyes can be prepared using techniques known to those of skill in the art or using methods known in the art in combination with methods described herein. In some cases, tandem dyes of the DHP bridged compounds can be prepared with a commercially available NHS ester of an acceptor dye according to manufacturer's protocols. The tandem dyes can be water-soluble.

[00251] In some embodiments, instead of being attached directly to the compound acceptor dyes, chromophores, fluorophores, functional moieties, and binding partners can be attached to the compounds of the present disclosure through a linker moiety using the method of direct modification as shown in FIG. 6, Scheme 6.

[00252] In some embodiments, the present disclosure provides a tandem dye comprising: a fluorescent compound or labeled specific binding partner according to the disclosure; and a donor or an acceptor chromophore covalently linked to the fluorescent compound or labeled specific binding partner.

[00253] Table 2 shows normalized emission of UV DHP bridged fluorenyl tandem conjugates 38-46 upon excitation at 355 nm in PBS lx. The emission max for UV - conjugate compound 37 without acceptor dye was 415 nm. The emission maxima range for tandem conjugate compounds ranged from 538 nm to 925 nm. Thus, a single small molecule DHP bridged dye according to the disclosure can be used to prepare several different tandem dyes and antibody conjugates in which a single excitation wavelength at

355 nm can result in multiple emission wavelengths in a range of from 415 nm to 925 nm.

VL Methods of Detecting an Analyte

[00254] The present disclosure provides methods for detecting a target analyte in a sample, the method comprising: providing a sample that is suspected of containing a target analyte; and contacting the sample with a specific binding partner conjugated to a fluorescent compound or tandem dye of the present disclosure, wherein the binding partner is capable of specifically interacting with the target analyte.

[00255] A light source is applied to the sample that can excite the fluorescent compound, labeled specific binding partner or tandem dye according to the disclosure; and light emitted from the conjugated fluorescent compound is detected. In the typical assay, water-soluble fluorescent compounds, labeled specific binding partners or tandem dyes of the present disclosure are excitable with a light having wavelength between about 300 nm to about 880 nm and the emitted light is typically between about 300 to about 930.

[00256] Alternatively, excitation light can have a wavelength between about 300 nm to about 600 nm, about 300 to about 500, or about 300 to about 420 nm and the emitted light can have a wavelength between about 300 to about 600, or about 350 nm to about 550 nm. The excitation and emitted light wavelengths will be dependent upon the

and

groups attached to the DHP moiety. The fluorescent compounds of the present disclosure may have an excitation spectrum tuned to the UV or another laser depending on design of the compound.

[00257] In the method of the present disclosure, the small molecule fluorescent compound can be any water-soluble fluorescent compound of the present disclosure as disclosed herein. [00258] A method is provided for detecting a target analyte in a sample comprising: providing a sample that is suspected of containing the analyte; and contacting the sample with a specific binding partner conjugated to a fluorescent compound or tandem dye according to the disclosure, wherein the binding partner is capable of interacting with the target analyte. The binding partner may be a protein, peptide, affinity ligand, antibody, antibody fragment, carbohydrate, lipid, nucleic acid, or an aptamer. When the binding partner is an antibody, the method may be configured for flow cytometry; the water- soluble fluorescent compound may be bound to a substrate; the analyte may be a protein expressed on a cell surface; the method may be configured as an immunoassay; or the method may further comprise providing additional specific binding partners for detecting additional analytes simultaneously.

Sample

[00259] The sample in the methods of the present disclosure can be, for example, blood, bone marrow, spleen cells, lymph cells, bone marrow aspirates (or any cells obtained from bone marrow), urine (lavage), serum, saliva, cerebral spinal fluid, urine, amniotic fluid, interstitial fluid, feces, mucus, or tissue (e.g., tumor samples, disaggregated tissue, disaggregated solid tumor). In certain embodiments, the sample is a blood sample. In some embodiments, the blood sample is whole blood. The whole blood can be obtained from the subject using standard clinical procedures. In some embodiments, the sample is a subset of one or more cells of whole blood (e.g., erythrocyte, leukocyte, lymphocyte (e.g., T cells, B cells orNK cells), phagocyte, monocyte, macrophage, granulocyte, basophil, neutrophil, eosinophil, platelet, or any cell with one or more detectable markers). In some embodiments, the sample can be from a cell culture.

[00260] The subject can be a human (e.g., a patient suffering from a disease), a commercially significant mammal, including, for example, a monkey, cow, or horse. Samples can also be obtained from household pets, including, for example, a dog or cat. In some embodiments, the subject is a laboratory animal used as an animal model of disease or for drug screening, for example, a mouse, a rat, a rabbit, or guinea pig.

Analyte

[00261] An “analyte” or “target analyte” as used herein, refers to a substance, e.g., molecule, whose presence and/or abundance/concentration is determined by some analytical procedure. For example, in the present disclosure, an analyte can be a protein, peptide, nucleic acid, lipid, carbohydrate small molecule, or a target-associated biomolecule.

[00262] The target analyte may 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, a structural microfilament, microtubule, and intermediate filament proteins, organelle- specific markers, proteasomes, transmembrane proteins, surface receptors, nuclear pore proteins, protein/peptide translocases, protein folding chaperones, signaling scaffolds, ion channels and the like. The protein can be an activatable protein or a protein differentially expressed or activated in diseased or aberrant cells, including but not limited to transcription factors, DNA and/or RNA-binding and modifying proteins, nuclear import and export receptors, regulators of apoptosis or survival and the like.

[00263] The compounds, labeled specific binding partner, or tandem dye according to the present disclosure may be a water-soluble fluorescent dye. The fluorescent compound, labeled specific binding partner, or tandem dye according to the present disclosure may exhibit 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, >40 mg/mL, >50 mg/mL, > 80 mg/mL, or >100 mg/mL.

[00264] The compounds, labeled specific binding partner, or tandem dye according to the present disclosure may exhibit a maximum excitation wavelength (Xex) of >300 nm, >400 nm, >500 nm, >600 nm, >700 nm, >800 nm, >850 nm, or within a range of between about 300 nm to about 1,000 nm, about 300 nm to about 880 nm, about 300 nm to about 600 nm, about 300 to about 500, or about 300 to about 420 nm. The DHP bridged compounds, labeled specific binding partner comprising a DHP bridged compound or tandem dye comprising a DHP bridged compound according to the present disclosure may exhibit an emission maxima (Xem) of >350 nm, > 450 nm, >550 nm, >650, >750 or within a range of between about 300 to about 880, about 300 to about 600, or about 350 nm to about 550 nm.

Assays [00265] The DHP bridged dyes, tandem dyes, labeled specific binding partners, compositions, methods and systems as described herein may find use in a variety of applications, including diagnostic and research applications, in which the labelling, detection and/or analysis of a target of interest is desirable. Such applications include methodologies such as, for example, cytometry, microscopy, immunoassays (e.g. competitive or non-competitive), fluorescence in situ hybridization (FISH), cell tracing, receptor labeling, fluorescence spectroscopy, assessment of a free analyte, assessment of receptor bound ligand, and so forth. The compositions, system and methods described herein may be usefill in analysis of any of a number of samples, including but not limited to, biological fluids, cell culture samples, and tissue samples. In certain aspects, the compositions, system and methods described herein may find use in methods where analytes are detected in a sample, if present, using fluorescent labels, such as in fluorescent activated cell sorting or analysis, immunoassays, immunostaining, and the like. In certain instances, the compositions and methods find use in applications where the evaluation of a sample for the presence of a target analyte is of interest. In some cases, the methods and compositions find use in any assay format where the detection and/or analysis of a target from a sample is of interest, including but not limited to, flow cytometry, fluorescence microscopy, in-situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separation assays and fluorochrome purification chromatography. In certain instances, the methods and compositions find use in any application where the fluorescent labelling of a target molecule is of interest. The subject compositions may be adapted for use in any convenient applications where pairs of specific binding members find use, such as biotin- streptavidin and hapten-anti-hapten antibody.

[00266] Assay systems utilizing a binding partner and a fluorescent label to quantify bound molecules are well known. Examples of such systems include flow cytometers, scanning cytometers, imaging cytometers, fluorescence microscopes, and confocal fluorescent microscopes.

[00267] In some embodiments, flow cytometry is used to detect fluorescence. A number of devices suitable for this use are available and known to those skilled in the art.

Examples include Beckman Coulter Navios, Gallios, Aquios, and CytoFLEX flow cytometers. [00268] In other embodiments, an assay is used. The assay can be an immunoassay. Examples of immunoassays useful in the disclosure include, but are not limited to, fluoroluminescence assay (FLA), and the like. The assays can also be carried out on protein arrays. When the binding partners are antibodies, antibody or multiple antibody sandwich assays can also be used. A sandwich assay refers to the use of successive recognition events to build up layers of various binding partners and reporting elements to signal the presence of a particular analyte. Examples of sandwich assays are disclosed in U.S. Pat. No. 4,486,530 and in the references noted therein.

VII.KITS

[00269] The disclosure provides a kit comprising at least one DHP bridged compound, labeled specific binding partner, or tandem dye according to the present disclosure.

Aspects of the invention further include kits for use in practicing the subject methods and compositions. The compositions of the invention can be included as reagents in kits either as starting materials or provided for use in, for example, the methodologies described above.

[00270] A kit can include a DHP bridged compound, labeled specific binding partner, or tandem dye as described herein and a container. Any convenient containers can be utilized, such as tubes, bottles, or wells in a multi-well strip or plate, a box, a bag, an insulated container, and the like. In some instances, the subject kits can include one or more components selected from a DHP bridged compound, labeled specific binding partner, or tandem dye according to the present disclosure, a fluorophore, a chromophore, a specific binding member, a specific binding member conjugate, a support bound specific binding member, a cell, a support, a biocompatible aqueous elution buffer, and/or instructions for use. In some embodiments of the kit, the DHP bridged compound, or tandem dye according to the present disclosure is covalently linked to a specific binding partner. The kit can include a sheet of instructions.

[00271] In some instances, the subject kits can be “labeling kits” that include a DHP bridged compound or tandem dye according to the present disclosure comprising a sidechain chemoselective functional group (also referred to as a “conjugation tag”) such as, for example, a NHS ester of a DHP bridged compound and the like, to which any convenient target moiety of interest (e.g., a donor or acceptor dye, fluorophore, chromophore, a specific binding partner, a support) can be conjugated. The chemoselective functional group may include a reactive group (e.g., biotin) that targets specific functional groups on biomolecules (e.g., proteins or antibodies), such as, for example, primary amines, sulfhydryls, carboxyls, or carbohydrates. The chemoselective functional group (“conjugation tag”) can be one used in “click chemistry” reactions.

[00272] In certain instances, the conjugation tag includes a maleimide functional group and the target moiety includes a thiol functional group, or vice versa. In some instances, the conjugation tag includes an alkyne functional group (e.g., a cyclooctyne group) and the target moiety includes an azide functional group, or vice versa, which can be conjugated via Click chemistry. In certain instances, the conjugation tag includes an alkene functional group (e.g., a cyclooctene group) and the target moiety includes a tetrazine functional group, or vice versa, which can be conjugated via inverse-demand Diels-Alder cycloaddition reaction. In some instances, the conjugation tag includes an amine-reactive chemical group, such as, for example, a NHS ester (N-hydroxysuccinimde esters) or imidoester functional group and the target moiety includes a NH₂ functional group, or vice versa. In some instances, the conjugation tag includes a biotin-binding protein (e.g., Avidin, Streptavidin, or NeutrAvidin) and the target moiety includes a biotin molecule, or vice versa, which can non-covalently interact.

EXAMPLES

Example 1. Synthesis of Compound A

[00273] Compound A was prepared in an analogous fashion to that described in WO 2017/180998.

[00274] 2, 7-dibromo-trans-9, 10-dihydrophenanthrene-9, 10-diol (DHP-OH) was prepared as follows. In a conical flask (2000 L), 26 g of NaBH₄ was added into a stirring water-ethanol mixture (120 mL+780 mL). To this solution, about 24 g of 2,7- dibromophenanthrene,9, 10-dione was added portion-wise quickly (in 5 min). The reaction mix was stirred for a day. The color of the solution changed from orange red to pale yellow to white by the end of the reaction. The reaction was stopped, and the reaction mixture neutralized with dilute HC1 acid. After the neutralization, the white precipitate was filtered and washed with excess water. The white precipitate (DHP-OH) was washed with very cold (<-15° C.) ethanol (100 mL) and methanol (100 mL).

[00275] 3, 3 ’-((2, 7-dibromo-9, 10-dihydrophenanthrene-9, 10-diyl)bis(oxy))bis(propane-

1-sulfonic acid) (DHP-OpropylSO₃H) (compound 17) was prepared as follows. In a 2 neck, round bottom flask, DHP-OH (3.6 g) and 18C₆ (500 mg) were dissolved in 120 mL of THF. The solution was purged with nitrogen (20 min) and NaH (2 g) was added while nitrogen purging continued. The color of the solution changed from colorless to pale pink, dark pink, brown and dark green in 10-15 min. In another round bottom flask, 12 g of 1,3 propane sulfone was dissolved in 20 mL of THE and nitrogen purged. This sulfone solution was added to the DHP-OH solution by addition funnel over a period of 20-30 minutes. The reaction was stirred at room temperature for 4-5 hrs. The solvents were evaporated and the precipitate dissolved in water. Acetone was added to obtain white precipitate of DHP-OSO₃H in the form of disodium salt. Filter the precipitate and redissolve in water (minimal amount) neutralize with HC1 and precipitate again in acetone. Repeated precipitation (2-3 times) followed by centrifugation gives DHP- OSO₃H as white solid.

[00276] DHP-OSO₂Cl was prepared as follows. 5 g of DHP-OSO₃H was taken in a round bottom flask and mixed with 25 mL of DMF. To this about 10 mL of SOCI₂ was added dropwise and the mixture allowed to stir for overnight. Next morning, reaction mixture was poured into 200 mL water and precipitate was filtered and dried.

[00277] DHP-OSO₂CI was mixed with 2.2 equivalent of PEG amine (f= 15) in dichloromethane/TEA mixture. After 3 h sonication reaction the crude product was extracted in dichloromethane followed by column chromatography (silica gel, MeOH- CHCl₃) to collect to obtain purified compound A.

Example 2. Synthesis of Compound B

[00278] Compound B was prepared in the same manner as Compound A but PEG amine (f= 10) was used instead of PEG amine (f= 15).

Example 3. Synthesis of Compound 4

[00279] Compound 4 was prepared as shown in FIG. 1, Scheme 1. In a flask compound A (217 mg, 0.1 mmoles) andN,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)aniline (93 mg, 0.25 mmoles) were dissolved in 3.9 mL of DMF. To the mixture CsF (380mg, 2.5 mmoles) dissolved in 1.3 mL of H₂O was added. The mixture was purged with N₂ for 5 minute and then Pd(OAc)₂ (2.7 mg, 0.012 mmoles) was added. The flask was sealed, and mixture stirred at 80 °C for 13 hours. After that the reaction mix was filtered over celite and washed with 1/1 CHCl₃/MeOH. The filtrate was collected, concentrated and purified with an automated chromatography system using neutral Silica as stationary phase and CHCl₃/MeOH as mobile phase. Product 4, 160 mg (64%) was collected as yellow waxy material with abs max at 381 nm and emission max at 451 nm in PBS.

[00280]¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 1.98 (m, 4H), 2.91 (m, 4H), 3.08 (t, J= 4.7 Hz, 4H),3.37 (s, 6H), 3.42(t, J= 3.3 Hz, 4H), 3.47-3.76 (m, 137H), 4.55 (s, 2H), 7.14-7.16 (m, 12H), 7.28-7.30 (m, 8H), 7.54-7.57 (m, 6H), 7.63-7.65 (m, 2H), 7.86-7.88 (d, J=8 Hz, 2H).

Example 4. Synthesis of Compound 3

[00281] Compound 3 was prepared following a similar experimental protocol as Compound 4 but 2-(9,9-dimethyl-9H-fluoren-2-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane was used in place of N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)aniline. Product 3, 109 mg (52%) was collected as waxy material with abs max at 351 nm and emission max at 429 nm in PBS.

[00282]¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 1.60-1.61 (d, J= 3.6 Hz, 12H), 2.03 (m, 4H), 2.98 (m, 4H), 3.11 (t, J= 3.7 Hz, 4H), 3.40 (s, 6H), 3.42(m, 4H), 3.47-3.85 (m, 127H), 4.66 (s, 2H), 7.35-7.41 (m, 4H ), 7.50 (d, , J= 6.3 Hz, 2H), 7.68-7.70 (m, 4H), 7.76-7.80 (m, 6H), 7.85 (d, J= 7.9 Hz, 2H), 7.98 (d, J=7.8 Hz, 2H).

Example 5. Synthesis of Compound 1

[00283] Compound 1 was prepared following a similar experimental protocol as Compound 4 but 2-(3,5-difluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was used in place of N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline and Compound B was used in place of Compound A. Product 1, 152mg (79 %) was collected as waxy material with abs max at 321 nm and emission max at 378 nm in PBS.

[00284]¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 1.98-2.04 (m, 4H), 2.89-3.00 (m, 4H), 3.13 (t, J= 4.6 Hz, 4H), 3.38 (s, 6H), 3.48 (t, J= 4.2 Hz 4H), 3.54-3.77 (m, 94H), 4.58 (s, 2H), 6.81-6.84 (m, 2H ), 7.20 (d, , J= 5.8 Hz, 2H), 7.58 (d, , J= 2.5 Hz, 2H), 7.64-7.66 (dd, J¹ = 1.4 Hz, J² = 7.5 Hz 2H), 7.92 (d, J= 7.5 Hz, 2H). Example 6. Synthesis of Compound 2

[00285] Compound 2 was prepared following a similar experimental protocol as Compound 4 but 4,4,5,5-tetramethyl-2-(naphthalen-1-yl)-1,3,2-dioxaborolane was used in place of N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline. Product 2, 66 mg (32%) was collected as waxy material with abs max at 322 nm and emission max at 395 nm in PBS.

[00286]¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 1.99-2.09 (m, 4H), 2.88-3.04 (m, 4H), 3.13 (m, 4H), 3.37 (s, 6H), 3.39-3.83 (m, 115H), 4.62 (s, 2H), 7.47-7.63 (m, 12H), 7.90 (d, , J= 7.6 Hz, 2H), 7.94 (d, J= 7.2 Hz, 2H), 8.00 (d, J= 8.9 Hz 2H).

Example 7. Synthesis of Compound 6

[00287] Compound 6 was prepared following a similar experimental protocol as Compound 4 but (4-fluorophenyl) boronic acid was used in place ofN,N-diphenyl-4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline. Product 6, 220 mg (86%) was collected as waxy material with abs max at 320 nm and emission max at 378 nm in PBS. [00288]¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 1.97-2.02 (m, 4H), 2.91-2.96 (m, 4H), 3.09 (t, J= 5.2 Hz, 4H), 3.37 (s, 6H), 3.43 (t, J= 3.6 Hz 4H), 3.49-3.80 (m, 127H), 4.57 (s, 2H), 7.16 (d, J= 8.9 Hz, 4H), 7.5-7.57 (m, 2H), 7.61-7.65 (m, 6H), 7.89 (d, J= 8.2 Hz, 2H).

Example 8. Synthesis of Compound 5

[00289] Compound 5 was prepared following a similar experimental protocol as Compound 4 but 4,4,5,5-tetramethyl-2-(naphthalen-2-yl)-1,3,2-dioxaborolane was used in place of N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline. Product 5, 270 mg (99%) was collected as waxy material with abs max at 330nm and emission max at 405nm in PBS.

Example 9. Synthesis of Compound 9

[00290] Compound 9 was prepared following a similar experimental protocol as Compound 4 but 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazole was used in place of N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline. Product 9, 115 mg (48%) was collected as waxy material with abs max at 322 nm and emission max at 381 nm in PBS.

[00291]¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 2.01-2.06 (m, 4H), 2.92-3.03 (m, 4H), 3.11-3.13 (m, 4H), 3.37 (s, 6H), 3.43-3.90 (m, 133H), 4.61 (s, 2H), 7.67-7.74 (m, 8H), 7.94 (d, J= 7.3 Hz, 2H), 8.07 (s, 2H), 8.19 (s, 2H).

Example 10. Synthesis of Compound 10

[00292] Compound 10 was prepared following a similar experimental protocol as Compound 4 but 2-(3-fluoro-4-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was used in place of N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline and Compound B was used in place of Compound A. Product 10, 148mg (95 %) was collected as waxy material with abs max at 321 nm and emission max at 395 nm in PBS.

[00293]¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃)

1.93-2.05 (m, 4H), 2.88-2.99 (m, 4H), 2.99-3.10 (m, 4H), 3.37 (s, 6H), 3.44 (t, J= 4.9 Hz, 4H), 3.49-3.81 (m, 93H), 3.95 (s, 6H), 4.56 (s, 2H), 7.07 (t, J= 9.2 Hz, 2H), 7.40-7.43 (m, 4H), 7.55 (d, J= 1.4 Hz, 2H), 7.60 (dd, J¹ = 7.9 Hz, J² = 1.8 Hz ,2H), 7.88 (d, J= 8.2 Hz, 2H). Example 11. Synthesis of Compound 11

[00294] Compound 11 was prepared following a similar experimental protocol as Compound 4 but 2-(2-fluoro-4-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was used in place of N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline and Compound B was used in place of Compound A. Product 11, 145mg (78 %) was collected as waxy material with abs max at 312 nm and emission max at 386 nm in PBS. [00295] .¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 1.92-2.05 (m, 4H), 2.89-3.00 (m, 4H), 2.99-3.10 (m, 4H), 3.37 (s, 6H), 3.43 (m, 4H), 3.46-3.80 (m ,114H), 3.86 (s, 6H), 4.54 (s, 2H), 6.72 (dd, J¹ = 12.6 Hz, J² = 2.5 Hz ,2H), 6.72 (dd, J¹ = 8.5 Hz, J² = 2.7 Hz ,2H), 7.45 (t, J= 9.2 Hz, 2H), 7.51-7.62 (m, 4H), 7.87 (d, J= 8.4 Hz, 2H).

Example 12. Synthesis of Compound 12a

[00296] Compound 12a was prepared following a similar experimental protocol as Compound 4 but 2-(3,5-difluoro-2-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane was used in place of N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)aniline and Compound B was used in place of Compound A. Product 12a, 204mg (78 %) was collected as waxy material.¹H-NMR spectra confirmed the monosubstituted structure.¹HNMR (500 MHz, CDCl₃) 1.98-2.08 (m, 4H), 2.90-3.03 (m, 4H), 3.15-3.19 (m, 4H), 3.37 (s, 6H), 3.48-3.85 (m ,104H), 4.42 (s, 1H), 4.56 (s, 1H), 6.86-6.91 (m, 1H), 6.96-6.98 (m, 1H), 7.51-7.55 (m, 2H), 7.60-7.62 (m, 3H), 7.90 (d, J= 8.5 Hz, 1H).

Example 13. Synthesis of Compound 12

[00297] In a flask compound 12a (135 mg, 0.078 mmoles) and N,N-diphenyl-4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (35 mg, 0.093 mmoles) were dissolved in 2 mL of DMF. To the mixture CsF (236 mg, 1.55 mmoles) dissolved in 0.7 mL of H₂O was added. The mixture was purged with N₂ for 5 minute and then Pd(OAc)₂ (1.8 mg, 0.008 mmoles) was added. The flask was sealed, and mixture stirred at 80 °C for 13 hours. After that the reaction mix was filtered over celite and washed with 1/1 CHCl₃/MeOH. The filtrate was collected, concentrated and purified with an automated chromatography system using neutral Silica as stationary phase and CHCl₃/MeOH as mobile phase. Product 12, 130 mg (88%) was collected as yellow waxy material with abs max at 380 nm and emission max at 451 nm in PBS.

[00298] .¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 1.98-2.08 (m, 4H), 2.87-3.03 (m, 4H), 3.07-3.21 (m, 4H), 3.38 (s, 6H), 3.41-3.86 (m ,108H), 4.55-4.57 (m, 2H), 6.85-6.92 (m, 1H), 6.95-6.99 (m, 1H), 7.90 (t, J= 7.9 Hz, 1H), 7.13-7.17 (m, 4H), 7.26-7.31 (m, 7H), 7.52-7.66 (m, 6H), 7.86-7.92 (m, 2H).

Example 14. Synthesis of Compounds 14-16

Synthesis of Compound 14

[00299] Compounds 14 -16 were prepared as shown in FIG. 2, Scheme 2. In a sealed flask 4-bromo-2-fluorophenol (126 mg, 0.66 mmoles), tert-butyl 3-(2-(2-(2- (tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (BroadPharm BP20636) (260mg, 0.6 mmoles) were dissolved in 8 mL of MeCN. To the mixture K₂CO₃ (331 mg, 2.4 mmoles) was added. The mixture was purged with N₂ for 5 minutes, sealed, and stirred at 70 °C for 15 hours. After that the reaction mix was dried, the residue dissolved inj chloroform and washed with diluted brine. The organic layer was collected, filtered over Na₂SO₄ and concentrated to dryness to afford Product 14, 260 mg (96%).¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 1.44 (s, 9H), 2.49 (t, J = 6.5 Hz, 2H), 3.59-3.67 (m, 6H), 3.69-3.87 (m, 4H), 3.84-3.87 (m, 2H), 4.16-4.18 (m, 2H), 6.88 (t, J= 8.8 Hz, 1H), 7.17 (d, J= 8.5 Hz , 1H), 7.22 (d, J= 9.8 Hz , 1H).

Synthesis of Compound 15

[00300] Compound 14 (0.24g, 0.53 mmoles) was dissolved in 5 mL of dichloromethane and TFA (0.5 mL) added. The mixture was stirred at room temperature for 1 hour. After that the reaction mix was dried, the residue dissolved in dichloromethane and washed with diluted brine. The organic layer was collected, filtered over Na₂SO₄ and concentrated to dryness to afford Product 15, 230 mg (96%).¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 2.64 (t, J= 5.9 Hz, 2H), 3.64-3.69 (m, 6H), 3.72-3.74 (m, 2H), 3.77 (t, J= 6.2 Hz, 2H), 3.87 (t, J= 5.0 Hz, 2H), 3.77 (t, J= 4.5 Hz, 2H), 6.88 (t, J= 8.5 Hz, 1H), 7.17 (d, J= 8.4 Hz , 1H), 7.22 (dd, J¹⁼ 2.2 Hz , J²= 10.3 Hz, 1H).

Synthesis of Compound 16

[00301] Compound 15 (239g, 0.606 mmoles) and bis(pinacolato) diboron (215 mg, 0.85 mmoles) were dissolved in 1 mL DMSO. The mixture was purged with N₂ for 15 minutes then KO Ac (356 mg, 3.64 mmoles) was introduced into the reaction mixture. After purging for 5 minutes Pd(dppf)Cl2 (19 mg, 0.026 mmoles) was added. The reaction mix was left to stir at 90 °C for 12h under N₂. Then the mix was cooled down to room temperature, diluted with CHCl₃ (50 mL) and washed with H₂O (3x 60 mL). The organic fraction was collected and filtered over a celite pad. The solvent was evaporated to afford a dark oily product. To the collected material 10 mL of Hexanes were added and the mixture sonicated for 2 minutes, and the hexanes removed and discarded. This procedure was repeated three times and after that 169 mg (63%) of Product 16 was collected. Purity was confirmed by¹H-NMR.¹H NMR (500 MHz, CDCl₃) 1.33 (s, 12H), 2.63 (t, J= 6.0 Hz, 2H), 3.65-3.69 (m, 6H), 3.72-3.74 (m, 2H), 3.77 (t, J= 5.6 Hz, 2H), 3.88 (t, J= 4.6 Hz, 2H), 4.22 (t, J= 5.5 Hz, 2H), 6.97(t, J= 8.3 Hz, 1H), 7.49 (t, J= 11.4 Hz , 2H).

Example 15. Synthesis of Compound 18

[00302] Compound 18 was prepared as shown in FIG. 3, Scheme 3 following a similar experimental protocol as Compound 4 but intermediate Compound 16 was used in place of N,N-diphenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline and Compound 17 was used in place of Compound A. Product 18, 10mg (13 %) was collected, with abs max at 328 nm and emission max at 395 nm in PBS.

[00303]¹H-NMR spectra confirmed the desired structure.¹H NMR (500 MHz, CDCl₃) 1.66-1.71 (m, 4H), 2.27-2.32 (m, 4H), 2.41(t, J= 6.2 Hz, 4H), 3.48-3.69 (m, 26H), 3.80 (m, 4H), 4.24 (m, 4H), 4.56 (s, 2H), 7.27 (t, J= 8.4 Hz, 2H), 7.58 (d, J= 9.0 Hz, 2H), 7.66-7.75 (m, 6H), 7.98 (d, J= 8.4 Hz, 2H).

Example 16. Synthesis of Compound 19

[00304] Compound 19 was prepared following the same experimental protocol as Compound 4 except Compound 16 was used in place of N,N-diphenyl-4-(4, 4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)aniline and Compound B was used in place of Compound A. Product 19, 10mg (7 %) was collected as waxy material with abs max at 329 nm and emission max at 395nm in PBS.

[00305]¹H-NMR spectra confirmed the desired structure.¹H -NMR spectra confirmed the desired structure.¹HNMR (500 MHz, CDCl₃) 1.98-2.03 (m, 4H), 2.59-2.62 (m, 4H), 2.89-3.01 (m, 4H), 3.13-3.15 (m, 4H), 3.59 (s, 6H), 3.47-3.82 (m, 118H), 3.91 m, 6H), 4.25-4.30 (m, 4H), 4.58 (bs, 2H), 7.12-7.16 (m, 2H), 7.37-7.43 (m, 4H), 7.55-7.57 (m, 2H), 7.60 (d, J= 8.5 Hz, 2H), 7.87 (d, J= 7.9 Hz ,2H).

Example 17. Synthesis of Compounds 7b, and 7

Synthesis of Compound 7b

[00306] Compound 7b was prepared as shown in FIG. 4, Scheme 4. In a flask Compound B (500mg, 0.31 mmoles) and tert-butyl (4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)phenyl)carbamate (208 mg, 0.65 mmoles) were dissolved in 2 mL of DMF. To the mixture CsF (1.65g, 10.9 mmoles) dissolved in 8mL of H₂O was added. The mixture was purged with N₂ for 5 minute and then Pd(OAc)₂ (8.3 mg, 0.037 mmoles) was added. The flask was sealed, and mixture stirred at 80 °C for 13 hours. After that the reaction mix was filtered over celite and washed with 1/1 CHCl₃/MeOH. The filtrate was collected, concentrated and the crude Product Compound 7b was used as such for the next step.

Synthesis of Compound 7

[00307] Compound 7 was prepared as shown in FIG. 4, Scheme 4. In a flask 500mg of compound 7b was taken and dissolved using 6mL MeOH:water (1 : 1) mixture. To this 6mL of concentrated HC1 was added and allowed to stir for 90 minutes. Later it was basified using excess K₂CO₃ and extracted using CHCl₃. After drying 455mg of the

Product 7 was obtained. Yield 90%. λ_abs - 345 nm λ_em - 490 nm

Example 18. Synthesis of Compounds 23a-d & 8a-e

Synthesis of DHP-Naphthalimide (NI) Compounds

[00308] The general procedure for the synthesis of water-soluble DHP-NI compounds is illustrated in FIG. 5, Scheme 5 and involved a Suzuki coupling reaction between the pegylated 550-DHP-BA compound 24 and Naphthalimide derivatives 23a-23d. Synthesis of DHP bridged compounds was carried out in a 3/1 H₂O /DMF solvent system with a concentration of 100mg/ml. Into a Schlenk flask, Compound 24 and naphthalimide derivatives 23a-23d were dissolved completely in DMF and then CsF dissolved in H₂O was added. The reaction mixture was purged with N₂. After 5 minutes, a Pd(OAc)₂ solution was added and the reaction mixture was heated at 82°C for 16 hrs. The reaction mixture was cooled down to room temperature, diluted with chloroform, extracted with water and dried with sodium sulfate. The solvents were evaporated, and the residue was purified by column chromatography eluting with a gradient composed of Chloroform/ Methanol using a small-size silica column with a Yamazen automated system. Synthesis of compound 8a

[00309] Compound 8a (f = 10) was prepared from 24 (0.1g, 0.059mmol) and 23a (0.041 g, 0.129 mmol) using the general procedure as shown in FIG. 5, Scheme 5 to yield 0.071 g of yellow semisolid Product compound 8a (63%, MW = 1928). Purity was confirmed by¹H -NMR spectroscopy.¹H NMR (500 MHz, CDCl₃) δ 1.05 (t, J = 7.4 Hz, 6H), 1.81 (sx, J= 7.5 Hz, 4H), 2.06 (m, 4H), 2.92-3.09 (m, 4H), 3.15 (t, J= 4.6 Hz, 4H), 3.32-3.41 (m, 6H), 3.42-4.03 (m, 82H), 4.13 - 4.26 (m, 4H), 4.67 (s, 2H), 7.57 - 7.69 (m, 4H), 7.81 (dt, J= 11.4, 7.7 Hz, 4H), 8.06 (d, J= 7.8 Hz, 2H), 8.38 (d, J= 8.3 Hz, 2H), 8.74 - 8.63 (m, 4H).

Synthesis of compound 8b

[00310] Compound 8b (f = 10) was prepared from 24 (0.1g, 0.059mmol) and 23b (0.045 g, 0.129 mmol) using the general procedure as shown in FIG. 5, Scheme 5 to yield 0.028 g of semisolid Product compound 8b (24%, MW = 1994). Purity was confirmed by¹H-NMR spectroscopy.¹H NMR (500 MHz, CDCl₃) δ 2.06-2.10 (m, 4H), 3.02 (hept, J= 7.1 Hz, 4H), 3.17 (t, J= 5.0 Hz, 4H), 3.43 - 3.32 (m, 6H), 3.95 - 3.43 (m, 93H), 4.70 (s, 2H), 7.42 - 7.33 (m, 4H), 7.51 (t, J= 7.5 Hz, 2H), 7.59 (t, J= 7.6 Hz, 4H), 7.72 - 7.63 (m, 4H), 7.92 - 7.81 (m, 4H), 8.09 (d, J= 7.9 Hz, 2H), 8.45 (d, J= 8.5 Hz, 2H), 8.73 (dd, J= 9.8, 7.3 Hz, 4H).

Synthesis of compound 8c

[00311] Compound compound 8c (f = 10) was prepared from 24 (0.1g, 0.059mmol) and 23c (0.049 g, 0.129 mmol) using the general procedure as shown in FIG. 5, Scheme 5 to yield 0.024 g of semisolid Product compound 8c (24%, MW = 2054).¹H NMR (500 MHz, CDCl₃) δ 2.08 (t, J= 6.4 Hz, 4H), 3.02 (q, J= 7.9 Hz, 4H), 3.16 (t, J= 4.8 Hz, 4H), 3.31 - 3.41 (m, 6H), 3.43 - 3.88 (m, 82H), 3.89 (s, 6H), 4.69 (s, 2H), 7.05 - 7.14 (m, 4H), 7.22 - 7.32 (m, 4H), 7.62 - 7.72 (m, 4H), 7.78 - 7.90 (m, 4H), 8.09 (d, J= 7.9 Hz, 2H), 8.44 (d, J= 8.4 Hz, 2H), 8.72 (dd, J= 9.9, 7.3 Hz, 4H).

Synthesis of compound 8d

[00312] Compound 8d (f = 10) was prepared from 24 (0.1g, 0.059mmol) and 23d (0.048 g, 0.129 mmol) using the general procedure as shown in FIG. 5, Scheme 5 to yield 0.017 g of semisolid Product compound 8d (14%, MW = 2046).¹H NMR (500 MHz, CDCl₃) 2.07 (q, J= 7.7 Hz, 4H), 3.02 (hept, J= 7.0 Hz, 4H), 3.17 (t, J= 4.9 Hz, 4H), 3.41 - 3.31 (m, 6H), 3.95 - 3.43 (m, 88H), 4.70 (s, 2H), 7.55 - 7.48 (m, 4H), 7.72 - 7.63 (m, 4H), 7.94 - 7.83 (m, 8H), 8.10 (d, J= 7.9 Hz, 2H), 8.49 (d, J= 8.5 Hz, 2H), 8.78 - 8.69 (m, 4H).

Synthesis of compound 8e

[00313] Compound 8e (f = 10) was prepared from 24 (0.1g, 0.059mmol), 23c (0.022 g, 0.059 mmol) and 23d (0.022 g, 0.059 mmol) using the general procedure as shown in FIG. 5, Scheme 5 to yield 0.040 g of semisolid Product compound 8e (33%, MW = 2050).¹HNMR (500 MHz, CDCl₃) 2.08 (d, J= 8.8 Hz, 4H), 3.02 (h, J= 6.9 Hz, 4H), 3.16 (t, J= 4.7 Hz, 4H), 3.40 - 3.32 (m, 6H), 3.43 - 3.83 (m, 88H), 3.89 (s, 3H), 4.70 (s, 2H), 7.07- 7.12 (m, 2H), 7.26 - 7.29 (m, 2H), 7.52 (d, J= 8.4 Hz, 2H), 7.62 - 7.72 (m, 4H), 7.80 - 7.92 (m, 6H), 8.09 (dd, J= 8.0, 3.2 Hz, 2H), 8.46 (ddt, J= 22.6, 8.5, 1.4 Hz, 2H), 8.69 - 8.77 (m, 4H).

Example 19. Synthesis of DHP Bridged Dihydrophenanthrenyl Compounds

[00314] Synthesis of water-soluble DHP bridged DHP compounds involves a Suzuki coupling reaction between a DHP-sulfonamide PEG, diboronic ester and a mono-bromo DHP-sulfonamide PEG, for example, as shown in FIG. 8, Scheme 8. DHP-sulfonamide PEG, diboronic ester 28 and mono-bromo DHP-sulfonamide PEG 36 were employed in the preparation of DHP bridged-sulfonamide PEG DHP compound 20, as provided below.

[00315] Synthesis of compound 28

[00316] DHP-sulfonamide PEG, diboronic ester compound 28 was prepared as shown in Scheme 9. Briefly, dibromo-functionalized DHP-sulfonamide compound B is mixed with DMSO under nitrogen and 3 equivalents of bispinacolatodiboron is added. The reagents are reacted with 12 equivalents of potassium acetate and 4 equivalents of Pd(dppf)Cl₂ catalyst for ~5 hours at 80 °C. The reaction mixture is cooled and extracted with CHCl₃/water. The organic layer is concentrated and purified by column chromatography (silica gel, MeOH-CHCl₃).

[00317] Synthesis of mono-bromo-DHP-sulfonamide PEG monomer compound 36

[00318] Synthesis of compounds 31-36 was performed as illustrated in FIG. 7, Scheme

[00319] Briefly, commercially available phenanthrene, 9, 10-dione compound 31 is reacted with N-bromosuccinimide using methane sulfonic acid overnight, the next day transferred to ice, and the precipitate was washed with water and dried. Product 2- bromophenanthrene,9, 10-dione compound 32 was used for next step as such.

[00320] F or example, 2-bromo-trans-9, 10-dihy drophenanthrene-9, 10-diol (DHP-OH) compound 33 can be prepared as follows. In a conical flask (2000L), about 26g of NaBH4 is added into a stirring water-ethanol mixture (120 mL + 780mL). To this solution, about 24g of 2-bromophenanthrene,9, 10-dione compound 32 is added portion- wise. The reaction mix is allowed stirring for a day. Stop the reaction and neutralize the reaction mixture with dil HC1 acid. After the neutralization, filter the white precipitate and wash with excess water. Thus obtained white precipitate can be washed with very cold (< -15°C) ethanol (100mL) and methanol (100mL).

[00321] 2-bromo-DHP-OSO₃H compound 34 can be prepared as follows. In a 2 neck round bottom flask, DHP-OH compound 33 (3.6 g) and 18C6 (500mg) are dissolved in 120mL of THF. The solution is purged with nitrogen (20min) and NaH (2g) is added. In another round bottom flask, 12g of 1,3 propane sulfone is dissolved in 20mL of THF and nitrogen purged. This sulfone solution is added to the DHP-OH solution by addition funnel over a period of 20-30 minutes. The reaction is stirred at RT for 4-5 hrs. The solvents are evaporated, and the precipitate dissolved in water. Acetone is added to obtain white precipitate of DPS compound 34 in the form of disodium salt. The precipitate is filtered, redissolved in water (minimal amount), neutralized with HC1 and precipitated again in acetone. The precipitation is repeated (2-3 times) followed by centrifugation gives 2-bromo-DHP-OSO₃H compound 34 as white solid.

[00322] 2-bromo-DHP-OSO₂Cl compound 35 can be prepared as follows. 5g of 2- bromo-DHP-OSO₃H compound 34 is added into a round bottom flask and mixed with 25mL of DMF. To this about lOmL of SOCl₂ is added dropwise and the mixture allowed to stir for overnight. Next morning, the reaction mixture is poured into ~200mL water and precipitate filtered and dried to provide 2-bromo-DHP-OSO₂Cl compound 35.

[00323] 2-bromo-DHP-sulfonamide PEG compound 36 can be prepared as follows.

DHP-OSO₂CI compound 35 is mixed with 2.2 equivalent of PEG amine in dichloromethane/TEA mixture. After 3 h sonication reaction the crude product is extracted in dichloromethane followed by column chromatography (silica gel, MeOH- CHCl₃) o provide 2-bromo-DHP-sulfonamide PEG compound 36. Compound 36 was characterized by¹H-NMR.

[00324]¹H NMR (CDCl₃, 300MHz) δ 7.73-7.75 (d, 1H, aromatic), 7.62-7.67 (d, 1H, aromatic), 7.29-7.55 (m, 5H, aromatic), 4.42- 4.47(m, 2H, -CH-CH), 3.5-3.75 (m, 105H, CH₂-O-CH₂), 3.37 (s, 6H, -OMe), 3.14-3.2 (m, 4H, -CH₂), 2.9-2.98 (m, 4H, -CH₂), 1.95- 2 (m, 4H, -CH₂) ppm.

[00325] Synthesis of DHP Bridged-sulfonamide PEGDHP compound 20

[00326] DHP bridged -sulfonamide PEG DHP compound 20 was prepared as shown in FIG. 8, Scheme 8. Briefly, in a round bottom flask the mono-bromo-DHP-sulfonamide PEG monomer compound 36 and DHP-sulfonamide PEG, diboronic ester compound 28 are dissolved in a DMF -water mixture and purged with nitrogen for 10 minutes. Under nitrogen, about 20 equivalents of CsF and of Pd(OAc)₂ (10 mol %) are mixed and heated at 80 °C. After the reaction, solvents are removed from the crude mixture via evaporation and the crude material is purified by column chromatography to provide DHP bridged - sulfonamide PEG DHP compound 20.

[00327]¹H NMR (CDCl₃, 300MHz), δ 7.74-7.96 (m, 15H, aromatic), 7.33-7.44 (m, 5H, aromatic), 4.53-4.68 (m, 6H, -CH-CH), 3.48-3.86 (m, 288H, -CH-CH), 3.37 (s,18H, - OMe), 3.01-3.14 (m, 24H, -CH₂), 2.04 (broad S, 12H, -CH₂) ppm.

[00328] FIG. 9 shows absorption spectrum of the DHP bridged-sulfonamide PEG DHP compound 20 having Abs maxima at 359 nm (left panel). Also shown are fluorescence excitation/emission spectra of the DHP bridged -sulfonamide PEG DHP compound 20 having an emission max at 419 nm (right panel).

[00329] Amino-derivatives of DHP bridged Compounds 21 and 22 were prepared as shown in FIG. 10, Scheme 10.

[00330] Briefly, DHP bridged-sulfonamide PEG DHP compound 20 was dissolved in anhydrous DMF to provide a solution. Under nitrogen atmosphere, the solution was transferred to a reaction flask containing cesium carbonate (100 eq.). tert-Butyl-3- iodopropyl-carbamate solution was diluted from a stock solution (10 mg/mL in anhydrous DMF), and 10 eq. was added. The sealed reaction flask was heated to 50 °C, and the reaction was continued for 1 h under stirring at 500 rpm. The reaction mixture was cooled to RT and the DMF was evaporated in a rotary evaporator under high vacuum.

The crude reaction mixture was diluted with chloroform and washed with 15% w/v brine solution. The organic layer was collected in a 250-mL conical flask, additional chloroform was added, and the mixture was washed three times with 30% w/v brine solution. The organic fraction was dried by adding anhydrous sodium sulfate and then filtered. The chloroform was evaporated in a rotary. Residual DMF was removed under a high vacuum pump. The dried compound 21 was washed with diethyl ether and sonicated for two minutes to eliminate any unreacted tert-butyl-3 -iodopropyl- carbamate. The dried NHBoc amino DHP bridged DHP compound 21 was characterized using¹H NMR.

[00331]¹H NMR (CDCl₃, 300MHz), δ 7.78-7.93 (m, 15H, aromatic), 7.33-7.43 (m, 5H, aromatic), 4.55-5.12 (m, 6H, -CH-CH), 2.88-3.65 (m, 317H, -CH-CH, NCH₂), 1.62- 1.97 (m, -CH₂), 1.41-1.43 (m, 44H, COOtbu), 1.18-1.25 (m, 10H, -CH₂) ppm. The proton signals at ~1.4 ppm indicate the existence of NH-Boc moieties in the compound.

[00332] The NHBoc amino DHP bridged DHP compound 21 was deprotected as shown in FIG. 9, Scheme 8 by dissolving in chloroform and adding TFA. The mixture was stirred at room temperature for 1 hour. After that the reaction mix was dried, the residue dissolved in chloroform and washed with diluted brine. The organic layer was collected, filtered over Na₂SO₄ and concentrated to dryness to afford amino DHP bridged DHP compound 22.

[00333]¹H NMR (CDCl₃, 300MHz), δ 6.91-8.47 (m, 20H, aromatic), 4.48-4.66 (m, 6H, -CH-CH), 2.69-3.65 (m, 317H, -CH-CH, NCH₂), 1.95-2.02 (m, -16, CH₂) ppm.

[00334] DHP Bridged Compound-Acceptor dye formation. Amino DHP bridged DHP compound 22 was dissolved in anhydrous DMSO. To this, acetonitrile and diisopropylethylamine were added. A solution of 2 eq. acceptor dye NHS ester (Dy495NHS; Dyomics GmbH) was prepared in anhydrous DMSO, and added to the amino DHP bridged DHP compound 22 solution. The mixture was stirred for two hours at room temperature, protected from light, then purified by LH20 to provide DHP timer- Dy495 tandem dye compound 25. The absorption spectrum of DHP timer-Dy495 tandem dye compound 25 is shown in FIG. 10. The absorption spectrum has two maxima at 370 nm and 500 nm (left panel). FIG. 10 also shows the fluorescence emission spectrum of DHP timer-Dy495 tandem dye 25 after excitation at 355 nm exhibiting an emission max at 532 nm (right panel).

[00335] Example 20. Synthesis of DHP bridged fluorenyl compounds

[00336] Inventive DHP bridged fluorenyl compounds were prepared from mono- bromo fluorenyl compounds, for example, as shown in FIG. 12, Scheme 11 illustrating preparation of DHP bridged fluorenyl compound 30 from mono-bromo fluorenyl compound 27 and DHP-sulfonamide PEG, diboronic ester compound 28.

[00337] Starting compound 26: 3,3'-(2-bromo-9H-fluorene-9,9-diyl)dipropionic acid was prepared as described from Chem. Comm. 2019, Vol. 55, 95, 14287-14290.

[00338] Synthesis of intermediate compound 27: di-tert-but1y,l1'- (2-bromo-9H- fluorene-9,9-diyl)bis(3-oxo-7,10,13-trioxa-4-azahexadecan-16-oate). 100 mg of compound 26 were placed in a 20mL vial and dried overnight in vacuum oven at 50°C. After that, the solid was dissolved in 0.2 mL of DMF and once all solid dissolved, 1 mL of MeCN was added. To the mixture 0.99 mL of DIPEA and 178 mg of TSTU were added and the mixture stirred for 70 minutes at room temperature in the dark. Then, 0.2 mL of amino-PEG3-t-butyl ester compound BP20697 (Broadpharm®) were added together with 50 μL of DIPEA and the mixture left to stir for 14 hours. The solvents were evaporated, and the residue purified by column chromatography eluting with a gradient composed of Chloroform/Methanol using a small-size silica column with a Yamazen automated system. Fractions containing the desired product were collected to afford 164 mg (70%) of product as semisolid materia¹H. -NMR spectra confirmed the desired structure.¹HNMR (500 MHz, CDCl₃): 1.37-1.45 (m, 22H), 2.35-2.47 (m, 8H), 3.20-3.29 (m, 4H), 3.37-3.42 (4H, m), 3.46-3.58 (m, 16H), 3.63 (t, J= 6.5 Hz, 4H), 5.66 (bs, 2H), 7.33-7.35 (m, 2H), 7.39-7.40 (m, 1H), 7.47-7.49 (m, 1H), 7.52-7.53 (m, 1H), 7.55-7.57 (m, 1H), 7.65-7.67 (m, 1H).

[00339] Synthesis of Compound 29. In a flask Compound 28 (53 mg, 0.0315 mmoles) and Compound 27 (60 mg, 0.066 mmoles) were dissolved in 1 mL of DMF. To the mixture CsF (120mg, 0.79 mmoles) dissolved in 0.55 mL of H₂O was added. The mixture was purged with N₂ for 5 minute and then Pd(OAc)₂ (0.85 mg, 0.0038 mmoles) was added. The flask was sealed, and the mixture stirred at 80 °C for 13 hours. After that, the reaction mix was filtered over celite and washed with 1/1 CHCl₃/MeOH. The filtrate was collected, concentrated, and purified with an automated chromatography system using neutral Silica as a stationary phase and CHCl₃/MeOH as a mobile phase. Product compound 29, 46 mg (47%) was collected as yellow waxy material.¹H-NMR spectra confirmed the desired structure compound 29.¹H NMR (500 MHz, CDCl₃) 1.42 (36H, s), 1.51-1.62 (m, 8H), 2.01-2.09 (m, 4H), 2.39-2.49 (m, 12H), 2.59-2.71 (m, 4H), 2.93-3.05 (m, 4H), 3.08-3.14 (m, 4H), 3.18-3.27 (m, 6H), 3.32-3.92 (m, 148H), 4.61-4.72 (bs, 2H), 6.10-6.32 (m, 4H), 7.30-7.97 (m, 20H).

[00340] Synthesis of Compound 30. In a 20mL vial, 46 mg of Compound 29 (14.8 μmol) were dissolved in 0.8 mL of DMF. CS₂CO₃ (145mg, 444 μmol) and tert-butyl (3- lodopropyl) carbamate (9.3mg, 33 μmol) were added and the mixture stirred for 2 hours at 50 °C. After that, the solvents were evaporated, the residue dissolved in chloroform, and the solution washed with diluted brine. The organic layer was collected, dried over Na₂SO₄, filtered and dried. 3mL of diethyl ether were added to the collected residue and the mix vortexed. After decanting diethyl ether, the residue was dried to afford 38mg of product.¹H-NMR confirmed the attachment of the tert-butyl carbamate to the sulfonamide groups.¹HNMR (500 MHz, CDCl₃) 1.41-1.43 (54H, m), 1.51-1.66 (m, 12H), 1.94-2.03 (m, 4H), 2.41-2.51 (m, 12H), 2.59-3.29 (m, 16H), 3.38-3.92 (m, 154H), 4.61-4.67 (bs, 2H), 5.90-6.18 (m, 4H), 7.30-7.94 (m, 20H).

[00341] Removal of the tert-butyl ester and tert-butyl carbamate groups was achieved in a single step as follows: the material was dissolved in 1 mL of chloroform and 0.5 mL of TEA was added. The mixture was stirred for 2 hours at room temperature and then dried to afford the deprotected product Compound 30 (40 mg).¹H-NMR analysis confirmed successfill tert-butyl groups removal in the 1.4 ppm region.

[00342] General procedure for tandem dye formation

[00343] In a 4mL vial containing a spinning bar, 0.9 mg of Compound 30 (0.308 μmol), previously dried overnight at 50 °C in vacuum oven, was dissolved in 90 μL of MeCN and 20 μL of DIPEA added. To the solution 1.1 equivalents of NHS acceptor dye or tetrafluorophenyl (TFP) acceptor dye was added as DMSO solution, and the mixture sonicated for 65 minutes in the dark to form the DHP bridged tandem dye.

[00344] General procedure for NHS active ester UV dye formation

[00345] Active NHS esters of the DHP bridged dyes and tandem dyes were prepared as shown in FIG. 6, Scheme 6. TSTU (4 equivalents) and 10 μL of DIPEA were added to the DHP bridged dye or DHP bridged tandem dye. The mixture was stirred at room temperature for 60 minutes and then dried using a rotavapor while maintaining the temperature below 39 °C. To remove residual DMSO and unreacted reagents, 2 mL of cold diethyl ether were added, the resulting mixture vortexed, and the solvent decanted. This procedure was repeated two more times and the final NHS activated DHP bridged tandem dyes dried under high vacuum. The resulting NHS activated DHP bridged tandem dye material was dissolved in 90 μL of dry DMSO to use for antibody conjugation.

[00346] To prepare UV conjugate compound 37 the same protocol for tandem preparation is followed but instead of adding acceptor dyes, 1.1 eq of m-PEG2-NHS ester compound BP-23656 (Broadpharm) was added.

[00347] General procedure for antibody conjugation

[00348] In an Eppendorf vial, 0.35 mg of a selected antibody in borate buffer (total volume about 65 μL) was added together with the desired aliquot of an NHS active DHP bridged tandem dye solution (about 14 μL). The mixture was placed in a shaker for 75 minutes and then purified using a size exclusion spin column (Zeba) to collect the DHP bridged tandem dye antibody conjugate.

[00349] Table 2 shows normalized emission of UV DHP bridged tandem dye-antibody conjugates 38-46 upon excitation at 355nm in PBS 1×. The emission max for UV DHP bridged conjugate compound 37 without acceptor dye was 415 nm. The emission maxima range for tandem conjugate compounds ranged from 538 nm to 925 nm.

[00350] Table 2. UV tandem antibody conjugates of Compound 30

[00351] A chart showing normalized emission spectra of UV DHP bridged fluorenyl tandem conjugates 38-46 and UV DHP bridged fluorenyl conjugate 37 upon excitation at 355nm in PBS lx is shown in FIG. 13. A single excitation wavelength at 355 nm results in multiple emission wavelengths from 415 nm to 925 nm.

[00352] An overlay of Absorption spectra of UV CF850 tandem (A) conjugate 45 and of reference dye CF850 (B) in PBS lx is shown in FIG. 14.

[00353] Emission spectra of UV Compound 30 donor molecule before (A) and after (B) attachment of acceptor dye CF850 to form UV CF850 tandem conjugate 45 is shown in FIG. 15 showing the donor emission quenching (PBS lx, ex at 355nm).

[00354] Emission spectra of UV CF850 tandem conjugate 45 after ex at 355nm (A) is shown in FIG. 16 with an em max at 887 nm and blank (B) in PBS lx.

[00355] Flow cytometry results from CD8 UV -CF850 tandem conjugate 45 in whole blood sample (Cytoflex LX bp 885/40) are shown in FIG. 17. The circled area corresponds to CDS positive cells.

CLAUSES

[00356] Clause 1. A compound comprising a structure according to Formula (I):

wherein each

and

is independently selected from the group consisting of a substituted or unsubstituted benzene, benzene derivative, monocyclic aryl group, polycyclic aryl group, monocyclic heteroaryl group, and polycyclic heteroaryl group; each T is independently selected from the group consisting of C, C(R¹), N, N(R¹), P, O, S, and Si(R¹); each X is independently C, Si, O, N, or P; each Y is independently C, Si, O, N, or P; each R¹ is independently selected from the group consisting of a water- solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, hydroxy, halogen, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfmamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

>

each R² is independently selected from the group consisting of a water- solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

each R³ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a water- solubilizing moiety, a chromophore, functional moiety, binding partner, E, and a PEG group; each optional R³' is independently selected from the group consisting of R¹, R², aryl, heteroaryl, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, halogen, a water-solubilizing moiety, functional moiety, a PEG group, -B(R³)₂, O-alkyl, O-aryl, NR'R', NHR', NH₂ -S-R', SO₃H, -SO₂R', -SO₂NR', -PR'₃, POR'₃, -SiR'₃, -ammonium, alkylammonium, and arylammonium; each R' is independently selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted aryl, optionally substituted with one or more independently selected PEG groups, water solubilizing moiety, linked water- solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group; each R⁴ is independently selected from the group consisting of H, alkyl, PEG, a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a chromophore, a functional group, a conjugation tag, a binding partner, a linked E, carboxylic amine, amine, carbamate, carboxylic acid, carboxylate ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, alkene, tetrazine, aldehyde, and thiol, or protected groups thereof; each optional R⁴' is selected from the group consisting of R¹, R², R³ and R³', optionally wherein two R⁴' together form an unsubstituted or substituted cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, cycloalkoxy, aryl, or heteroaryl having 3 to 9 ring members; each R⁷ is independently selected from the group consisting of H, hydroxyl, C₁- C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂haloalkyl, C₁- C₁₂ alkoxy, C₂-C₁₈ (hetero)aryloxy, C₂-C₁₈ (hetero)arylamino, C₂-C₁₂ carboxylic acid, C₂- C₁₂ carboxylate ester, and C₁-C₁₂ alkoxy, a functional group, a chemoselective functional group, conjugation tag, linked conjugation tag, E, a linker, sulfonic acid, sulfonate, C₁- C₁₂ alkyl sulfonate, sulfonamide; at least one of R¹, R², R³, R³', R⁴, or R⁴' comprises an independently selected water-solubilizing moiety or linked water-solubilizing moiety; each Q is independently a bond, NH, NR⁴, C₁-C₁₂ alkylene, CHR⁴, and CH₂; each Z is independently CH₂, CHR⁴, O, NR⁴, or NH; each W¹ is independently a water-solubilizing moiety;

L¹, L², and L³ are each independently selected linker moieties; each E is independently selected from the group consisting of a chromophore, a functional moiety, a substrate, a conjugation tag, and a binding partner; each f is independently an integer from 0 to 50; each m is independently 0 or 1; each n is independently 0, 1, 2, 3, or 4; each s is independently 1 or 2; and each t is independently 0, 1, 2, or 3.

[00357] Clause 2. A compound comprising a structure according to Formula (I):

wherein | each

and

is independently selected from the group consisting of a substituted or unsubstituted benzene, benzene derivative, monocyclic aryl group, polycyclic aryl group, monocyclic heteroaryl group, and polycyclic heteroaryl group; each T is independently selected from the group consisting of C, C(R¹), N, N(R¹), P, O, S, and Si(R¹); each X is independently C, Si, O, N, or P; each Y is independently C, Si, O, N, or P; each R¹ and R² is independently selected from the group consisting of a water- solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfmamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

each R³ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a water- solubilizing moiety, a chromophore, functional moiety, binding partner, E, and a PEG group; each optional R³' is independently selected from the group consisting of R¹, R², aryl, heteroaryl, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, halogen, a water-solubilizing moiety, functional moiety, a PEG group, -B(R³)₂, O-alkyl, O-aryl, NR'R', NHR', NH₂ -S-R', SO₃H, -SO₂R', -SO₂NR', -PR'₃, POR'₃, -SiR'₃, -ammonium, alkylammonium, and arylammonium; each R' is independently selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted aryl, optionally substituted with one or more independently selected PEG groups, water solubilizing moiety, linked water- solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group; each R⁴ is independently selected from the group consisting of H, alkyl, PEG, a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a chromophore, a functional group, a conjugation tag, a binding partner, a linked E, carboxylic amine, amine, carbamate, carboxylic acid, carboxylate ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, alkene, tetrazine, aldehyde, and thiol, or protected groups thereof; each optional R⁴' is selected from the group consisting of R¹, R², R³ and R³', optionally wherein two R⁴' together form an unsubstituted or substituted cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, cycloalkoxy, aryl, or heteroaryl having 3 to 9 ring members; each R⁷ is independently selected from the group consisting of H, hydroxyl, C₁- C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C3-C₁₂ cycloalkyl, C₁-C₁₂haloalkyl, C₁- C₁₂ alkoxy, C₂-C₁₈ (hetero)aryloxy, C₂-C₁₈ (hetero)arylamino, C₂-C₁₂ carboxylic acid, C₂- C₁₂ carboxylate ester, and C₁-C₁₂ alkoxy, a functional group, a chemoselective functional group, conjugation tag, linked conjugation tag, E, a linker, sulfonic acid, sulfonate, C₁- C₁₂ alkyl sulfonate, sulfonamide; at least one of R¹, R², R³, R³', R⁴, or R⁴' comprises an independently selected water-solubilizing moiety or linked water-solubilizing moiety; each Q is independently a bond, NH, NR⁴, C₁-C₁₂ alkylene, CHR⁴, and CH₂; each Z is independently CH₂, CHR⁴, O, NR⁴, or NH; each W¹ is independently a water-solubilizing moiety;

L¹, L², and L³ are each independently selected linker moieties; each E is independently selected from the group consisting of a chromophore, a functional moiety, a substrate, a conjugation tag, and a binding partner; each f is independently an integer from 0 to 50; each m is independently 0 or 1; each n is independently 0, 1, 2, 3, or 4; each s is independently 1 or 2; and each t is independently 0, 1, 2, or 3.

[00358] Clause 3. A compound according to clause 1 or 2, wherein the compound comprises a structure according to any one of Formulas (IIa), (IIb), (IIc), (lid), or (IIe):

; or

[00359] Clause 4. A compound according to any one of clauses 1 to 3, wherein the compound comprises a structure according to any one of Formulas (IIIa), (IIIb), (IIIc), or (IIId):

[00360] Clause 5. The compound of any one of clauses 1, 2 and 4, wherein the compound comprises a structure according to any one of Formulas (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf):

[00361] Clause 6. The compound of any one of clauses 1, 2 and 4, wherein the compound comprises a structure according to any one of Formulas (Va), (Vb), (Vc), or

[00362] Clause 7. The compound of any one of clauses 1 to 5, wherein T is independently selected from the group consisting of C, C(R¹), N, and N(R¹).

[00363] Clause 8. The compound of any one of clauses 1 to 5, wherein T is independently selected from the group consisting of C and C(R¹).

[00364] Clause 9. The compound of any one of clauses 1 to 5, wherein T is independently selected from the group consisting of C, C(R¹), P, O, S, and Si(R¹).

[00365] Clause 10. The compound of any one of clauses 1 to 9, where both X and Y are independently C or Si.

[00366] Clause 11. The compound of any one of clauses 1 to 10, wherein both X and

Y are C.

[00367] Clause 12. The compound of any one of clauses 1 to 9, wherein X and Y are independently selected from the group consisting of C, O, N, and P.

[00368] Clause 13. The compound of any one of clauses 1 to 9 and 12, wherein X and

Y are independently selected from the group consisting of C and O.

[00369] Clause 14. The compound of any one of clauses 1 to 9 and 12, wherein X and

Y are independently selected from the group consisting of C and N.

[00370] Clause 15. The compound of any one of clauses 1 to 9 and 12, wherein X and

Y are independently selected from the group consisting of C and P. [00371] Clause 16. The compound of any one of clauses 1 to 15, wherein each of

and

is independently selected from the group consisting of

172

, wherein / each of

and

is independently optionally substituted with (R³ )n; and p=l, 2, 3, 4, 5, 6, or 7.

[00372] Clause 17. The compound of any one of clauses 1 to 16, wherein

and

are each independently selected from the group consisting of:

, optionally wherein R² is C₁-C₆ alkyl, phenyl, or phenyl substituted with halogen, C₁-C₆ alkoxy, or CN.

[00373] Clause 18. The compound of any one of clauses 1 to 17, wherein

and

are the same. [00374] Clause 19. The compound of any one of clauses 1 to 17, wherein

and

are different.

[00375] Clause 20. The compound of any one of clauses 1 to 19, wherein

and

are not optionally substituted DHP groups.

[00376] Clause 21. The compound of any one of clauses 1 to 19, wherein

and

are not optionally substituted fluorenyl groups.

[00377] Clause 22. The compound of any one of clauses 1 to 19, wherein

and

are independently selected from optionally substituted DHP groups or optionally substituted fluorenyl groups .

[00378] Clause 23. The compound of any one of clauses 1 to 16 and 18 to 22, wherein R³' is independently selected from the group consisting of halogen, NH₂, C₁-C₆ alkyl, C₁- C₆ alkoxy, a PEG group, PEG carboxylic acid, phenyl, or phenyl substituted with halogen, C₁-C₆ alkoxy, or CN

,

[00379] Clause 24. The compound of any one of clauses 1 to 23, wherein R¹ is independently selected from the group consisting of a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, sulfonamide-PEG, phosphoramide-PEG, alkoxy sulfonic acid,

; optionally wherein R¹ is independently selected from the group consisting of sulfonamide-PEG, alkoxy sulfonic acid,

[00380] Clause 25. The compound of any one of clauses 1 to 4 and 6 to 24, wherein the compound comprises a a structure selected from the group consisting of:

[00381] Clause 26. The compound of any one of clauses 1 to 25, wherein R² is hydrogen.

[00382] Clause 27. The compound of any one of clauses 1 to 24, wherein R¹ and R² are the same.

[00383] Clause 28. The compound of any one of clauses 1 to 24, wherein R¹ and R² are the different.

[00384] Clause 29. The compound of clause 25, wherein G is independently propyl, phenyl, 4-methoxyphenyl, or 4-cy anophenyl.

[00385] Clause 30. The compound of any one of clauses 1 to 29, wherein the compound is a small molecule dye.

[00386] Clause 31. The compound of any one of clauses 1 to 29, wherein the compound is water-soluble.

[00387] Clause 32. The compound of any one of clauses 1 to 30, wherein the compound is a fluorescent compound.

[00388] Clause 33. The compound of any one of clauses 1 to 33, wherein the compound exhibits excitation maxima in a range of from about 300 to about 810 nm, or about 300 to about 650 nm, or about 300 to about 565 nm, or about 300 to about 500 nm, or about 320 to about 420 nm.

[00389] Clause 34. The compound of any one of clauses 1 to 34, wherein the compound can emit at an ultraviolet, violet, blue, yellow, green, red, or near infrared (NIR) wavelength.

[00390] Clause 35. The compound of any one of clauses 1 to 34, wherein the compound is a water-soluble fluorescent compound and small molecule dye.

[00391] Clause 36. A tandem dye comprising the compound of any one of clauses 1 to

51.

[00392] Clause 37. The tandem dye of clause 36, wherein the compound is a donor dye.

[00393] Clause 38. The tandem dye of clause 36, wherein the compound is an acceptor dye.

[00394] Clause 39. The tandem dye of any of clauses 36 to 38, wherein one or more, two or more, three or more, four or more, or from 1-30, 2-20, 3-15, or 4-10 fluorescent dye are bonded to the compound of any one of clauses 1 to 35. [00395] Clause 40. A labeled specific binding partner comprising the compound of any one of clauses 1 to 35, and one or more, two or more, three or more, four or more, or from 1-30, 2-20, 3-15, or 4-10 binding partners bonded to the compound of any one of clauses 1 to 35.

[00396] Clause 41. A labeled specific binding partner comprising the tandem dye of any one of clauses 36 to 39, and one or more, two or more, three or more, four or more, or from 1-30, 2-20, 3-15, or 4-10 binding partners bonded to the tandem dye of any one of clauses 36 to 39.

[00397] Clause 42. The tandem dye of clause 39 or labeled specific binding partner of clause 41, wherein the bond is a covalent bond.

[00398] Clause 43. The tandem dye of clause 39 or labeled specific binding partner of clause 41, wherein the bond is a non-covalent bond.

[00399] Clause 44. The labeled specific binding partner of clause 40, wherein the bond is a covalent bond.

[00400] Clause 45. The compound of clause 40, wherein the bond is a non-covalent bond.

[00401] Clause 46. The labeled specific binding partner of clause 40 or tandem dye of clause 41, wherein the specific binding partner is elected from the group consisting of a protein, peptide, affinity ligand, antibody, antibody fragment, carbohydrate, lipid, nucleic acid, and an aptamer.

[00402] Clause 47. The labeled specific binding partner or tandem dye of clause 46, wherein the specific binding partner is an antibody.

[00403] Clause 48. The labeled specific binding partner or tandem dye of clause 47, wherein the specific binding partner is an antibody specific for a target analyte.

[00404] Clause 49. A method for detecting a target analyte in a sample comprising: providing a sample that is suspected of containing the analyte; and contacting the sample with a specific binding partner conjugated to a water-soluble fluorescent compound of clause 35, or tandem dye according to any one of clause 36 to 39, wherein the binding partner is capable of interacting with the target analyte.

[00405] Clause 50. The method of clause 49, wherein the specific binding partner is elected from the group consisting of a protein, peptide, affinity ligand, antibody, antibody fragment, carbohydrate, lipid, nucleic acid, and an aptamer. [00406] Clause 51. The method of clauses 49 or 50, wherein the specific binding partner is an antibody.

[00407] Clause 52. The method of any one of clauses 49 to 51, wherein the specific binding partner is an antibody specific for a target analyte.

[00408] Clause 53. The method of any one of clauses 49 to 52, wherein the method is configured for flow cytometry.

[00409] Clause 54. A compound of any one of clauses 1 to 35, labeled specific binding partner of clause 40 or 41, or tandem dye of any one of clauses 36 to 39, wherein the compound, tandem dye, or labeled specific binding partner is bound to a substrate.

[00410] Clause 55. The method of any one of clauses 40 to 43, wherein the method is configured as an immunoassay.

[00411] Clause 56. A kit comprising at least one compound of any one of clauses 1 to 35, tandem dye of any one of clauses 36 to 39, or labeled specific binding partner of clause 40 or 41, optionally wherein the kit further comprises a container, buffer, and/or instructions.

[00412] Clause 57. The kit of clause 56, wherein the compound, tandem dye, or labeled specific binding partner comprise a conjugation tag for attachment of an acceptor dye, donor dye, support, or binding partner to the compound or tandem dye.

Claims

WE CLAIM:

1. A fluorescent compound comprising a structure according to Formula (I):

wherein each

and

is independently selected from the group consisting of a substituted or unsubstituted benzene, benzene derivative, monocyclic aryl group, polycyclic aryl group, monocyclic heteroaryl group, and polycyclic heteroaryl group; each T is independently selected from the group consisting of C, C(R¹), N, N(R¹), P, O, S, and Si(R¹); each X is independently C or Si; each Y is independently C or Si; each R¹ is independently selected from the group consisting of a water- solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, hydroxy, halogen, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, alkoxy sulfonic acid, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

and

each R² is independently selected from the group consisting of a water- solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, carbonyl, acyl, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, phosphonate, alkoxy sulfonamide PEG, alkoxy sulfonate, alkoxy sulfonic acid, alkyl sulfonate, alkyl sulfonate salt,

and

each R³ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a water-solubilizing moiety, a chromophore, functional moiety, binding partner, E, a PEG group, a PEG ester, and a PEG carboxylic acid; each optional R³' is independently selected from the group consisting of R¹, R², substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, halogen, a water-solubilizing moiety, functional moiety, a PEG group, PEG carboxylic acid, alkyl amido PEG ester, alkyl amido PEG carboxylic acid, - B(R³)₂, 0-alkyl, O-aryl, arylalkoxy, alkylamide, alkylamidoPEG, aryl-CN, NR'R', NHR', NH₂, -S-R', SOSH, -SO₂R', -SO₂NR', -PR'₃, POR'₃, -SiR'₃, - ammonium, alkylammonium, and arylammonium; each R' is independently selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted aryl, optionally substituted with one or more independently selected PEG groups, water solubilizing moiety, linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, binding partner, linked binding partner, a functional group, a linked functional group; each R⁴ is independently selected from the group consisting of H, alkyl, a PEG group, a linked PEG group, a water-solubilizing moiety, a linked water- solubilizing moiety, a linker moiety, a chromophore, a functional group, a conjugation tag, a binding partner, a linked E, carboxylic amine, amine, carbamate, carboxylic acid, carboxylate ester, maleimide, activated ester, N- hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, alkene, tetrazine, aldehyde, and thiol, or protected groups thereof; each optional R⁴' is selected from the group consisting of R¹, R², R³ and R³', optionally wherein two R⁴' together form an unsubstituted or substituted cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, cycloalkoxy, aryl, or heteroaryl having 3 to 9 ring members; each R⁷ is independently selected from the group consisting of H, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C3-C₁₂ cycloalkyl, C₁- C₁₂ haloalkyl, C₁-C₁₂ alkoxy, C₂-C₁₈ (hetero)aryloxy, C₂-C₁₈ (hetero)arylamino, C₂-C₁₂ carboxylic acid, C₂-C₁₂ carboxylate ester, and C₁-C₁₂ alkoxy, a functional group, a chemoselective functional group, conjugation tag, linked conjugation tag, E, a linker, sulfonic acid, sulfonate, C₁-C₁₂ alkyl sulfonate, sulfonamide; at least one of R¹, R², R³, R³', R⁴, or R⁴' comprises an independently selected water-solubilizing moiety or linked water-solubilizing moiety; each Q is independently a bond, NH, NR⁴, C₁-C₁₂ alkylene, CHR⁴, and CH₂; each Z is independently CH₂, CHR⁴, O, NR⁴, or NH; each W¹ is independently a water-solubilizing moiety;

L¹, L², and L³ are each independently selected linker moieties; each E is independently selected from the group consisting of a chromophore, a functional moiety, a substrate, a conjugation tag, and a binding partner; each f is independently an integer from 0 to 50; each m is independently 0 or 1; each n is independently 0, 1, 2, 3, or 4; each s is independently 1 or 2; and each t is independently 0, 1, 2, or 3.

2. The compound of claim 1, comprising a structure according to any one of Formulas (IIa), (IIb), (lie), (IId), or (IIe):

3. The compound of claim 1, comprising a structure according to any one of Formulas (IIIa), (IIIb), (IIIc), or (IIId):

.

4. The compound of claim 1, comprising a structure according to any one of Formulas

(IVa), (IVb), (IVc), (IVd), (IVe), or (IVf):

5. The compound of claim 1, comprising a structure according to any one of Formulas (Va), (Vb), (Vc), or (Vd):

6. The compound of claim 1, wherein each

and

is independently selected from the group consisting of

206

wherein each

and

is independently optionally substituted with (R^3')_n; and p=1, 2, 3, 4, 5, 6, or

7.

The compound of claim 1, wherein

and

are the same.

8. The compound of claim 1, wherein

are different.

9. The compound of claim 1, selected from the group consisting of:

wherein each compound is independently optionally substituted with (R^3')_n; and each G is independently propyl, phenyl, 4-methoxyphenyl, or 4-cy anophenyl.

10. The compound of any one of claims 1-9, further comprising a binding partner bonded to said compound.

11. The compound of claim 10, wherein the binding partner is selected from the group consisting of an antibody, antigen-binding fragment of an antibody, protein, peptide, affinity ligand, sugar, lipid, nucleic acid, or aptamer.

12. The compound of claim 11, wherein the binding partner is an antibody.

13. A fluorescent dye or a binding partner covalently bonded to a compound of any one of claims 1-12.

14. A tandem dye comprising a compound of any one of claims 1-13.

15. A method for detecting an analyte in a sample comprising: providing a sample that is suspected of containing the analyte; contacting the sample with a binding partner conjugated to a compound or tandem dye of any one of claims 1-14.

16. The method of claim 15, wherein the binding partner is selected from the group consisting of an antibody, antigen-binding fragment of an antibody, protein, peptide, affinity ligand, sugar, lipid, nucleic acid, or aptamer.

17. The method of claim 15, wherein the binding partner is an antibody optionally wherein: a. the method is configured for flow cytometry; b. the compound is bound to a substrate; c. the analyte is a protein expressed on a cell surface; d. the method is configured as an immunoassay; or e. the method further comprises providing additional binding partners for detecting additional analytes simultaneously.

18. A kit comprising at least one compound or tandem dye according to any one of claims 1 to 14, optionally wherein the kit further comprises a container, buffer, and/or instructions, further optionally wherein the compound or dye comprises a conjugation tag for attachment of an acceptor dye, donor dye, support, or binding partner to the compound or tandem dye.